WO2024254193A1

WO2024254193A1 - Tandem guide agents and compositions and uses thereof

Info

Publication number: WO2024254193A1
Application number: PCT/US2024/032622
Authority: WO
Inventors: Karl BARBER; Franciscus POELWIJK; Joao Vieira RODRIGUES; Roy ZIBLAT
Original assignee: Generate Biomedicines, Inc.
Priority date: 2023-06-06
Filing date: 2024-06-05
Publication date: 2024-12-12

Abstract

Provided herein are tandem guide agents and tandem guide complexes, and methods and compositions of using the same.

Description

TANDEM GUIDE AGENTS AND COMPOSITIONS AND USES THEREOF

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63/506568 filed on June 06, 2023 the entire contents each of which is hereby incorporated by reference in its entirety.

BACKGROUND

[0002] Polynucleotide synthesis and assembly, e.g., for generating large DNA sequences, is essential for a number of applications. However, current methods for DNA synthesis and assembly suffer from issues such as high cost and/or low-quality DNA products. For example, the per-base cost of generating libraries of longer DNA sequences currently exceeds that of the constituent oligonucleotides.

SUMMARY OF THE INVENTION

[0003] The present disclosure provides, among other things, tandem guide agents and tandem guide complexes and methods of using the same. The present disclosure encompasses a recognition that tandem guide agents and tandem guide complexes as described herein have beneficial characteristics useful for a number of applications where two materials are to be brought together in close proximity. For example, in some embodiments, provided tandem guide agents and tandem guide complexes are useful for in vitro assembly of polynucleotide sequences (e.g., DNA sequences), production of protein microarrays, self-assembly of micro- and nanomaterials, among others. In some embodiments, provided tandem guide agents and tandem guide complexes are based on CRISPR-Cas systems.

[0004] In some embodiments, the present disclosure provides in vitro methods that comprise: obtaining or providing a tandem guide complex or components thereof, wherein the tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, and contacting the tandem guide complex with a first polynucleotide sequence and a second polynucleotide sequence, wherein the tandem guide complex is capable of binding to the first polynucleotide sequence and the second polynucleotide sequence.

[0005] In some embodiments, the present disclosure provides in vitro methods that comprise: contacting a first polynucleotide sequence and a second polynucleotide sequence with a tandem guide complex, wherein the tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, wherein the first variant Cas protein binds the first polynucleotide sequence and the second variant Cas protein binds the second polynucleotide sequence, and assembling the first polynucleotide sequence and the second polynucleotide sequence to form an assembled polynucleotide that comprises the first polynucleotide sequence and the second polynucleotide sequence.

[0006] In some embodiments, the first polynucleotide sequence and/or the second polynucleotide sequence are present in solution. In some embodiments, the first polynucleotide sequence and/or the second polynucleotide sequence are associated with a surface. In some embodiments, the first polynucleotide sequence and the second polynucleotide sequence are each present in solution. In some embodiments, the first polynucleotide sequence and the second polynucleotide sequence are each associated with a surface. In some embodiments, the surface associated with the first polynucleotide and the surface associated with the second polynucleotide arc different.

[0007] In some embodiments, the present disclosure provides in vitro methods that comprise: obtaining or providing a surface associated with a first polynucleotide sequence, contacting the first polynucleotide with a tandem guide complex and a second polynucleotide sequence, wherein the tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, and wherein the first valiant Cas protein binds the first polynucleotide sequence and the second variant Cas protein binds the second polynucleotide sequence, and assembling the first polynucleotide sequence and the second polynucleotide sequence to form an assembled polynucleotide that comprises the first polynucleotide sequence and the second polynucleotide sequence. In some embodiments, the second polynucleotide sequence is present in solution. In some embodiments, the second polynucleotide sequence is associated with a second, different surface. In some embodiments, the surfaces comprise or consist of planar surfaces, beads (e.g., microbeads), and/or cell surfaces. [0008] In some embodiments, the contacting brings the first polynucleotide sequence and the second polynucleotide sequence in close proximity of one another. In some embodiments, the contacting brings the first polynucleotide sequence and the second polynucleotide sequence within a proximity of about 200 nm or less of one another. In some embodiments, the proximity of the first polynucleotide sequence and the second polynucleotide sequence are measured and/or characterized using a proximity assay, such as, e.g., an AlphaLIS A proximity assay.

[0009] In some embodiments, contacting the first polynucleotide sequence and the second polynucleotide sequence with the tandem guide complex occurs simultaneously.

[0010] In some embodiments, contacting comprises contacting the first polynucleotide sequence and the tandem guide complex and subsequently contacting the second polynucleotide sequence, such that the tandem guide complex binds to both the first polynucleotide sequence and the second polynucleotide sequence.

[0011] In some embodiments, the first polynucleotide sequence and/or the second polynucleotide sequence comprise sticky ends. In some embodiments, the first polynucleotide sequence and/or the second polynucleotide sequence are restriction digested. In some embodiments, the first polynucleotide sequence and/or the second polynucleotide sequence are restriction digested prior to the contacting step.

[0012] In some embodiments, the first polynucleotide sequence and/or the second polynucleotide sequence are not limited to a particular length.

[0013] In some embodiments, the first polynucleotide sequence and/or the second polynucleotide sequence have a length of 5 to 5000 nucleotides. In some embodiments, the first polynucleotide sequence and/or the second polynucleotide sequence have a length of 10 to 1000 nucleotides. In some embodiments, the first polynucleotide sequence and/or the second polynucleotide sequence have a length of 25 to 500 nucleotides. [00141 In some embodiments, assembling comprises ligating the first polynucleotide sequence and the second polynucleotide sequence to form the assembled polynucleotide.

[0015] In some embodiments, assembling comprises annealing the first polynucleotide sequence and the second polynucleotide sequence to each other followed by amplification to form the assembled polynucleotide. In some embodiments, amplification comprises polymerase chain reaction (PCR), rolling circle amplification (RCA), isothermal amplification, DNA polymerase-mediated extension, or a combination thereof.

[0016] In some embodiments, provided methods further include detecting a sequence corresponding to the assembled polynucleotide. In some embodiments, detecting includes amplification of an assembled polynucleotide, such as by, e.g., PCR, RCA, isothermal amplification, DNA polymerase-mediated extension, or a combination thereof.

[0017] In some embodiments, the first polynucleotide sequence, the second polynucleotide sequence and/or the assembled polynucleotide comprise or consist of DNA and/or RNA. In some embodiments, the first polynucleotide sequence comprises or consists of DNA. In some embodiments, the second polynucleotide sequence comprises or consists of DNA. In some embodiments, the assembled polynucleotide sequence comprises or consists of DNA. In some embodiments, the first polynucleotide sequence, the second polynucleotide sequence and the assembled polynucleotide comprise or consist of DNA. In some embodiments, the first polynucleotide sequence comprises or consists of RNA. In some embodiments, the second polynucleotide sequence comprises or consists of RNA. In some embodiments, the assembled polynucleotide sequence comprises or consists of RNA. In some embodiments, the first polynucleotide sequence, the second polynucleotide sequence and the assembled polynucleotide comprise or consist of RNA. In some embodiments, the first polynucleotide sequence, the second polynucleotide sequence and/or the assembled polynucleotide comprise or consist of a combination of DNA and RNA.

[0018] In some embodiments, provided methods further comprise: contacting the assembled polynucleotide with an additional tandem guide complex and an additional polynucleotide sequence, wherein the additional tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, wherein the first variant Cas protein of the additional tandem guide complex binds the assembled polynucleotide and the second variant Cas protein of second tandem guide complex binds the additional polynucleotide sequence, and assembling the additional polynucleotide sequence and the assembled polynucleotide, thereby further extending the assembled polynucleotide.

[0019] In some embodiments, the additional polynucleotide sequence has a length of 5 to 5000 nucleotides. In some embodiments, the additional polynucleotide sequence has a length of 10 to 1000 nucleotides. In some embodiments, the additional polynucleotide sequence has a length of 25 to 500 nucleotides.

[0020] In some embodiments, provided methods are multiplex methods. In some embodiments, the contacting and assembling steps are repeated, thereby iteratively extending the assembled polynucleotide.

[0021] In some embodiments, the present disclosure provides in vitro methods comprising: obtaining or providing two or more tandem guide complexes, wherein each tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, contacting the two or more tandem guide complexes with (i) a polynucleotide scaffold comprising two or more scaffold sequences that are positioned adjacently in series along the polynucleotide scaffold, and (ii) two or more assembly polynucleotide sequences, wherein the first variant Cas protein of each tandem guide complex binds a scaffold sequence and the second variant Cas protein of each tandem guide complex binds an assembly polynucleotide sequence, wherein the contacting brings the two or more assembly polynucleotide sequences into close proximity to one another, and assembling the two or more assembly polynucleotide sequences to form an assembled polynucleotide.

[0022] In some embodiments, the two or more assembly polynucleotide sequences and the polynucleotide scaffold are present in solution.

[0023] In some embodiments, contacting the two or more tandem guide complexes with the polynucleotide scaffold and the two or more assembly polynucleotide sequences occurs simultaneously. In some embodiments, the two or more tandem guide complexes are contacted sequentially with the polynucleotide scaffold and assembly polynucleotide sequences. In some embodiments, provided is a polynucleotide scaffold that is then contacted with two or more tandem guide complexes, followed by the two or more assembly polynucleotide sequences. In some embodiments, the two or more tandem guide complexes are contacted first with the polynucleotide scaffold, such that the tandem guide complexes associate with the polynucleotide scaffold, and then contacted with two or more assembly polynucleotide sequences.

[0024] In some embodiments, the two or more assembly polynucleotide sequences are provided simultaneously (e.g., in the same solution). In some embodiments, the two or more assembly polynucleotide sequences are themselves provided sequentially.

[0025] In some embodiments, one or more of the assembly polynucleotide sequences comprise sticky ends. In some embodiments, one or more of the assembly polynucleotide sequences are restriction digested. In some embodiments, all of the assembly polynucleotide sequences are restriction digested prior to the contacting step. In some embodiments one or more of the assembly polynucleotide sequences are restriction digested prior to the contacting step (e.g., contacting with tandem guide complexes and/or polynucleotide scaffold).

[0026] In some embodiments, the assembly polynucleotide sequences are not limited to a particular length.

[0027] In some embodiments, the assembly polynucleotide sequences have a length of 5 to 5000 nucleotides. In some embodiments, the assembly polynucleotide sequences have a length of 10 to 1000 nucleotides. In some embodiments, the assembly polynucleotide sequences have a length of 25 to 500 nucleotides.

[0028] In some embodiments, assembling comprises ligating the two or more assembly polynucleotide sequences to form the assembled polynucleotide.

[0029] In some embodiments, assembling comprises annealing the two or more assembly polynucleotide sequences to each other followed by amplification to form the assembled polynucleotide. In some embodiments, amplification comprises polymerase chain reaction (PCR), rolling circle amplification (RCA), isothermal amplification, DNA polymerase-mediated extension, or a combination thereof. [00301 In some embodiments, provided methods further include detecting a sequence corresponding to the assembled polynucleotide. In some embodiments, detecting includes amplification of an assembled polynucleotide, such as by, e.g., PCR, RCA, isothermal amplification, DNA polymerase-mediated extension, or a combination thereof.

[0031] In some embodiments, the present disclosure provides in vitro methods comprising: obtaining or providing a plurality of tandem guide complexes, wherein each tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, and contacting the plurality of tandem guide complexes with one or more compositions that comprise a plurality of polynucleotides, wherein each tandem guide complex is capable of binding to two different polynucleotides among the plurality of polynucleotides.

[00321 In some embodiments, each polynucleotide of the plurality of polynucleotides comprises a detectable label. In some embodiments, each polynucleotide of the plurality of polynucleotides comprises a unique barcode sequence.

[0033] In some embodiments, the present disclosure provides in vitro methods comprising: contacting a plurality of tandem guide complexes with one or more compositions that comprise a plurality of polynucleotides, wherein each tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, and wherein each tandem guide complex binds to two different polynucleotides among the plurality of polynucleotides, and assembling the two different polynucleotides bound by each tandem guide complex, thereby forming a plurality of assembled polynucleotides.

[0034] In some embodiments, a plurality of polynucleotides comprises at least 3 polynucleotides, at least 10 polynucleotides, at least 100 polynucleotides, at least 1,000 polynucleotides, at least 10,000 polynucleotides, at least 100,000 polynucleotides, at least 1,000,000 polynucleotides, or more.

[0035] In some embodiments, a plurality of assembled polynucleotides comprises at least 3 assembled polynucleotides, at least 10 assembled polynucleotides, at least 100 assembled polynucleotides, at least 1,000 assembled polynucleotides, at least 10,000 assembled polynucleotides, at least 100,000 assembled polynucleotides, at least 1,000,000 assembled polynucleotides, or more.

[0036] In some embodiments, the two different polynucleotides are each associated with a surface, and the contacting brings the surfaces into close proximity to one another.

[0037] In some embodiments, the contacting brings the surfaces within a proximity of about 200 nm or less of one another. In some embodiments, the proximity of the surfaces is measured and/or characterized. In some embodiments, the proximity of the surfaces is measured and/or characterized using a proximity assay, such as, e.g., an AlphaLISA proximity assay. In some embodiments, the proximity of the surfaces is measured and/or characterized using microscopy. In some embodiments, the surfaces comprise or consist of planar surfaces, beads (e.g., microbeads), and/or cell surfaces.

[0038] In some embodiments, the two different polynucleotides arc each present in solution. In some embodiments, the plurality of polynucleotides are present in solution.

[0039] In some embodiments, the two different polynucleotides are provided simultaneously (e.g., in the same solution). In some embodiments, the different polynucleotides are themselves provided sequentially.

[0040] In some embodiments, two different polynucleotides comprise sticky ends. In some embodiments, two different polynucleotides are restriction digested. In some embodiments, the plurality of polynucleotides are restriction digested prior to the contacting step. In some embodiments two different polynucleotides are restriction digested prior to the contacting step (e.g., contacting with tandem guide complex).

[0041] In some embodiments, the plurality of polynucleotides are not limited to a particular length. In some embodiments, the plurality of polynucleotides each have a length of 5 to 5000 nucleotides. In some embodiments, the plurality of polynucleotides each have a length of 10 to 1000 nucleotides. In some embodiments, the plurality of polynucleotides each have a length of 25 to 500 nucleotides.

[0042] In some embodiments, the two different polynucleotides are not limited to a particular length. In some embodiments, the two different polynucleotides each have a length of 5 to 5000 nucleotides. In some embodiments, the two different polynucleotides each have a length of 10 to 1000 nucleotides. In some embodiments, the two different polynucleotides each have a length of 25 to 500 nucleotides.

[0043] In some embodiments, the present disclosure provides in vitro methods comprising: obtaining or providing a surface associated with a first plurality of polynucleotides, contacting the first plurality of polynucleotides with a plurality of tandem guide complexes and a second plurality of polynucleotides, wherein each tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, and wherein the first variant Cas protein binds a polynucleotide of the first plurality of polynucleotides and the second variant Cas protein binds a polynucleotide of the second plurality of polynucleotides, and assembling the polynucleotides bound by each tandem guide complex, thereby forming a plurality of assembled polynucleotides.

[0044] In some embodiments, the second plurality of polynucleotides and/or the plurality of tandem guide complexes are present in solution.

[0045] In some embodiments, the plurality of assembled polynucleotides are each associated with the surface. In some embodiments, each polynucleotide of the first plurality of polynucleotides is associated with a defined position on the surface. In some embodiments, each assembled polynucleotide of the plurality of assembled polynucleotides is associated with a defined position on the surface. In some embodiments, the surface comprises or consists of a planar surface. In some embodiments, the surface comprises or consists of a bead (e.g., a microbead) and/or a cell surface.

[0046] In some embodiments, contacting the first plurality of polynucleotides with a plurality of tandem guide complexes and a second plurality of polynucleotides occurs simultaneously.

[0047] In some embodiments, the contacting comprises contacting the first plurality of polynucleotides and the plurality of tandem guide complexes and subsequently contacting the second plurality of polynucleotides. [00481 In some embodiments, the first plurality of polynucleotides and/or second plurality of polynucleotides are not limited to a particular length. In some embodiments, the first plurality of polynucleotides and/or second plurality of polynucleotides each have a length of 5 to 5000 nucleotides. In some embodiments, the first plurality of polynucleotides and/or second plurality of polynucleotides each have a length of 10 to 1000 nucleotides. In some embodiments, the first plurality of polynucleotides and/or second plurality of polynucleotides each have a length of 25 to 500 nucleotides.

[0049] In some embodiments, the second plurality of polynucleotides have sticky ends. In some embodiments, the second plurality of polynucleotides are restriction digested. In some embodiments, the second plurality of polynucleotides are restriction digested prior to the contacting step.

[0050] In some embodiments, assembling comprises ligating the polynucleotides bound by each tandem guide complex to form the plurality of assembled polynucleotides.

[0051] In some embodiments, assembling comprises annealing polynucleotides bound by each tandem guide complex to each other followed by amplification to form the plurality of assembled polynucleotides. In some embodiments, the amplification comprises polymerase chain reaction (PCR), rolling circle amplification (RCA), isothermal amplification, DNA polymerase-mediated extension, or a combination thereof.

[0052] In some embodiments, provided methods further comprise detection of sequences corresponding to the plurality of assembled polynucleotides. In some embodiments, detecting includes amplification of assembled polynucleotides, such as by, e.g., PCR, RCA, isothermal amplification, or a combination thereof.

[0053] In some embodiments, the plurality of polynucleotides and/or the plurality of assembled polynucleotides comprise or consist of DNA and/or RNA. In some embodiments, the plurality of polynucleotides and/or the plurality of assembled polynucleotides comprise or consist of DNA. In some embodiments, the plurality of polynucleotides and/or the plurality of assembled polynucleotides comprise or consist of RNA. In some embodiments, the plurality of polynucleotides and/or the plurality of assembled polynucleotides comprise or consist of a combination of DNA and RNA.

[0054] In some embodiments, a plurality of polynucleotides comprises at least 3 polynucleotides, at least 10 polynucleotides, at least 100 polynucleotides, at least 1,000 polynucleotides, at least 10,000 polynucleotides, at least 100,000 polynucleotides, at least 1,000,000 polynucleotides, or more.

[0055] In some embodiments, a plurality of assembled polynucleotides comprises at least 3 assembled polynucleotides, at least 10 assembled polynucleotides, at least 100 assembled polynucleotides, at least 1,000 assembled polynucleotides, at least 10,000 assembled polynucleotides, at least 100,000 assembled polynucleotides, at least 1,000,000 assembled polynucleotides, or more.

[0056] In some embodiments, provided methods are multiplex methods. In some embodiments, the method further comprises: contacting the plurality of assembled polynucleotides with an additional plurality of tandem guide complexes and an additional plurality of polynucleotides, wherein each tandem guide complex of the additional plurality comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, wherein the first variant Cas protein of the additional tandem guide complex binds an assembled polynucleotide and the second variant Cas protein of the additional tandem guide complex binds a polynucleotide of the additional plurality, and assembling the additional polynucleotide and the assembled polynucleotide, thereby further extending the plurality of assembled polynucleotides.

[0057] In some embodiments, the contacting and assembling steps are repeated, thereby iteratively extending the plurality of assembled polynucleotides.

[0058] In some embodiments, the present disclosure provides in vitro methods comprising: obtaining or providing a first surface associated with a first polynucleotide and a second surface associated with a second polynucleotide, contacting the first surface and the second surface with a tandem guide complex, wherein the tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, and wherein the first variant Cas protein binds the first polynucleotide and the second variant Cas protein binds the second polynucleotide, thereby assembling the first and second surfaces.

[0059] In some embodiments, the present disclosure provides in vitro methods comprising: obtaining or providing a plurality of surfaces, wherein each surface is associated with a unique polynucleotide, and contacting the plurality of surfaces with a plurality of tandem guide complexes, wherein each tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, and wherein each tandem guide complex binds to two unique polynucleotides, each associated with a different surface among the plurality of surfaces, thereby bringing together two different surfaces into close proximity.

[0060] In some embodiments, the surfaces consist or comprise planar surfaces, beads (e.g., microbeads), and/or cell surfaces.

[0061] In some embodiments, the present disclosure provides tandem guide agents for use in methods described herein. In some embodiments, a tandem guide agent or each tandem guide agent of the plurality for use in methods described herein comprise: a first unit comprising a first spacer (or a first targeting sequence) and a first scaffold, and a second unit comprising a second spacer (or a second targeting sequence) and a second scaffold. It is to be understood that the terms “spacer” and “targeting sequence” are equivalent herein and can be used interchangeably.

[0062] In some embodiments, a provided tandem guide agent further comprises a linker between the first unit and the second unit (e.g., between the first scaffold and the second spacer or between the first spacer and the second scaffold).

[0063] In some embodiments, the first unit comprises in 5’ to 3’ order: the first spacer and the first scaffold and/or the second unit comprises in 5’ to 3’ order: the second spacer and the second scaffold. In some embodiments, the first unit comprises in 3’ to 5’ order: the first spacer and the first scaffold and/or the second unit comprises in 3’ to 5’ order: the second spacer and the second scaffold. In some embodiments, the first unit comprises in 5’ to 3’ order: the first spacer and the first scaffold and/or the second unit comprises in 3’ to 5’ order: the second spacer and the second scaffold. In some embodiments, the first unit comprises in 3’ to 5’ order: the first spacer and the first scaffold and/or the second unit comprises in 5’ to 3’ order: the second spacer and the second scaffold.

[0064] In some embodiments, the first unit comprises a sgRNA and/or the second unit comprises a sgRNA.

[0065] In some embodiments, the first scaffold and second scaffold bind to the same variant Cas protein. In some embodiments, the first scaffold and second scaffold bind to different variant Cas proteins.

[0066] In some embodiments, the first variant Cas protein and/or second variant Cas protein is a variant Class 2 Cas protein. In some embodiments, the first variant Cas protein and/or second variant Cas protein is a variant Type II, Type V or Type VI Cas protein.

[0067] In some embodiments, the first variant Cas protein and/or second variant Cas protein is a variant Cas9 protein, a variant Cas 12 protein, a variant Cas 13 protein, and/or a variant Cas 14 protein. In some embodiments, the first variant Cas protein and/or second variant Cas protein is inactive. In some embodiments, the first variant Cas protein and/or second variant Cas protein is a nickase. In some embodiments, the first variant Cas protein and/or second variant Cas protein is PAM-less.

[0068] In some embodiments, the linker is a polynucleotide linker, a non-polynucleotide covalent linker, or a non-covalent linker. In some embodiments, the linker is a poly-adenosine linker. In some embodiments, the poly-adenosine linker comprises 5 to 50 adenosine residues. In some embodiments, the poly-adenosine linker comprises 10 to 15 adenosine residues.

[0069] In some embodiments, the first unit comprises a first spacer (or a first targeting sequence) and a first scaffold, and the second unit comprises a second spacer (or a second targeting sequence) and a second scaffold. In some embodiments, a tandem guide agent further comprises a linker between the first unit and the second unit (e.g., between the first scaffold and the second spacer or between the first spacer and the second scaffold).

[0070] In some embodiments, the present disclosure provides compositions comprising one or more components of technologies described herein. In some embodiments, a composition comprises a tandem guide agent as described herein. In some embodiments, a composition comprises a tandem guide complex as described herein.

[0071] In some embodiments, the present disclosure provides self-assembling compositions. In some embodiments, a self-assembling composition comprises one or more surfaces associated with a polynucleotide, and one or more tandem guide complexes as described herein. In some embodiments, a self-assembling composition comprises two or more surfaces each associated with a polynucleotide, and one or more tandem guide complexes as described herein, wherein the one or more tandem guide complexes bind to the polynucleotide on each of the surfaces, thereby self-assembling. In some embodiments, the one or more surfaces comprise a planar surface, a bead (e.g., a microbead), and/or a cell surface.

[0072] These, and other aspects of the invention, are described in more detail below and in the claims.

BRIEF DESCRIPTION OF THE DRAWING

[0073] The Drawing included herein, which is composed of the following Figures, is for illustration purposes only and not for limitation.

[0074] FIG. 1A depicts a schematic of the domains of an exemplary tandem guide agent. From 5’ to 3’ are: a first spacer (or a first targeting sequence), a first scaffold, a linker (e.g., a poly A linker), a second spacer (or a second targeting sequence), and a second scaffold. FIG. IB depicts a linear schematic of an exemplary tandem guide agent. From left to right (5’ to 3’) a first spacer and scaffold (red line), a linker (black line), and a second spacer and scaffold (blue line). FIG. 1C depicts a schematic of an exemplary tandem guide complex comprising a tandem guide agent complexed with two variant Cas proteins (e.g., inactive variants, e.g., dCas9). From left to right (5’ to 3’) an exemplary tandem guide complex comprises a first spacer/scaffold (red line) complexed with a first variant Cas protein (e.g., inactive variant, e.g., dCas9) (which binds target A), a linker (black line) and a second spacer/scaffold (blue line) complexed with a second variant Cas protein (e.g., inactive variant, e.g., dCas9) (which binds target B). [0075] FIG. 2A depicts a schematic of an exemplary proximity assay (AlphaLISA assay) to monitor dual DNA target binding by a tandem guide complex. Excitation of the donor bead will induce emission of the acceptor bead only when donor and acceptor beads are in close proximity (-200 nm). FIG. 2B provides AlphaLISA data demonstrating that an exemplary tandem guide complex (dCas9-tgRNA) specifically binds to target DNA sequences. From left to right, the first six configurations assess binding of a donor-labeled target DNA to an acceptor- labeled Cas9-tgRNA complex. Columns 1 and 2 depict binding to a first target DNA sequence (with the acceptor label attached to the first gRNA or second gRNA of the tandem guide agent, respectively), columns 3 and 4 depict binding to a second target DNA sequence (with the acceptor label attached to the first gRNA or second gRNA of the tandem guide agent, respectively) and columns 5 and 6 depict binding to a non-target control DNA sequence (with the acceptor label attached to the first gRNA or second gRNA of the tandem guide agent, respectively). The last two configurations assess simultaneous binding of donor and acceptor labeled target DNA sequences to a tandem guide complex. Column 7 depicts binding of a tandem guide complex to a first donor labeled target DNA sequence and a second acceptor labeled target DNA sequence (as depicted in FIG. 2A) and column 8 depicts binding with a nontarget donor labeled control DNA sequence replacing the donor labeled target DNA sequence. The panel on the right shows a zoom-in of columns 7 and 8. AlphaLISA signal for control configurations 5, 6, and 8 containing non-target DNA represent background. Observation that the signal for configurations 1, 2, 3, 4, and 7 is higher than background demonstrates that the dCas9- tgRNA complex is functional.

[0076] FIG. 3 provides results from an AlphaLISA experiment analyzing binding of an exemplary tandem guide complex (dCas9-tgRNA) to target DNA sequences, with varying concentrations of an exemplary tandem guide agent (tgRNA). From left to right: the first four columns represent simultaneous binding of donor and acceptor labeled target DNA sequences (“blue” and “red” DNA sequences bound to donor and acceptor beads, respectively) to varying concentrations of a dCas9-tgRNA. Specifically, dCas9-tgRNA at concentrations of 5 pg, 500 ng, 50 ng, and 5 ng were analyzed. The following groups all represent background controls, specifically: the second four columns represent binding of varying concentrations of dCas9- tgRNA where the tgRNA includes a gRNA with a non-target control specificity, the following eight columns represent binding of varying concentrations of two different tgRNAs without dCas9 protein and the final column represents binding of dCas9 protein in the absence of tgRNA. [0077] FIG. 4 depicts a schematic for flow cytometry-based charaterization of an exemplary tandem guide complex (dCas9-tgRNA) binding to two target DNA sequences (left side of figure) and provides data from an exemplary flow cytometry-based assay (top right of figure).

[0078] FIG. 5A and FIG. 5B depict schematics of exemplary programmable DNA assembly via proximity ligation of two DNA fragments brought together by an exemplary tandem guide complex (dCas9-tgRNA). FIG. 5C provides results of exemplary assembly of two DNA fragments via proximity ligation.

[0079] FIG. 6 depicts an exemplary schematic of multiplexed CASsembly of pooled target polynucleotides (DNA) sequences of interest using a pool of tandem guide complexes. [0080] FIGs. 7A-7D depict schematics and results of a pilot pooled CASsembly using tgRNA and target DNA pools. FIG. 7A provides an overview of an exemplary pooled CASsembly pilot. A DNA pool encoding 8 target sequences (or “barcodes”) and 2 non-target sequences was synthesized. tgRNA sequences were designed to link together target sequences (or “barcodes”) 1&2, 3&4, 5&6 and 7&8. No tgRNA sequences were included to link the non-target sequences to any target or non-target sequences. FIG. 7B provides design of target DNA sequences. Odd numbered DNA sequences were amplified with primers 1 A and 2; even numbered with IB and 2; assembled DNA sequences after CASsembly were amplified using primers 1A and IB. FIG. 7C provides pie charts with percentage of NGS reads in which each barcode was identified, for CASsembly and “ligation control” conditions. FIG. 7D provides a bar graph with percentage of NGS reads for each barcode in which the barcode was identified with its correct anticipated partner, as dictated by tgRNA sequences. CASsembly and “ligation control” conditions are shown for comparison. [0081] FIG. 8 depicts a schematic of an exemplary CASsembly method that employs a polynucleotide scaffold.

[0082] FIG. 9A-9C depicts schematics of exemplary components for a CASsembly method that employs nickase variant Cas proteins. FIG. 9A depicts exemplary polynucleotide components including an exemplary first polynucleotide target (dsDNA target 1), an exemplary second polynucleotide target (dsDNA target 2), and an exemplary tandem guide agent(tgRNA). FIG. 9B depicts a schematic of an exemplary tandem guide complex that includes a tandem guide agent polynucleotide (tgRNA) and two exemplary nickase variant Cas proteins. FIG. 9C depicts a schematic of an exemplary CASsembly method that employs an exemplary tandem guide complex that includes nickase variant Cas proteins; the nickase variant Cas proteins themselves are not shown to facilitate understanding of tgRNA-DNA binding.

[0083] FIG. 10 depicts a schematic of tgRNA-mediated self-assembly of a proteinencoding DNA on a solid surface.

[0084] FIG. 11 depicts schematics of two exemplary self-assembling applications of tandem guide agents (e.g., tgRNAs). On the left is depicted a schematic of self-assembling cellular scaffolds, where a tandem guide agent is used to bring together a first cell (cell 1) and a second cell (cell 2) that are each functionalized with DNA. On the right is depicted a schematic of self-assembling materials (e.g., biomaterials), where a surface of each material is functionalized with DNA and a tandem guide agent is used to bring the two surfaces together.

[0085] FIG. 12A depicts a schematic of a library of oligonucleotides (oligos) 230 mer in length that were designed to encode 20 base pair (bp) target regions (for tgRNAs) that moved incrementally by 1 bp in each strand. Target sequences were positioned along both sense and antisense strands. 156 oligos each encoding the incremental 1 bp shift in target region were used per barcode on each DNA strand (312 total oligos per barcode). FIG. 12B depicts CASsembly of DNA sequences with dCas9-tgRNA complexes. FIG. 12C shows experimental results with correct pairing of each of the 312 oligos associated with one target region with the 312 oligos associated with a second target region. The left panel shows results obtained using CASsembly (experiment). The right panel shows results obtained using direct ligation (control). CERTAIN DEFINITIONS

[0086] In general, terminology used herein is in accordance with its understood meaning in the art, unless clearly indicated otherwise. Explicit definitions of certain terms are provided below; meanings of these and other terms in particular instances throughout this specification will be clear to those skilled in the art from context.

[0087] In order that the present invention may be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms arc set forth throughout the specification.

[0088] The indefinite articles “a” and “an” refer to at least one of the associated noun, and are used interchangeably with the terms “at least one” and “one or more.” The conjunctions “or” and “and/or” are used interchangeably as non-exclusive disjunctions.

[0089] About: The term “about”, when used herein in reference to a value, refers to a value that is similar, in context to the referenced value. In general, those skilled in the art, familiar with the context, will appreciate the relevant degree of variance encompassed by “about” in that context. For example, in some embodiments, the term “about” may encompass a range of values that within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less of the referred value.

[0090] Agent: As used herein, the term “agent”, may refer to a compound, molecule, or entity of any chemical class including, for example, a small molecule, polypeptide, polynucleotide (e.g., DNA and/or RNA), saccharide, lipid, metal, or a combination or complex thereof. In some embodiments, the term “agent” may refer to a compound, molecule, or entity that comprises a polynucleotide. In some embodiments, the term may refer to a compound or entity that comprises one or more polynucleotide moieties. In some embodiments, an agent may refer to a compound, molecule, or entity that comprises RNA.

[0091] Associated: Two entities are physically “associated” with one another, as that term is used herein, if the presence of one is correlated with that of the other. In some embodiments, two or more entities are physically “associated” with one another if they interact, directly or indirectly, so that they are and/or remain in physical proximity with one another. In some embodiments, two or more entities that are physically associated with one another are covalently linked to one another; in some embodiments, two or more entities that are physically associated with one another are not covalently linked to one another but are non-covalently associated, for example by means of hydrogen bonds, van der Waals interaction, hydrophobic interactions, magnetism, and combinations thereof.

[0092] Composition: Those skilled in the art will appreciate that the term “composition”, as used herein, may be used to refer to a discrete physical entity that comprises one or more specified components. In general, unless otherwise specified, a composition may be of any form - e.g., gas, gel, liquid, solid, etc.

[0093] Hybridization: The term “hybridization” as used herein refers to a reaction in which one or more polynucleotides interact to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self-hybridizing strand, or any combination of these. A sequence capable of hybridizing with a given sequence is referred to as the “complement” of the given sequence.

[0094] In vitro: The term “zn vitro” as used herein refers to events that occur in an artificial environment, e.g., in a test tube or reaction vessel, in cell culture, etc., rather than within a multi-cellular organism.

[0095] Isolated: The term “isolated” as used herein refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting) and/or otherwise previously associated, and/or (2) designed, produced, prepared, and/or manufactured by the hand of man. In some embodiments, a substance may be considered to be “isolated” if it is (or has been caused to be) free of or separated from about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of other components (e.g., components with which it was previously associated). In some embodiments, isolated agents are about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is “pure” if it is substantially free of other components. In some embodiments, as will be understood by those skilled in the ail, a substance may still be considered “isolated” or even “pure”, after having been combined with certain other components such as, for example, one or more carriers or excipients (e.g., buffer, solvent, water, etc.); in such embodiments, percent isolation or purity of the substance is calculated without including such carriers or excipients.

[0096] Nucleic acid: As used herein, in its broadest sense, refers to any compound and/or substance that is or can be incorporated into a polynucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into a polynucleotide chain via a phosphodiester linkage. As will be clear from context, in some embodiments, “nucleic acid” refers to an individual nucleic acid residue (e.g., a nucleotide and/or nucleoside); in some embodiments, “nucleic acid” refers to a polynucleotide chain comprising individual nucleic acid residues. In some embodiments, a “nucleic acid” is or comprises RNA; in some embodiments, a “nucleic acid” is or comprises DNA. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleic acid residues. In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleic acid analogs. In some embodiments, a nucleic acid analog differs from a nucleic acid in that it does not utilize a phosphodiester backbone. For example, in some embodiments, a nucleic acid is, comprises, or consists of one or more “peptide nucleic acids”, which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. Alternatively or additionally, in some embodiments, a nucleic acid has one or more phosphorothioate and/or 5’-N-phosphoramidite linkages rather than phosphodiester bonds. In some embodiments, a nucleic acid is, comprises, or consists of one or more natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxy cytidine). In some embodiments, a nucleic acid is, comprises, or consists of one or more nucleoside analogs (e.g., 2- aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3 -methyl adenosine, 5- methylcytidine, C5-propynyl-cytidine, C5-propynyl-uridine, 2-aminoadenosine, C5- bromouridine, C5-fluorouridine, C5-iodouridine, 2-aminoadenosine, 7-deazaadenosine, 7- deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, methylated bases, intercalated bases, and combinations thereof). In some embodiments, a nucleic acid comprises one or more modified sugars (e.g., 2’-fluororibose, ribose, 2’- deoxyribose, arabinose, and hexose) as compared with those in natural nucleic acids. Nucleic acid sequences provided herein are intended to encompass nucleic acids containing any combination of natural or modified RNA and/or DNA, including, but not limited to, such nucleic acids having modified nucleobases.

[0097] Polynucleotide: The term “polynucleotide” as used herein refer to a polymer of at least three nucleic acid residues (also called “nucleotide bases” or “nucleotides”). In some embodiments, a polynucleotide comprises DNA. In some embodiments, a polynucleotide comprises RNA. In some embodiments, a polynucleotide is single stranded. In some embodiments, a polynucleotide is double stranded. In some embodiments, a polynucleotide comprises both single and double stranded portions. In some embodiments, a polynucleotide comprises a backbone that comprises one or more phosphodiester linkages. In some embodiments, polynucleotides can be chimeric mixtures or derivatives or modified versions thereof, single- stranded or double- stranded. In some such embodiments, modifications can occur at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, its hybridization parameters, etc. In some embodiments, a polynucleotide comprises double or single stranded genomic DNA, RNA, any synthetic and genetically manipulated polynucleotide, and/or sense and/or antisense polynucleotides. In some embodiments, nucleic acids containing modified bases.

[0098] Conventional IUPAC notation is used in nucleotide sequences presented herein, as shown in Table 1, below (see also Cornish-Bowden, Nucleic Acids Res. 13(9):3021-30 (1985), incorporated by reference herein). It should be noted, however, that “T” denotes “Thymine or Uracil” in those instances where a sequence may be encoded by either DNA or RNA, for example in certain CRISPR/Cas guide molecules.

[0099] Table 1. 1UPAC nucleic acid notation

roiooi Protein: As used herein, the term “protein” refers to a polypeptide (i.e., a string of at least three amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a “protein” can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Polypeptides may contain L- amino acids, D- amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term “peptide” is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are Cas proteins, biologically active portions thereof, and/or characteristic portions thereof.

[0101] Reference: As used herein, the term “reference” describes a standard or control relative to which a comparison is performed. For example, in some embodiments, an agent, animal, individual, population, sample, sequence or value of interest is compared with a reference or control agent, animal, individual, population, sample, sequence or value. In some embodiments, a reference or control is tested and/or determined substantially simultaneously with the testing or determination of interest. In some embodiments, a reference or control is a historical reference or control, optionally embodied in a tangible medium. Typically, as would be understood by those skilled in the art, a reference or control is determined or characterized under comparable conditions or circumstances to those under assessment. Those skilled in the art will appreciate when sufficient similarities are present to justify reliance on and/or comparison to a particular possible reference or control. In some embodiments, a reference is a negative control reference; in some embodiments, a reference is a positive control reference. [0102] Surface: As used herein, the term “surface” refers to any surface that is capable of being associated with a polynucleotide. In some embodiments, a surface is a planar surface. In some embodiments, a surface is on a bead (e.g., microbeads). In some embodiments, a surface is on a cell (e.g., plasma membrane, cell wall). In some embodiments, a surface is a non-planar surface. In some embodiments, a surface is on a liposome. In some embodiments, a surface is on a lipid nanoparticle. In some embodiments, a surface is on a substrate. In some embodiments, a surface is hard. In some embodiments, a surface is porous. Additional suitable surfaces that can associate with a polynucleotide will be apparent to a skilled artisan based on the present disclosure and the aforementioned references, and the disclosure is not limited in this respect.

[0103] Variant: As used herein in the context of molecules, e.g., nucleic acids, proteins, or small molecules, the term “variant” refers to a molecule that shows significant structural identity with a reference molecule but differs structurally from the reference molecule, e.g., in the presence or absence or in the level of one or more chemical moieties as compared to the reference entity. In some embodiments, a variant also differs functionally from its reference molecule. In general, whether a particular molecule is properly considered to be a “variant” of a reference molecule is based on its degree of structural identity with the reference molecule. As will be appreciated by those skilled in the art, any biological or chemical reference molecule has certain characteristic structural elements. A variant, by definition, is a distinct molecule that shares one or more such characteristic structural elements but differs in at least one aspect from the reference molecule. To give but a few examples, a polypeptide may have a characteristic sequence element comprised of a plurality of amino acids having designated positions relative to one another in linear or three-dimensional space and/or contributing to a particular structural motif and/or biological function; a nucleic acid may have a characteristic sequence element comprised of a plurality of nucleotide residues having designated positions relative to on another in linear or three-dimensional space. In some embodiments, a variant polypeptide or nucleic acid may differ from a reference polypeptide or nucleic acid as a result of one or more differences in amino acid or nucleotide sequence and/or one or more differences in chemical moieties (e.g., carbohydrates, lipids, phosphate groups) that arc covalently components of the polypeptide or nucleic acid (e.g., that are attached to the polypeptide or nucleic acid backbone). In some embodiments, a variant polypeptide or nucleic acid shows an overall sequence identity with a reference polypeptide or nucleic acid that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98%. In some embodiments, a variant polypeptide or nucleic acid does not share at least one characteristic sequence element with a reference polypeptide or nucleic acid. In some embodiments, a reference polypeptide or nucleic acid has one or more biological activities. In some embodiments, a variant polypeptide or nucleic acid shares one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid lacks one or more of the biological activities of the reference polypeptide or nucleic acid. In some embodiments, a variant polypeptide or nucleic acid shows a reduced level of one or more biological activities as compared to the reference polypeptide or nucleic acid. In some embodiments, a polypeptide or nucleic acid of interest is considered to be a “variant” of a reference polypeptide or nucleic acid if it has an amino acid or nucleotide sequence that is identical to that of the reference but for a small number of sequence alterations at particular positions. Typically, fewer than about 20%, about 15%, about 10%, about 9%, about 8%, about 7%, about 6%, about 5%, about 4%, about 3%, or about 2% of the residues in a variant arc substituted, inserted, or deleted, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises about 10, about 9, about 8, about 7, about 6, about 5, about 4, about 3, about 2, or about 1 substituted residues as compared to a reference. Often, a variant polypeptide or nucleic acid comprises a very small number e.g., fewer than about 5, about 4, about 3, about 2, or about 1) number of substituted, inserted, or deleted, functional residues (i.e., residues that participate in a particular biological activity) relative to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises not more than about 5, about 4, about 3, about 2, or about 1 addition or deletion, and, in some embodiments, comprises no additions or deletions, as compared to the reference. In some embodiments, a variant polypeptide or nucleic acid comprises fewer than about 25, about 20, about 19, about 18, about 17, about 16, about 15, about 14, about 13, about 10, about 9, about 8, about 7, about 6, and commonly fewer than about 5, about 4, about 3, or about 2 additions or deletions as compared to the reference.

[0104] All literature and similar' material cited in this application, including, but not limited to, patents, patent applications, articles, books, treatises, and web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety. In the event that one or more of the incorporated literature and similar' materials differs from or contradicts this application, including but not limited to defined terms, term usage, described techniques, or the like, this application controls. The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0105] The present disclosure recognizes limitations of methods currently employed for bringing polynucleotides together in certain applications, particularly in vitro applications. Many applications require homology between DNA fragments in order to bring two pieces of DNA together in spatial proximity. For example, multiplexed DNA assembly is commonly performed using overlap-extension PCR, in which pairs of shorter DNA sequences with complementary ends anneal and are extended via DNA polymerase to iteratively assemble longer gene fragments in a thermocycler. However, spurious annealing between DNA sequences within a mixed pool can occur due to partial homology, yielding undesired products. This is particularly problematic for multiplexed DNA assembly of higher similar molecules (e.g., antibody variant sets). The present disclosure provides tandem guide agents and tandem guide complexes that can be used, inter alia, to address these issues. In preferred embodiments, provided tandem guide agents and tandem guide complexes are based on CRISPR-Cas systems.

[0106] As described further below, provided tandem guide agents and/or tandem guide complexes can bind to and co-localize polynucleotides (e.g., DNA), including polynucleotides that lack sequence homology to each other. The present disclosure encompasses a recognition that provided tandem guide agents (e.g., tgRNAs) and tandem guide complexes may be useful for any applications in which two separate DNA molecules are desired in close proximity. The present disclosure provides the insight that co-localization of polynucleotides using such tandem guide agents and tandem guide complexes are particularly beneficial for in vitro applications, such as multiplexed applications.

I. Tandem Guide Agents

[0107] The present disclosure provides tandem guide agents that bind to at least two distinct target nucleotide sequences. In some embodiments, a tandem guide agent comprises at least two units, each comprising a spacer and a scaffold. In some embodiments, a tandem guide agent comprises a first unit comprising (i) a first spacer (or a first targeting sequence) and (ii) a first scaffold, and a second unit comprising (i) a second spacer (or a second targeting sequence) and (ii) a second scaffold. In some embodiments, a tandem guide agent further comprises a linker between the first and second units (e.g., between the first scaffold and the second spacer or between the first spacer and the second scaffold). FIG. 1A and FIG. IB provide exemplary schematics of a tandem guide agent. In some embodiments, a scaffold sequence serves as a binding scaffold for a Cas protein. Without wishing to be limited by theory, two spacers encoded within a tandem guide agent (e.g., tgRNAs) act as a bridge to bring together two polynucleotide sequences (e.g., DNA sequences) of interest. In some embodiments, each spacer sequence of a tandem guide agent can hybridize with a target sequence.

[0108] In some embodiments, the present disclosure provides CRISPR-based tandem guide agents that comprise two units linked together, where each unit comprises a guide RNA (gRNA). Guide RNAs compatible with any class 2 Cas protein may be used in tandem guide agents of the present disclosure. Those of skill in the art will appreciate that, although structural differences may exist between gRNAs for use with Cas proteins from different prokaryotic species, the principles by which gRNAs operate are generally consistent. Skilled artisans will appreciate that a given tandem guide agent can include any suitable gRNA structure, including a chimeric gRNA or single-guide RNA (sgRNA), or a gRNA that includes one or more chemical modifications and/or sequential modifications (substitutions, additional nucleotides, truncations, etc.).

[0109] In some embodiments, a tandem guide agent comprises at least two units, each unit comprising (i) a spacer (or a targeting sequence) and (ii) a scaffold. In some embodiments, a tandem guide agent comprises at least two gRNAs, each comprising a crispr RNA (crRNA) and a transactivating crRNA (tracrRNA), e.g., when using a variant Cas9 protein. In some embodiments, a tandem guide agent comprises at least two gRNAs, each comprising a crispr RNA (crRNA) but no transactivating crRNA (tracrRNA), e.g., when using a variant Casl2a protein. It is to be understood that references to “crRNA” and “tracrRNA” herein encompass the sequences and structures of the crRNAs and tracrRNAs that are associated with a particular variant Cas protein in nature but also engineered functional forms thereof, e.g., where certain sequences or structures have been modified, e.g., via addition, deletion or substitution or certain nucleic acid residues or by chemical modification. This includes for examples engineered forms where certain sequences or structures have been deleted, where certain sequences or structures have been replaced with other sequences or structures, etc.

[0110] In some embodiments, a tandem guide agent comprises a gRNA where the tracrRNA and crRNA components are separate. In some embodiments, a tandem guide agent comprises two crRNAs that are covalently linked to one another (optionally via a linker) and two tracrRNAs that non-covalently associate with the crRNAs.

[0111] In some embodiments, a tandem guide agent comprises a gRNA where the tracrRNA and crRNA components are covalently linked to form a chimeric or single-guide RNA (sgRNA). In some embodiments, a sgRNA includes an additional loop of nucleotides connecting the 3’ end of the crRNA to the 5’ end of the tracrRNA, e.g., when using a variant Cas9 protein. [0112] In some embodiments, a gRNA is a single guide RNA (sgRNA). In some embodiments, tandem guide agent comprises a first unit comprising a first sgRNA and a second unit comprises a second sgRNA. In some embodiments, a tandem guide agent further comprises a linker. [01131 In some embodiments, a tandem guide agent comprises two gRNAs each comprising a crispr RNA (crRNA) but no transactivating crRNA (tracrRNA), e.g., when using a valiant Casl2a protein. In some embodiments, the two crRNA components are covalently linked and no tracrRNA components are present.

[0114] In some embodiments, a tandem guide agent comprises a first gRNA comprising tracrRNA and crRNA components that are covalently linked to form a chimeric or single-guide RNA (sgRNA) (e.g., for use with a variant Cas9 protein) and a second gRNA comprising a crispr RNA (crRNA) but no transactivating crRNA (tracrRNA) (e.g., for use with a variant Casl2a protein). In some embodiments, the first and second gRNAs of the tandem guide agent are covalently linked, e.g., the 3’ end of the tracrRNA from the first gRNA is covalently linked (optionally via a linker) to the 5’ end of the crRNA from the second gRNA.

[01151 While gRNAs are typically RNA molecules it is well known in the art that chemically modified RNA molecules including DNA/RNA hybrid molecules can be used as gRNAs and this also applies in the context of tandem guide agents of the present disclosure. [0116] In some embodiments, a tandem guide agent or portion thereof comprises RNA. In some embodiments, a tandem guide agent or portion thereof comprises chemically modified RNA. In some embodiments, a tandem guide agent or portion thereof comprises a DNA/RNA hybrid molecule.

[0117] In some embodiments, one or both units and/or a linker of a tandem guide agent comprises RNA. In some embodiments, one or both units of a tandem guide agent and/or a linker comprises chemically modified RNA. In some embodiments, one or both units and/or a linker of a tandem guide agent comprises a DNA/RNA hybrid molecule.

[0118] In some embodiments, a tandem guide agent is a tandem guide RNA (tgRNA) that comprises two gRNAs, each comprising a crRNA and a tracrRNA. In some embodiments, a tandem guide agent is a tandem guide RNA (tgRNA) that comprises two gRNAs, each comprising a crRNA but no tracrRNA. In some embodiments, a tandem guide agent is a tandem guide RNA (tgRNA) that comprises two gRNAs, one comprising a crRNA and a tracrRNA and one comprising a crRNA but no tracrRNA. In some embodiments, at least one of the gRNAs of the tgRNA have the crRNA and tracrRNA covalently linked to one another. In some embodiments, both of gRNAs of the tgRNA have the crRNA and tracrRNA covalently linked to one another. In some embodiments, for at least one of the gRNAs of the tgRNA, the crRNA and tracrRNA are separate and associate non-covalently. In some embodiments, both of gRNAs of the tgRNA have the crRNA and tracrRNA separate and associate non-covalently.

[0119] In some embodiments, a tandem guide agent is a tandem guide RNA (tgRNA) that comprises at least two gRNA (e.g., sgRNA or crRNA) sequences joined by a linker (e.g., a flexible linker). In some embodiments, a tgRNA comprises two Cas protein binding sites (scaffold sequences) and two spacer sequences that are each complementary to a distinct target polynucleotide sequence. In some embodiments, a tgRNA comprises: a first spacer (or a first targeting sequence), a first gRNA scaffold, a linker, a second spacer (or a second targeting sequence), and a second gRNA scaffold.

[0120] In some embodiments, a tandem guide agent is a tandem guide RNA (tgRNA) that comprises two gRNAs, where the spacers of the two gRNAs are different and the scaffolds of the two gRNAs are the same.

[0121] In some embodiments, a tandem guide agent is a tandem guide RNA (tgRNA) that comprises two gRNAs, where the spacers of the two gRNAs arc different and the scaffolds of the two gRNAs are also different. The present disclosure encompasses a recognition that including two different scaffolds may facilitate amplification of tgRNA templates.

[0122] Principles for designing gRNAs, including scaffolds spacers, are known in the ait. See, e.g., Qi et al., (2013) Cell 152(5): 1173- 1183, Dang et al., (2015) Genome Biology 16, 280, Doudna et al., (2018) Science 362(6416):839-842, Zhang et al., (2015) Cell 163(3):759-771 , and Zhang et al., (2017) Science 358(6366): 1019- 1027 . In some embodiments, a scaffold comprises a polynucleotide sequence that is capable of binding a Cas protein, e.g., a Cas9 protein.

[0123] In some embodiments, a scaffold for a variant Cas9 protein may comprise or consist of a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence of SEQ ID NO: 23. [01241 GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAA CTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 23)

[0125] In some embodiments, a scaffold for a variant Cas9 protein may comprise or consist of a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence of SEQ ID NO: 24.

[0126] GTTTCAGAGCTATGCTGGAAACAGCATAGCAAGTTGAAATAAGGCTAG TCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC (SEQ ID NO: 24)

[0127] It will be understood that the exemplary scaffolds disclosed herein are provided to illustrate non-limiting embodiments embraced by the present disclosure. Additional suitable scaffold sequences for variant Cas9 proteins and variant non-Cas9 proteins will be apparent to the skilled artisan based on the present disclosure and the aforementioned references, and the disclosure is not limited in this respect.

[0128] In some embodiments, a spacer (or a targeting sequence) is a polynucleotide that is about 15-25 nucleotides in length. In some embodiments, a spacer is a targeting sequence of a crRNA. In some embodiments, a crRNA comprises a 15-25 nucleotide targeting sequence. In some embodiments, a crRNA comprises an approximately 20 nucleotide targeting sequence. Spacers (or targeting sequences) can be designed to target any desired target sequence(s).

Principles for designing spacers (or targeting sequences) are known in the art. In theory, any unique 15-25 nucleotide sequence can be used as a targeting sequence.

[0129] In the context of tandem guide agents, a target sequence is a sequence to which a targeting sequence or spacer is designed to have complementarity such that hybridization can occur between a target sequence and a portion of a tandem guide agent. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization. A target sequence may comprise any polynucleotide, such as DNA and/or RNA.

[0130] In some embodiments, a tandem guide agent comprises a linker. In some embodiments, a linker connects units (e.g., gRNA units, e.g., sgRNA units) of a tandem guide agent to one another. In some embodiments, a tandem guide agent comprises two gRNA units that are joined by a linker. [01311 In some embodiments, a linker is characterized in that it tends not to adopt a rigid three-dimensional structure, but rather provides flexibility. A variety of different linker elements that can appropriately be used when engineering polynucleotides are known in the ail.

[0132] In some embodiments, a linker is not cleavable. The present disclosure encompasses a recognition that tandem guide agents that include a stable linker (e.g., one that is not cleaved) are particularly suited for applications and methods described herein.

[0133] In some embodiments, a linker is a polynucleotide linker. In some embodiments, a polynucleotide linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more nucleic acids in length. In some embodiments, a linker is 2 to 40 nucleotides in length. In some embodiments, a linker is 3 to 30 nucleotides in length. In some embodiments, a linker is 4 to 25 nucleotides in length. In some embodiments, a linker is 5 to 20 nucleotides in length. In some embodiments, a linker is 5 to 20 nucleotides in length. In some embodiments, a linker is 10 to 15 nucleotides in length. In some embodiments, a linker is 8 to 14 nucleotides in length. In some embodiments, a linker is no more than 30 nucleotides in length.

[0134] In some embodiments, a linker is a polyA linker. In some embodiments, a polyA linker is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more adenine bases in length. In some embodiments, a polyA linker is 2 to 40 adenine bases in length. In some embodiments, a polyA linker is 3 to 30 adenine bases in length. In some embodiments, a polyA linker is 4 to 25 adenine bases in length. In some embodiments, a polyA linker is 5 to 20 adenine bases in length. In some embodiments, a polyA linker is 5 to 20 adenine bases in length. In some embodiments, a polyA linker is 10 to 15 adenine bases in length. In some embodiments, a polyA linker is 8 to 14 adenine bases in length. In some embodiments, a polyA linker is 10 adenine bases in length. In some embodiments, a polyA linker is 12 adenine bases in length. In some embodiments, a polyA linker is 30 adenine bases in length. In some embodiments, a polyA linker is no more than 30 adenine bases in length. [01351 In some embodiments, a linker is a non-polynucleotide covalent linker. In some embodiments, a non-polynucleotide covalent linker is or comprises ethylene glycol based (E_x) or ethylene glycol derived (E_xy, P_x) linkers. Other suitable linkers are known in the ail, for example, Pils et al., (2000) Nucleic Acids Res. 28(9): 1859-1863. In some embodiments, a linker is or comprises a linkage produced by click chemistry. In some embodiments, a linker is or comprises a linkage produced by a copper catalyzed alkyne-azide cycloaddition (CuAAC) reaction. In some embodiments, a linker is or comprises a linkage produced by strain promoted azide alkyne cycloaddition (SPAAC). Linkers and/or linkages produced by click-chemistry are known in the ail, for example El-Sagheer and Brown et al., (2010) Chemical Soc. Rev. 39:1388- 1405. In some embodiments, a cross-linker is used to link a 5’ end of a first unit (e.g., gRNA) and a 3’ end of a second unit (e.g., gRNA), and the 3’ or 5’ ends of each unit (gRNA) to be linked are modified with functional groups that react with the reactive groups of the cross-linker.

[0136] In some embodiments, a linker is a non-covalent linker. In some embodiments, a non-covalent linker comprises a region of complementary nucleic acid sequences, such that the two units (e.g., gRNAs) come together through hydrogen base-pair bonding. In some embodiments, a tandem guide agent comprises a non-covalent linker, wherein the units stably associate through hydrogen base-pair bonding of complementary nucleic acid sequences.

[0137] In some embodiments, a first scaffold and a second scaffold of a tandem guide agent bind to the same variant Cas protein. In some embodiments, a first scaffold and a second scaffold of a tandem guide agent bind to different variant Cas proteins.

[0138] In some embodiments, a tandem guide agent comprises a unit (e.g., a gRNA) comprising in 5’ to 3’ order: a spacer and a scaffold. In some embodiments, a tandem guide agent comprises a gRNA comprising in 5’ to 3’ order: a crRNA and a tracrRNA. In some embodiments, a tandem guide agent is for complexing with a variant Cas9 protein and the tandem guide agent comprises a unit (e.g., a gRNA) comprising in 5’ to 3’ order: a spacer and a scaffold.

[0139] In some embodiments, a tandem guide agent comprises two units (e.g., a sgRNAs), each comprising in 5’ to 3’ order: a spacer and a scaffold. In some embodiments, a tandem guide agent comprises two sgRNAs each comprising in 5’ to 3’ order: a crRNA and a tracrRNA. In some embodiments, a tandem guide agent is for complexing with a variant Cas9 protein and the tandem guide agent comprises two sgRNAs each comprising in 5’ to 3’ order: a spacer and a scaffold.

[0140] In some embodiments, a tandem guide agent comprises in 5’ to 3’ order: a first spacer (or a first targeting sequence), a first scaffold, a linker, a second spacer (or a second targeting sequence), and a second scaffold. In some embodiments, a tandem guide agent is for complexing with a variant Cas9 protein.

[0141] In some embodiments, a tandem guide agent comprises one unit (e.g., a gRNA) comprising in 5’ to 3’ order: a spacer and a scaffold and a second unit (e.g., a gRNA) comprising in 3’ to 5’ order: a spacer and a scaffold. In some embodiments, a tandem guide agent comprises one unit (e.g., a gRNA) comprising in 5’ to 3’ order: a spacer and a scaffold and a second unit (e.g., a gRNA) comprising in 5’ to 3’ order: a scaffold and a spacer. In some embodiments, a tandem guide agent is for complexing with a variant Cas9 protein and a second, different Cas protein.

[0142] In some embodiments, a tandem guide agent comprises two units (e.g., gRNAs), the first unit comprising in 5’ to 3’ order: a crRNA and a tracrRNA and the second unit comprising in 3’ to 5’ order: a crRNA and a tracrRNA. In some embodiments, a tandem guide agent comprises two units (e.g., gRNAs), the first unit comprising in 5’ to 3’ order: a crRNA and a tracrRNA and the second unit comprising in 5’ to 3’ order: a tracrRNA and a crRNA. In some embodiments, a tandem guide agent is for complexing with a variant Cas9 protein and a second, different variant Cas protein (e.g., a variant Cas 12 protein).

[0143] In some embodiments, a tandem guide agent comprises in 5’ to 3’ order: a first spacer (or a first targeting sequence), a first scaffold, a linker, a second scaffold, and a second spacer (or a second targeting sequence). In some embodiments, a tandem guide agent is for complexing with a variant Cas9 protein and a second, different variant Cas protein (e.g., a variant Cas 12 protein). [01441 In some embodiments, a tandem guide agent comprises in 5’ to 3’ order: a first scaffold, a first spacer (or a first targeting sequence), a linker, a second spacer (or a second targeting sequence) and a second scaffold. In some embodiments, a tandem guide agent is for complexing with a variant Cas9 protein and a second, different variant Cas protein (e.g., a variant Casl2 protein).

[0145] In some embodiments, a tandem guide agent comprises a unit (e.g., a gRNA) comprising in 5’ to 3’ order: a scaffold and a spacer. In some embodiments, a tandem guide agent comprises a gRNA comprising in 5’ to 3’ order: a tracrRNA and a crRNA.

[0146] In some embodiments, a tandem guide agent is for complexing with a variant Cas 12 protein and the tandem guide agent comprises a unit (e.g., a gRNA) comprising in 5’ to 3’ order: a scaffold and a spacer.

[01471 In some embodiments, a tandem guide agent comprises two units (e.g., gRNAs), each comprising in 5’ to 3’ order: a scaffold and a spacer. In some embodiments, a tandem guide agent comprises two gRNAs each comprising a crRNA. In some embodiments, a tandem guide agent is for complexing with a variant Cas 12 protein and the tandem guide agent comprises two gRNAs each comprising in 5’ to 3’ order: a scaffold and a spacer.

[0148] In some embodiments, a tandem guide agent is for use with a variant Cas 12 protein and the tandem guide agent comprises a second unit (e.g., second sgRNA) comprising 5’ to 3’ order: a second spacer and a second scaffold.

[0149] In some embodiments, a tandem guide agent comprises in 5’ to 3’ order: a first scaffold, a first spacer (or a first targeting sequence), a linker, a second scaffold and a second spacer (or a second targeting sequence).

[0150] In some embodiments, the present disclosure provides a plurality of tandem guide agents. In some embodiments, the present disclosure provides a library of tandem guide agents. [0151] Various methods for production of tandem guide agents can be utilized in accordance with the present disclosure. Tandem guide agents can be produced by methods known in the art. For example, in some embodiments, a polynucleotide corresponding to a tandem guide agent or portion thereof can be produced by in vitro transcription, for example, using a DNA template. A plasmid DNA used as a template for in vitro transcription to generate a polyribonucleotide described herein is also within the scope of the present disclosure.

[0152] In some embodiments, a tandem guide agent of the present disclosure is provided and/or used in complex with a Cas protein, e.g., as a tandem guide complex.

II. Cas Proteins

[0153] Various types of Cas proteins can be used to practice the technologies disclosed herein. In some embodiments, provided technologies include one or more Cas proteins and/or nucleic acids encoding the same. Cas proteins, as referred to herein, can include any Cas protein that exhibits specific association (or “targeting”) to a nucleic acid target site.

[0154] Cas proteins according to the present disclosure comprise, but are not limited to, Class 2 Cas proteins. In general, Class 2 Cas proteins have effectors that are single multi-domain proteins. Typically, Class 2 Cas are derived from and/or originated in bacteria. Provided technologies can include Cas proteins from any appropriate prokaryotic species, including but not limited to Cas proteins from S. pyogenes, S. aureus, C. jejun, N. meningitidis, or 5. thermophilus, or variants, or combinations thereof.

[0155] In some embodiments, a Cas protein comprises domains that are all derived from a single species. In some embodiments, a Cas protein includes one or more domains from one species and one or more domains from a different species.

[0156] In some embodiments, a Cas protein for use in accordance with the present disclosure is a Class 2 Cas protein. Class 2 Cas proteins are also divided into specific types that depend on the specific Cas endonuclease responsible for cleavage and its mechanism of action. Type 11 includes Cas9 proteins and is characterized into subtypes 11-A, 11-B, and 11-C; Type V includes Cas 12 and Cas 14 proteins and is characterized into subtypes V-A, V-B, V-E, V-K, V-F; and Type VI includes Cas 13 proteins and is characterized into subtypes VI- A and VI-B. Some types/subtypes of Class 2 Cas proteins target DNA while others target RNA or both DNA and RNA. [01571 In some embodiments, a Cas protein targets DNA. In some embodiments, a Cas protein targets RNA. In some embodiments, a Cas protein targets both DNA and RNA.

[0158] Cas proteins according to the present disclosure include, but are not limited to, Cas nucleases such as Cas9, Cas 12 (e.g., Cas 12a), Cas 14, as well as nucleases derived or obtained therefrom. In some embodiments, a Cas protein is a Cas9, or a variant thereof. In some embodiments, a Cas protein is a Cas 12, or a variant thereof. In some embodiments, a Cas protein is a Cas13, or a variant thereof. In some embodiments, a Cas protein is a Casl4, or a variant thereof.

[0159] In some embodiments, the present disclosure provides nucleic acids encoding a Cas protein. Nucleic acids encoding Cas proteins are known in the art. For example, exemplary nucleic acids encoding Cas9 proteins are described in Cong et al., (2013) Science 399(6121 ):819-823 ; Wang et al., (2013) Cell 153(4):910-918; Mali et al., (2013) Science 399(6121):823-826; Jinek et al., (2012) Science 337(6096):816-821.

[0160] In some embodiments, a Cas9 protein according to the present disclosure may comprise or consist of a sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a sequence of SEQ ID NO: 29 (shown below). In some embodiments, a Cas9 protein according to the present disclosure may comprise or consist of a modified sequence of SEQ ID NO: 29. In some embodiments, modification of a sequence of SEQ ID NO: 29 produces an inactive variant Cas9 protein, as described herein. In some embodiments, modification of a sequence of SEQ ID NO: 29 comprises a D10A and/or H840A mutation(s). In some embodiments, a sequence of SEQ ID NO: 29 comprises a tag (e.g., FLAG tag, Glutathione-S -transferase tag, histidine tag, Strep-tag, or maltose-binding protein tag).

[0161] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDK KHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEG DLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGE KKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFL AAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFF DQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPH QIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETIT PWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEG MRKPAFLSGEQKKA1VDLLFKTNRKVTVKQLKEDYFKK1ECFDSVE1SGVEDRFNASLGT YHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKR RRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQV SGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQK GQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDIN RLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNA KLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKL IREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFV YGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGET GEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDP KKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYK EVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKG SPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAEN IIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD (SEQ ID NO: 29).

[0162] In some embodiments, a Cas9 protein or variant Cas9 protein according to the present disclosure may comprise or consist of a commercially available Cas9 protein or variant Cas9 protein. In some embodiments, a variant Cas9 protein according to the present disclosure may comprise or consist of dCas9-3XFLAG™-Biotin Protein (Sigma- Aldrich, Catalog No. DCAS9PROT).

[0163] It will be understood that exemplary Cas9 proteins, amino acid sequences, and nucleic acid sequences disclosed herein are provided to illustrate non-limiting embodiments embraced by the present disclosure. Additional suitable Cas proteins and sequences for variant Cas9 proteins and variant non-Cas9 proteins will be apparent to a skilled artisan based on the present disclosure, and the disclosure is not limited in this respect. [01641 In some embodiments, a Cas protein for use in the context of the present disclosure is a variant Cas protein. A Cas protein may be described as a variant when at least one structural or functional feature is altered, such as, for example, when it is inactive, has altered cleavage and/or nuclease activity, and/or altered PAM specificity. In some embodiments, the present disclosure provides nucleic acids encoding a variant Cas protein.

[0165] In some embodiments, provided technologies include two or more variant Cas proteins. In some embodiments, provided technologies include a first variant Cas protein and a second variant Cas protein.

[0166] In some embodiments, a variant Cas protein is a Type II Cas protein. In some embodiments, a variant Cas protein is a Type V Cas protein. In some embodiments, a variant Cas protein is a Type VI Cas protein.

[01671 In some embodiments, a Type II variant Cas is a variant Cas9 protein. In some embodiments, a Type V variant Cas is a variant Casl2 protein, e.g., a variant Cas 12a protein. In some embodiments, a Type V variant Cas is a variant Cas 14 protein. In some embodiments, a Type VI variant Cas is a variant Cas 13 protein. In some embodiments, a Type VI variant Cas is a variant Cas 13a protein. In some embodiments, a Type VI variant Cas is a variant Cas 13b protein. [0168] In some embodiments, a variant Cas protein is a variant Cas9 protein. In some embodiments, a variant Cas protein is a variant Casl2 protein, e.g., a variant Casl2a protein. In some embodiments, a variant Cas protein is a variant Cas 13 protein. In some embodiments, a variant Cas protein is a variant Cas 14 protein.

[0169] In some embodiments, a variant Cas protein targets DNA. In some embodiments, a variant Cas protein targets RNA. In some embodiments, a variant Cas protein targets both DNA and RNA.

[0170] In some embodiments, a variant Cas protein has altered cleavage activity. Cleavage activity of a Cas protein is typically mediated by one or more Cas nuclease domain(s), e.g., endonuclease domain(s). For example, cleavage activity of S. pyogenes Cas9 is mediated by coordinated functions of two nuclease domains, RuvC and HNH. Without wishing to be bound by theory, the RuvC and HNH domains function together to generate blunt-ended, double- strand breaks (DSBs) by cleaving opposite strands of double- stranded DNA, with the HNH domain cleaving the target strand, i.e., the strand complementary to a gRNA, and the RuvC domain cleaving the non-targeting strand containing a Protospacer Adjacent Motif sequence (PAM sequence, NGG for 5. pyogenes Cas9).

[0171] In some embodiments, a variant Cas protein has altered nuclease domain function. In some embodiments, a variant Cas protein has a nuclease domain that is or has been engineered and/or mutated to alter nuclease domain function. In some embodiments, a variant Cas protein lacks nuclease domain function.

[0172] In some embodiments, a variant Cas protein is a nuclease-inactive Cas protein. The terms “inactive Cas”, “nuclease dead Cas”, “dead Cas”, and “dCas” as used herein all refer to a variant Cas protein that lacks cutting activity (e.g., lacks nuclease activity). Variant Cas proteins and nuclease domains thereof can be inactivated using one or more methods known to those skilled in the arts. In some embodiments, an inactive variant Cas protein retains sequencespecific nucleotide binding activity. In some embodiments, an inactive variant Cas protein binds to a target nucleic acid sequence.

[0173] The present disclosure is not bound to any particular method of Cas inactivation using any particular Cas protein or variant thereof and appreciates that a skilled artisan may employ one or more methods to inactivate one or more Cas proteins to produce a variant Cas protein of the present disclosure. It is further appreciated that nuclease domains may differ between Cas proteins, or have yet to be discovered, and that a skilled artisan, through the present disclosure, can mutate one or more nuclease domains of one or more Cas proteins to produce an inactive Cas protein described in the present disclosure.

[0174] In some embodiments, a variant Cas protein is an inactive Type 11 Cas protein. In some embodiments, a variant Cas protein is an inactive Type V Cas protein. In some embodiments, a variant Cas protein is an inactive Type VI Cas protein.

[0175] In some embodiments, a variant Cas protein is an inactive Cas9 protein. In some embodiments, a variant Cas protein is an inactive Cas 12 protein, e.g., an inactive Cas 12a protein. In some embodiments, a variant Cas protein is an inactive Cast 3 protein. In some embodiments, a variant Cas protein is an inactive Cas 14 protein.

[0176] In some embodiments, a variant Cas protein is rendered inactive via one or more mutations in and/or around one or more nuclease domains. For example, in some embodiments, a variant Cas protein is a variant Cas9 where both RuvC and HNH domains are mutated, such that the variant Cas protein lacks nuclease activity. In some embodiments, a variant Cas9 protein is rendered inactive via a mutation at one or more positions selected from D10, H840 and N863 in the case of a variant spCas9 protein or one or more corresponding positions of a Cas9 protein from a different bacterial species. In some embodiments, a variant Cas9 protein is inactive and comprises mutations at positions DIO and H840 in the case of a variant spCas9 protein or corresponding positions of a Cas9 protein from a different bacterial species. In some embodiments, a variant Cas9 protein is inactive and comprises D10A and H840A mutations in the case of a variant spCas9 protein or corresponding positions of a Cas9 protein from a different bacterial species. In some embodiments, a variant Cas 12a protein is inactive and comprises a mutation at position D908 in the case of a variant AsCasl2a protein or a corresponding position of a Cas 12a protein from a different bacterial species. In some embodiments, a variant Cas 12a protein is inactive and comprises a mutation at position D9O8A in the case of a variant AsCasl2a protein or a corresponding position of a Cas 12a protein from a different bacterial species. In some embodiments, a variant Cas 14 (Casl2f) protein is inactive and comprises a mutation at position D326 and/or D510 in the case of a variant Casl4al (Casl2fl) protein or a corresponding position of a Casl4 (Casl2f) protein from a different bacterial species. In some embodiments, a variant Casl4 (Casl2f) protein is inactive and comprises a mutation at positions D326A and/or D510A in the case of a variant Casl4al (Casl2fl) protein or a corresponding position of a Casl4 (Casl2f) protein from a different bacterial species.

[0177] In some embodiments, provided technologies include two variant Cas proteins, where the two Cas proteins have different nuclease domains. In some embodiments, provided technologies include two variant Cas proteins, where the two Cas proteins are or have been inactivated via different methods. [01781 In some embodiments, a variant Cas protein is a nicking enzyme or nickase. The term “nickase” as used herein refer to a variant Cas protein that produces a single-stranded break at or near a polynucleotide target site. In some embodiments, a nick, as used herein, refers to a single- stranded break of a polynucleotide sequence. In some embodiments, a nickase retains sequence-specific nucleotide binding activity. In some embodiments, a nickase binds to a target nucleic acid sequence.

[0179] In some embodiments, a nickase may nick a target strand of a nucleotide sequence. In some embodiments, a nickase may nick a non-target strand of a nucleotide sequence.

[0180] The present disclosure is not bound to any particular method of producing a Cas nickase or any particular Cas protein and appreciates that a skilled artisan may employ one or more methods to produce a Cas protein with nickase activity. It is further appreciated that nuclease domains may differ between Cas proteins, or have yet to be discovered, and that a skilled artisan, through the present disclosure, can mutate one or more nuclease domains of one or more Cas proteins to produce a Cas nickase described in the present disclosure.

[0181] In some embodiments, a variant Cas protein comprises an engineered and/or mutated nuclease domains that is a nickase. In some embodiments, a variant Cas protein that is a nickase comprises one functional nuclease domain and at least one inactive nuclease domain, such that the Cas protein is capable of creating a single stranded break, i.e., a nick, in a target polynucleotide.

[0182] In some embodiments, a variant Cas protein is a nickase and comprises a point mutation. In some embodiments, a variant Cas protein comprises a mutation that ablates HNH- mediated substrate cleavage and results in nickase activity. In some embodiments, a variant Cas protein comprises one or more mutations in the RuvC and/or HNH nuclease domains that result in nickase activity.

[0183] In some embodiments, a variant Cas9 protein is a nickase and comprises a mutation at one or more positions selected from D10, E762, H840, N854, N863 and D986 in the case of a variant spCas9 protein or corresponding positions of a Cas9 protein from a different bacterial species. In some embodiments, a variant Cas9 protein is a nickase and comprises one or more mutation selected from D10A, E762A, H840A, N854A, N863A and D986A in the case of a variant spCas9 protein or corresponding positions of a Cas9 protein from a different bacterial species.

[0184] In some embodiments, a variant Casl2a protein is a nickase comprising a mutation at position R1226 in the case of a variant AsCasl2a protein or a corresponding position of a Casl2a protein from a different bacterial species. In some embodiments, a variant Casl2a protein is a nickase comprising a mutation at position R1226A in the case of a variant AsCasl2a protein or a corresponding position of a Casl2a protein from a different bacterial species.

[0185] In some embodiments, a variant Cas9 protein is a nickase comprising a mutation at position DIO or H840 in the case of a variant spCas9 protein or corresponding positions of a Cas9 protein from a different bacterial species. In some embodiments, a nickase comprises a mutation at position DIO in the case of a variant spCas9 protein or corresponding positions of a Cas9 protein from a different bacterial species. In some embodiments, a nickase comprises a mutation at position H840 in the case of a variant spCas9 protein or corresponding positions of a Cas9 protein from a different bacterial species. In some embodiments, a nickase comprises a D10A substitution in the case of a variant spCas9 protein or corresponding positions of a Cas9 protein from a different bacterial species. In some embodiments, a nickase comprises a H840A substitution in the case of a variant spCas9 protein or corresponding positions of a Cas9 protein from a different bacterial species.

[0186] In some embodiments, a variant Cas9 protein is a nickase comprising a mutation at one or more of positions E762, H983 or D986 in the case of a variant spCas9 protein or corresponding positions of a Cas9 protein from a different bacterial species. In some embodiments, a Cas9 nickase comprises a mutation at position E762 or corresponding position from a different bacterial species. In some embodiments, a nickase comprises a mutation at position H983 or corresponding position from a different bacterial species. In some embodiments, a nickase comprises a mutation at position D986 or corresponding position from a different bacterial species. In some embodiments, a nickase comprises a E762A substitution or corresponding position from a different bacterial species. In some embodiments, a nickase comprises a H983A substitution or corresponding position from a different bacterial species. In some embodiments, a nickase comprises a D986A substitution or corresponding position from a different bacterial species.

[0187] In some embodiments, a variant Cas protein is a Type II Cas protein with nickase activity. In some embodiments, a Type II variant Cas protein nicks a target strand of a nucleotide sequence. In some embodiments, a Type II variant Cas protein nicks a non-target strand of a nucleotide sequence. In some embodiments, a variant Cas protein is a Type V Cas protein with nickase activity. In some embodiments, a Type V variant Cas protein nicks a target strand of a nucleotide sequence. In some embodiments, a Type V variant Cas protein nicks a non-target strand of a nucleotide sequence. In some embodiments, a variant Cas protein is a Type VI Cas protein with nickase activity. In some embodiments, a Type VI variant Cas protein nicks a target strand of a nucleotide sequence. In some embodiments, a Type VI variant Cas protein nicks a non-target strand of a nucleotide sequence.

[0188] In some embodiments, a variant Cas protein is a Cas9 nickase. In some embodiments, a Cas9 nickase nicks a target strand of a nucleotide sequence. In some embodiments, a Cas9 nickase nicks a non-target strand of a nucleotide sequence. In some embodiments, a variant Cas protein is a Casl2 nickase. In some embodiments, a Casl2 nickase nicks a target strand of a nucleotide sequence. In some embodiments, a Cas 12 nickase nicks a non-target strand of a nucleotide sequence. In some embodiments, a variant Cas protein is a Cas 13 nickase. In some embodiments, a Cas 13 nickase nicks a target strand of a nucleotide sequence. In some embodiments, a Casl3 nickase nicks a non-target strand of a nucleotide sequence. In some embodiments, a variant Cas protein is a Cas 14 nickase. In some embodiments, a Cas 14 nickase nicks a target strand of a nucleotide sequence. In some embodiments, a Cas 14 nickase nicks a non-target strand of a nucleotide sequence.

[0189] In some embodiments, a variant Cas protein has altered PAM specificity.

[0190] Typically, naturally-occurring Cas enzymes recognize a short sequence motif adjacent to target sites, e.g., a PAM, in foreign DNA to distinguish self from non-self. For example, Cas9 from Streptococcus pyogenes (spCas9) naturally recognizes target sites with a PAM consisting of the nucleotide NGG. In some embodiments, a Cas protein typically recognizes its optimal PAM by direct molecular interaction. For example, spCas9 typically recognizes its optimal NGG PAM by direct molecular readout of the guanine DNA bases via the amino acid side chains of R1333 and R1335.

[0191] In some embodiments, a variant Cas protein recognizes a different PAM sequence than a naturally-occurring Cas protein. In some embodiments, a variant Cas protein has engineered PAM sequence specificity (e.g., to a particular desired sequence).

[0192] In some embodiments, a variant Cas protein is a PAM-less Cas protein. The terms “variant Cas protein is PAM-less” and “PAM-less Cas protein” as used herein both refer to a variant Cas protein that has reduced, diminished, and/or changed dependency on a PAM for activity as described herein. In some embodiments, a variant Cas protein has a reduced reliance on a PAM.

[0193] In some embodiments, the activity of a PAM-less variant Cas protein is not dependent on one or more PAMs. In some embodiments, the activity of a PAM-less variant Cas protein has relaxed dependency on PAMs, e.g., reduced and/or diminished PAM dependent activity. In some embodiments, a PAM-less Cas variant protein requires PAMs comprising a single nucleotide.

[0194] In some embodiments, a variant Cas protein comprises one or more mutations that alter PAM specificity. In some embodiments, a variant Cas protein comprises one or more mutations that alter the specificity of a PAM sequence. In some embodiments, a variant Cas protein comprises one or more mutations that relax dependency on a PAM sequence. In some embodiments, a variant Cas protein comprises one or more mutations that eliminate the reliance on a PAM sequence.

[0195] In some embodiments, a PAM-less variant Cas9 protein is produced through modification of either R1333 or R1335 amino acid in the case of a PAM-less variant spCas9 protein or corresponding positions of a Cas9 protein from a different bacterial species. In some embodiments, a PAM-less variant spCas9 protein has ablated PAM dependent activity against sites with NGG, NAG, or NGA PAMs.

[0196] In some embodiments, a variant Cas protein is an inactive Type II Cas protein that is also PAM-less. In some embodiments, a variant Cas protein is an inactive Type V Cas protein that is also PAM-less. In some embodiments, a variant Cas protein is an inactive Type VI Cas protein that is also PAM-less.

[0197] In some embodiments, a variant Cas protein is a PAM-less inactive Cas9. In some embodiments, a variant Cas protein is a PAM-less inactive Cas 12, e.g., Casl2a. In some embodiments, a variant Cas protein is a PAM-less inactive Cas 13. In some embodiments, a variant Cas protein is a PAM-less inactive Casl4.

[0198] In some embodiments, a variant Cas protein is a PAM-less Type II Cas protein with nickase activity. In some embodiments, a variant Cas protein is a PAM-less Type V Cas protein with nickase activity. In some embodiments, a variant Cas protein is a PAM-less Type VI Cas protein with nickase activity.

[0199] In some embodiments, a variant Cas protein is a PAM-less Cas9 with nickase activity. In some embodiments, a variant Cas protein is a PAM-less Cas 12, e.g., Cas 12a with nickase activity. In some embodiments, a variant Cas protein is a PAM-less Cas 13 with nickase activity. In some embodiments, a variant Cas protein is a PAM-less Cas 14 with nickase activity. [0200] Various methods for production of Cas proteins can be utilized in accordance with the present disclosure. Methods for protein expression arc known in the art. In some embodiments, Cas proteins as described herein may be produced from nucleic acid molecules using molecular’ biological methods known to the ait. Nucleic acid molecules are inserted into a vector that is able to express the Cas protein(s) when introduced into an appropriate host cell. Any of the methods known to one skilled in the art for the insertion of DNA fragments into a vector may be used to construct expression vectors encoding Cas protein(s). These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombination (See Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory; Current Protocols in Molecular Biology, Eds. Ausubel, et al., Greene Publ. Assoc., Wiley- Interscience, NY).

III. Tandem Guide Complexes

[0201] In some embodiments, provided herein are tandem guide complexes that comprise or consist of a tandem guide agent (e.g., tgRNA) in complex with one or more Cas protein(s). In some embodiments, a tandem guide complex comprises or consists of a tandem guide agent (e.g., tgRNA) and two Cas proteins, i.e., a first Cas protein and a second Cas protein. FIG. 1C provides an exemplary schematic of a tandem guide complex.

[0202] In some embodiments, a tandem guide complex is capable of binding to one or more target DNA sequences. In some embodiments, a tandem guide complex is capable of binding to two or more target DNA sequences. In some embodiments, a tandem guide complex is capable of binding to two target DNA sequences.

[0203] In some embodiments, Cas proteins (e.g., variant Cas proteins, e.g., dCas) form a complex with a corresponding tandem guide agent (e.g., tgRNA) and the tandem guide agents provides the sequence specificity to the tandem guide complex via the guide sequences. In other words, the Cas proteins are guided to two target sites (e.g., stabilized at target sites) within one or more target nucleic acid(s) by virtue of their association with the tandem guide agent (e.g., tgRNA).

[0204] In some embodiments, a tandem guide complex comprises a tandem guide agent (e.g., tgRNA), a first variant Cas protein, and a second variant Cas protein, where the first variant Cas protein is guided to a first target site on a first nucleic acid via a first unit (e.g., first gRNA) of the tandem guide agent and the second variant Cas protein is guided to a second target site on a second nucleic acid via a second unit (e.g., second gRNA) of the tandem guide agent.

[0205] In some embodiments, a tandem guide complex is recruited to a target sequence(s) by base-pairing between the “guide” portions of the tandem guide agent (e.g., tgRNA) and the target DNA sequences. In some embodiments, for successful binding of Cas proteins, each target DNA sequence typically is adjacent to a correct Protospacer Adjacent Motif (PAM) sequence. In some embodiments, binding of a tandem guide complex (e.g., dCas-tgRNA complex) localizes the Cas proteins to the target polynucleotide sequences. In some embodiments, binding of a tandem guide complex to the target polynucleotide sequences promotes assembly of the polynucleotides and/or structures or surfaces associated therewith. [0206] In some embodiments, a tandem guide complex is capable of binding to one or more target polynucleotide sequences (e.g., DNA and/or RNA). In some embodiments, a tandem guide complex is capable of binding to two or more target polynucleotide sequences (e.g., DNA and/or RNA). In some embodiments, a tandem guide complex is capable of binding to two target polynucleotide sequences (e.g., DNA and/or RNA).

[0207] In some embodiments, a tandem guide complex is capable of binding to one or more target DNA sequences. In some embodiments, a tandem guide complex is capable of binding to two or more target DNA sequences. In some embodiments, a tandem guide complex is capable of binding to two target DNA sequences.

[0208] In some embodiments, a tandem guide complex comprises or consists of a tandem guide agent (e.g., tgRNA) as described above in complex with one or more variant Cas protein(s) as described above. In some embodiments, a tandem guide complex comprises or consists of a tandem guide agent (e.g., tgRNA) and two variant Cas proteins, i.e., a first Cas protein and a second Cas protein.

[0209] In some embodiments, a tandem guide complex comprises a tandem guide agent, a first variant Cas protein, and a second variant Cas protein. In some embodiments, first and second variant Cas proteins are a same type of variant Cas protein. In some embodiments, first and second variant Cas proteins are Type II. In some embodiments, first and second variant Cas proteins are Type V. In some embodiments, first and second variant Cas proteins arc Type VI. [0210] In some embodiments, a tandem guide complex comprises first and second variant Cas proteins that are different types of variant Cas protein. In some embodiments, a first variant Cas protein is Type II and a second variant Cas protein is Type V. In some embodiments, a first variant Cas protein is Type II and a second variant Cas protein is Type VI. In some embodiments, a first variant Cas protein is Type V and a second variant Cas protein is Type VI. [02111 In some embodiments, a tandem guide complex comprises first and second variant Cas proteins that are the same variant Cas protein. In some embodiments, first and second variant Cas proteins are variant Cas9 proteins. In some embodiments, first and second variant Cas proteins are variant Cas 12 proteins. In some embodiments, first and second variant Cas proteins are variant Cas 13 proteins. In some embodiments, first and second variant Cas proteins are variant Cas 14 proteins.

[0212] In some embodiments, a tandem guide complex comprises first and second variant Cas proteins that are different variant Cas proteins. In some embodiments, a first variant Cas protein is a variant Cas9 protein and a second variant Cas protein is a variant Cas 12 protein. In some embodiments, a first variant Cas protein is a variant Cas9 protein and a second variant Cas protein is a variant Cas 12 protein. In some embodiments, a first variant Cas protein is variant Cas9 protein and a second variant Cas protein is variant Cas 12 protein. In some embodiments, a first variant Cas protein is a variant Cas9 protein and a second variant Cas protein is a variant Cas 12 protein. In some embodiments, a first variant Cas protein is a variant Cas9 protein and a second variant Cas protein is a variant Cas 13 protein. In some embodiments, a first variant Cas protein is a variant Cas9 protein and a second variant Cas protein is a variant Cas 14 protein. In some embodiments, a first variant Cas protein is a variant Cas 12 protein and a second variant Cas protein is a variant Cas 13 protein. In some embodiments, a first variant Cas protein is a variant Cas 12 protein and a second variant Cas protein is a variant Cas 14 protein. In some embodiments, a first variant Cas protein is a variant Cas 13 protein and a second variant Cas protein is a variant Cas 14 protein.

[0213] In some embodiments, a tandem guide complex comprises first and second variant Cas proteins that are the same functional variants. In some embodiments, first and second variant Cas proteins are both inactive variant Cas proteins. In some embodiments, first and second variant Cas proteins are both nickases. In some embodiments, first and second variant Cas proteins are both PAM-less inactive variant Cas proteins. In some embodiments, first and second variant Cas proteins are both PAM-less nickases. [02141 In some embodiments, a tandem guide complex comprises first and second variant Cas proteins that are different functional variants. In some embodiments, a first variant Cas protein is an inactive variant Cas protein and a second variant Cas protein is a nickase. In some embodiments, a first variant Cas protein is an inactive variant Cas protein and a second variant Cas protein is a PAM-less variant Cas protein. In some embodiments, a first variant Cas protein is a nickase and a second variant Cas protein is a PAM-less variant Cas. In some embodiments, a first variant Cas protein is an inactive PAM-less variant Cas protein and a second variant Cas protein is a PAM-less nickase. In some embodiments, a first variant Cas protein is an inactive variant Cas protein and a second variant Cas protein is a PAM-less nickase. In some embodiments, a first variant Cas protein is an inactive PAM-less variant Cas protein and a second variant Cas protein is a nickase.

[02151 In some embodiments, a tandem guide complex comprises first and second variant Cas proteins that are different functional variants, but the same type of Cas protein. For example, in some embodiments, a first variant Cas protein is a Type II inactive variant Cas protein and a second variant Cas protein is a Type II Cas nickase.

[0216] In some embodiments, a tandem guide complex comprises first and second variant Cas proteins that are different types of Cas proteins and different functional valiants. In some embodiments, a first variant Cas protein is a Type II inactive variant Cas protein and a second variant Cas protein is a Type V Cas nickase. In some embodiments, a first variant Cas protein is a Type II inactive variant Cas protein and a second variant Cas protein is a Type VI Cas nickase. In some embodiments, a first variant Cas protein is a Type V inactive variant Cas protein and a second variant Cas protein is a Type VI Cas nickase. In some embodiments, a first variant Cas protein is a Type V inactive variant Cas protein and a second variant Cas protein is a Type II Cas nickase. In some embodiments, a first variant Cas protein is a Type VI inactive variant Cas protein and a second variant Cas protein is a Type II Cas nickase. In some embodiments, a first variant Cas protein is a Type VI inactive variant Cas protein and a second variant Cas protein is a Type V Cas nickase. In some embodiments, a first variant Cas protein is a Type II inactive variant Cas protein and a second variant Cas protein is a Type V PAM-less variant Cas protein. In some embodiments, a first variant Cas protein is a Type II inactive variant Cas protein and a second variant Cas protein is a Type VI PAM-less variant Cas protein. In some embodiments, a first variant Cas protein is a Type V inactive variant Cas protein and a second variant Cas protein is a Type VI PAM-less variant Cas protein. In some embodiments, a first variant Cas protein is a Type V inactive variant Cas protein and a second variant Cas protein is a Type II PAM-less variant Cas protein. In some embodiments, a first variant Cas protein is a Type VI inactive variant Cas protein and a second variant Cas protein is a Type II PAM-less variant Cas protein. In some embodiments, a first variant Cas protein is a Type VI inactive variant Cas protein and a second variant Cas protein is a Type V PAM-less variant Cas protein. In some embodiments, a first variant Cas protein is a Type II Cas nickase and a second variant Cas protein is a Type V PAM-less variant Cas protein. In some embodiments, a first variant Cas protein is a Type II Cas nickase and a second variant Cas protein is a Type VI PAM-less variant Cas protein. In some embodiments, a first variant Cas protein is a Type V Cas nickase and a second valiant Cas protein is a Type VI PAM-less variant Cas protein. In some embodiments, a first variant Cas protein is a Type V Cas nickase and a second variant Cas protein is a Type II PAM-less variant Cas protein. In some embodiments, a first variant Cas protein is a Type VI Cas nickase and a second variant Cas protein is a Type II PAM-less variant Cas protein. In some embodiments, a first variant Cas protein is a Type VI Cas nickase and a second variant Cas protein is a Type V PAM-less variant Cas protein.

[0217] In some embodiments, provided are methods for producing tandem guide complexes of the present disclosure. Tandem guide complexes can be produced by methods known in the ail. In some embodiments, tandem guide agents and Cas proteins are assembled as tandem guide complexes in the context of methods and systems of the present disclosure. In some embodiments, tandem guide agents and Cas proteins are provided separately and selfassemble to form tandem guide complexes in the context of methods and systems of the present disclosure.

IV. Methods and Uses of Tandem Guide Agents and Tandem Guide Complexes [02181 Traditionally, CRISPR-Cas systems have been used for applications in genetic editing and gene regulation (Adli, (2018) Nature Communications 9:1911). The present disclosure encompasses a recognition that provided tandem guide agents (e.g., tgRNAs) and tandem guide complexes may be used in any method and/or application in which two materials are desired to be brought together in close proximity. In some embodiments, tandem guide agents and tandem guide complexes are used in methods and applications that involve bringing two nucleotide sequences into close proximity. The present disclosure provides the insight that co-localization of polynucleotides using such tandem guide agents and tandem guide complexes has numerous potential applications and benefits, particularly for in vitro applications, including, but not limited to, multiplex applications. For example, as described herein, in some embodiments, provided tandem guide agents and tandem guide complexes enable in vitro assembly of polynucleotide (e.g., DNA) sequences. In some embodiments, tandem guide agents (e.g., tgRNAs) and tandem guide complexes can facilitate workflows such as multiplexed long DNA sequence assembly (e.g., “CASsembly”). In some embodiments, provided tandem guide agents and/or tandem guide complexes enable self-assembly of various surfaces through binding to target DNA sequences. In some embodiments, provided tandem guide agents (e.g., tgRNAs) can enable self-assembly of micro- and nano-materials, such as an assembly of various cell types with target DNA sequences attached to their surfaces, or multiple solid surfaces decorated with target DNAs. In some embodiments, provided tandem guide agents and complexes enable selfassembly of spatially arrayed protein-encoding DNA fragments for protein microarray fabrication.

[0219] In some embodiments, the present disclosure provides methods (e.g., in vitro methods) for bringing together two or more polynucleotide molecules and/or polynucleotide sequences spatially. In some embodiments, the present disclosure provides methods for bringing together two or more DNA molecules and/or DNA sequences spatially. In some embodiments, the present disclosure provides methods for bringing together two or more RNA molecules and/or RNA sequences spatially. [02201 In some embodiments, the present disclosure provides methods for bringing together two or more polynucleotide sequences that comprise: obtaining or providing a tandem guide complex as described herein or components thereof, contacting a tandem guide complex with a first polynucleotide sequence and a second polynucleotide sequence, where the tandem guide complex is capable of binding and/or binds to the first polynucleotide sequence and the second polynucleotide sequence, thereby bringing the sequences together.

[0221] In some embodiments, a tandem guide complex is provided or obtained as an assembled tandem guide complex(es). In some embodiments, contacting comprises substantially simultaneously contacting a tandem guide complex with polynucleotide sequences. In some embodiments, contacting comprises sequentially contacting a tandem guide complex with a first polynucleotide sequence and then a second polynucleotide sequence.

[02221 In some embodiments, components of a tandem guide complex are provided separately and assembled with the contacting (i.e., at substantially the same time as the contacting). For example, in some embodiments, a tandem guide agent, a first variant Cas protein, and/or a second variant Cas protein are provided separately and assemble with the contacting to form a tandem guide complex. In some embodiments, one or more of a tandem guide agent, a first variant Cas protein, a second variant Cas protein a first polynucleotide sequence, and a second polynucleotide sequence are provided separately and the contacting assembles a complex comprising a tandem guide complex that is associated with the first and second polynucleotide sequences.

[0223] In some embodiments, the ability of provided tandem guide agents and/or tandem guide complexes to bring nucleic acids into proximity can be demonstrated using a proximity assay. Proximity assays are known in the art and include commercially available proximity assays such as, e.g., an AlphaLISA proximity assay (Perkin Elmer, Waltham, MA).

[0224] In some embodiments, the present disclosure provides methods (e.g., in vitro methods) for bringing a plurality of pairs of polynucleotides together. In some embodiments, provided methods comprise: obtaining or providing a plurality of tandem guide complexes or components thereof, and contacting a plurality of tandem guide complexes with one or more compositions that comprise a plurality of polynucleotides, wherein each tandem guide complex is capable of binding to two different polynucleotides among the plurality of polynucleotides. In some embodiments, each tandem guide complex of the plurality binds to or is capable of binding to two unique polynucleotide sequences, thereby bringing a plurality of pairs of polynucleotides together.

[0225] In some embodiments, each polynucleotide of a plurality of polynucleotides comprises a detectable label. In some embodiments, each polynucleotide of a plurality of polynucleotides comprises a unique barcode sequence.

[0226] In some embodiments, a plurality of tandem guide complexes arc provided or obtained as a plurality of assembled tandem guide complexes. In some embodiments, contacting comprises substantially simultaneously contacting a plurality of tandem guide complexes with a plurality of polynucleotide sequences.

[0227] In some embodiments, components of a plurality of tandem guide complex are provided separately and assembled with the contacting. For example, in some embodiments, a plurality of tandem guide agents and a plurality of variant Cas proteins are provided separately and assemble with the contacting to form a tandem guide complex. In some embodiments, one or more of a plurality of tandem guide agents, a plurality of Cas proteins, and a plurality of polynucleotide sequences are provided separately and the contacting assembles a complex comprising a plurality of tandem guide complexes, where each is associated with a pair of polynucleotide sequences.

C ASsembly Methods

[0228] In some embodiments, the present disclosure provides methods for assembling polynucleotides, referred to herein as “CASsembly”. In some embodiments, the present disclosure provides methods for assembling large polynucleotides from smaller polynucleotides using tandem guide complexes. In some embodiments, provided methods are multiplex methods. In some embodiments, provided are methods to perform multiplexed assembly of large pieces of DNA using tandem guide complexes (e.g., dCas-tgRNAs). [02291 In some embodiments, provided tandem guide agents and/or tandem guide complexes can enable assembly of polynucleotide (e.g., DNA) sequences at room temperature, eliminating the necessity for high temperatures or thermocycling, potentially reducing sequence artifacts and improving resulting DNA library quality compared to PCR-based approaches. The present disclosure encompasses the recognition that provided CASsembly methods may be useful for polynucleotide (e.g., DNA) assembly from smaller pieces on scales ranging from one assembled molecule to millions.

[0230] In some embodiments, the present disclosure provides polynucleotide assembly methods that are performed, e.g., in solution. In some embodiments, the present disclosure provides in vitro methods that comprise: contacting a first polynucleotide sequence and a second polynucleotide sequence with a tandem guide complex or components thereof, and assembling the first polynucleotide sequence and the second polynucleotide sequence to form an assembled polynucleotide that comprises the first polynucleotide sequence and the second polynucleotide sequence. In some embodiments, the first polynucleotide sequence and/or the second polynucleotide sequence are present in solution. In some embodiments, the first polynucleotide sequence and the second polynucleotide sequence are each present in solution.

[0231] In some embodiments, the present disclosure provides methods for assembly of a plurality of polynucleotides in solution. In some embodiments, provided in vitro methods comprise: contacting a plurality of tandem guide complexes or components thereof with one or more compositions that comprise a plurality of polynucleotides, wherein each tandem guide complex binds to two different polynucleotides among the plurality of polynucleotides, and assembling the two different polynucleotides bound by each tandem guide complex, thereby forming a plurality of assembled polynucleotides. In some embodiments, each polynucleotide of a plurality of polynucleotides comprises a detectable label. In some embodiments, each polynucleotide of a plurality of polynucleotides comprises a unique barcode sequence.

[0232] In some embodiments, the present disclosure provides polynucleotide assembly methods that are performed on a solid surface. In some embodiments, the present disclosure provides in vitro methods that comprise: obtaining or providing a surface associated with a first polynucleotide sequence, contacting the first polynucleotide sequence with a tandem guide complex and a second polynucleotide sequence, and assembling the first polynucleotide sequence and the second polynucleotide sequence to form an assembled polynucleotide that comprises the first polynucleotide sequence and the second polynucleotide sequence.

[0233] In some embodiments, the second polynucleotide sequence is present in solution. In some embodiments, the second polynucleotide sequence is associated with a second, different surface, e.g., beads (e.g., microbeads).

[0234] In some embodiments, the present disclosure provides methods for assembly of a plurality of polynucleotides on a surface. In some embodiments, the present disclosure provides in vitro methods comprising: obtaining or providing a surface associated with a first plurality of polynucleotides, contacting the first plurality of polynucleotides with a plurality of tandem guide complexes and a second plurality of polynucleotides, and assembling the polynucleotides bound by each tandem guide complex, thereby forming a plurality of assembled polynucleotide pairs. In some embodiments, the surfaces comprise or consist of planar surfaces, beads (e.g., microbeads), and/or cell surfaces.

[0235] In some embodiments, each polynucleotide of a plurality of polynucleotides comprises a detectable label. In some embodiments, each polynucleotide of a plurality of polynucleotides comprises a unique barcode sequence.

[0236] In some embodiments, the plurality of polynucleotides are each associated the surface. In some embodiments, each polynucleotide of the first plurality of polynucleotides is associated with a defined position on the surface. In some embodiments, each assembled polynucleotide of the plurality of assembled polynucleotides is associated with a defined position on the surface.

[0237] In some embodiments, the second plurality of polynucleotide sequences are present in solution. In some embodiments, the second plurality of polynucleotide sequences are associated with a different surface(s), e.g., beads (e.g., microbeads).

[0238] In some embodiments, a tandem guide complex or plurality thereof is provided or obtained as an assembled tandem guide complex(es). In some embodiments, contacting comprises substantially simultaneously contacting a tandem guide complex or plurality thereof with polynucleotide sequences. In some embodiments, contacting comprises sequentially contacting a tandem guide complex or plurality thereof with a first polynucleotide sequence or first plurality of sequences and then a second polynucleotide sequence or second plurality of sequences.

[0239] In some embodiments, components of a tandem guide complex or plurality of tandem guide sequences are provided separately and assembled with the contacting (i.e., at substantially the same time as the contacting). For example, in some embodiments, tandem guide agent(s), first variant Cas protein(s), and/or second variant Cas protein(s) are provided separately and assemble with the contacting to form a tandem guide complex or plurality of tandem guide complexes. In some embodiments, tandem guide agent(s), variant Cas protein(s), and/or polynucleotide sequences are provided separately and the contacting assembles a tandem guide complex that is associated with polynucleotide sequences, or plurality thereof.

[0240] In some embodiments, the present disclosure provides methods for assembly of a plurality of polynucleotides along a scaffold. In some embodiments, provided methods comprise: obtaining or providing two or more tandem guide complexes or components thereof, contacting the two or more tandem guide complexes with (i) a polynucleotide scaffold comprising two or more scaffold sequences that are positioned adjacently in series along the polynucleotide scaffold, and (ii) two or more assembly polynucleotide sequences, wherein the first variant Cas protein of each tandem guide complex binds a scaffold sequence and the second variant Cas protein of each tandem guide complex binds an assembly polynucleotide sequence, wherein the contacting brings the two or more assembly polynucleotide sequences into close proximity to one another, and assembling the two or more assembly polynucleotide sequences to form an assembled polynucleotide. In some embodiments, the two or more assembly polynucleotide sequences and the polynucleotide scaffold are present in solution.

[0241] The present disclosure encompasses a recognition that assembly along a scaffold permits simultaneous assembly of any number of assembled polynucleotides. In some embodiments, provided methods simultaneously assemble at least 2 assembly polynucleotide sequences, at least 3 assembly polynucleotide sequences, at least 4 assembly polynucleotide sequences, at least 5 assembly polynucleotide sequences, at least 6 assembly polynucleotide sequences, at least 7 assembly polynucleotide sequences, at least 8 assembly polynucleotide sequences, at least 9 assembly polynucleotide sequences, at least 10 assembly polynucleotide sequences, at least 20 assembly polynucleotide sequences, at least 30 assembly polynucleotide sequences, at least 40 assembly polynucleotide sequences, at least 50 assembly polynucleotide sequences, at least 60 assembly polynucleotide sequences, at least 70 assembly polynucleotide sequences, at least 80 assembly polynucleotide sequences, at least 90 assembly polynucleotide sequences, at least 100 assembly polynucleotide sequences, at least 200 assembly polynucleotide sequences, at least 300 assembly polynucleotide sequences, at least 400 assembly polynucleotide sequences, at least 500 assembly polynucleotide sequences, at least 600 assembly polynucleotide sequences, at least 700 assembly polynucleotide sequences, at least 800 assembly polynucleotide sequences, at least 900 assembly polynucleotide sequences, at least 1,000 assembly polynucleotide sequences, or more.

[0242] In some embodiments, contacting the two or more tandem guide complexes with the polynucleotide scaffold and the two or more assembly polynucleotide sequences occurs simultaneously. In some embodiments, the two or more tandem guide complexes are contacted sequentially with the polynucleotide scaffold and assembly polynucleotide sequences. In some embodiments, provided is a polynucleotide scaffold that is then contacted with two or more tandem guide complexes, followed by the two or more assembly polynucleotide sequences. In some embodiments, the two or more tandem guide complexes are contacted first with the polynucleotide scaffold, such that the tandem guide complexes associate with the polynucleotide scaffold, and then contacted with two or more assembly polynucleotide sequences.

[0243] In some embodiments, the two or more assembly polynucleotide sequences are provided simultaneously (e.g., in the same solution). In some embodiments, the two or more assembly polynucleotide sequences are themselves provided sequentially.

[0244] In some embodiments, a tandem guide complex binds to two unique polynucleotide sequences upon contacting. In embodiments where there are a plurality of tandem guide complexes and polynucleotides, each tandem guide agent of the plurality binds to two polynucleotide sequences. The specificity of the binding is mediated by the gRNA units of the tandem guide agent (e.g., tgRNA), each gRNA having homology to a unique polynucleotide sequence (also referred to as the targeting sequences and targeted sequences).

[0245] In some embodiments, provided methods assemble of plurality of polynucleotides, where a plurality of polynucleotides comprises at least 3 polynucleotides, at least 5 polynucleotides, at least 10 polynucleotides, at least 25 polynucleotides, at least 50 polynucleotides, at least 75 polynucleotides, at least 100 polynucleotides, at least 200 polynucleotides, at least 300 polynucleotides, at least 400 polynucleotides, at least 500 polynucleotides, at least 600 polynucleotides, at least 700 polynucleotides, at least 800 polynucleotides, at least 900 polynucleotides, at least 1000 polynucleotides, at least 2,000 polynucleotides, at least 3,000 polynucleotides, at least 4,000 polynucleotides, at least 5,000 polynucleotides, at least 6,000 polynucleotides, at least 7,000 polynucleotides, at least 8,000 polynucleotides, at least 9,000 polynucleotides, at least 10,000 polynucleotides, at least 20,000 polynucleotides, at least 30,000 polynucleotides, at least 40,000 polynucleotides, at least 50,000 polynucleotides, at least 60,000 polynucleotides, at least 70,000 polynucleotides, at least 80,000 polynucleotides, at least 90,000 polynucleotides, at least 100,000 polynucleotides, at least 200,000 polynucleotides, at least 300,000 polynucleotides, at least 400,000 polynucleotides, at least 500,000 polynucleotides, at least 600,000 polynucleotides, at least 700,000 polynucleotides, at least 800,000 polynucleotides, at least 900,000 polynucleotides, at least 1,000,000 polynucleotides, or more.

[0246] In some embodiments, provided methods produce a plurality of assembled polynucleotides that comprises at least 10 assembled polynucleotides, at least 25 assembled polynucleotides, at least 50 assembled polynucleotides, at least 75 assembled polynucleotides, at least 100 assembled polynucleotides, at least 200 assembled polynucleotides, at least 300 assembled polynucleotides, at least 400 assembled polynucleotides, at least 500 assembled polynucleotides, at least 600 assembled polynucleotides, at least 700 assembled polynucleotides, at least 800 assembled polynucleotides, at least 900 assembled polynucleotides, at least 1000 assembled polynucleotides, at least 2,000 assembled polynucleotides, at least 3,000 assembled polynucleotides, at least 4,000 assembled polynucleotides, at least 5,000 assembled polynucleotides, at least 6,000 assembled polynucleotides, at least 7,000 assembled polynucleotides, at least 8,000 assembled polynucleotides, at least 9,000 assembled polynucleotides, at least 10,000 assembled polynucleotides, at least 20,000 assembled polynucleotides, at least 30,000 assembled polynucleotides, at least 40,000 assembled polynucleotides, at least 50,000 assembled polynucleotides, at least 60,000 assembled polynucleotides, at least 70,000 assembled polynucleotides, at least 80,000 assembled polynucleotides, at least 90,000 assembled polynucleotides, at least 100,000 assembled polynucleotides, at least 200,000 assembled polynucleotides, at least 300,000 assembled polynucleotides, at least 400,000 assembled polynucleotides, at least 500,000 assembled polynucleotides, at least 600,000 assembled polynucleotides, at least 700,000 assembled polynucleotides, at least 800,000 assembled polynucleotides, at least 900,000 assembled polynucleotides, at least 1,000,000 assembled polynucleotides, or more.

[0247] In some embodiments, a tandem guide complex promotes assembly by bringing the polynucleotide sequences to be assembled into close proximity of one another. In some embodiments, the contacting with a tandem guide complex brings the polynucleotide sequences to be assembled within a proximity of about 200 nm or less of one another. In some embodiments, the proximity of the polynucleotide sequences to be assembled are measured and/or characterized using a proximity assay. Proximity assays are known in the art and include commercially available assays, such as, e.g., an AlphaLISA proximity assay (PerkinElmer, Waltham, MA).

[0248] In some embodiments, a tandem guide complex or plurality thereof are provided or obtained as an assembled tandem guide complex(es). In some embodiments, contacting comprises substantially simultaneously contacting a tandem guide complex with polynucleotide sequences. In some embodiments, contacting comprises sequentially contacting a tandem guide complex with a first polynucleotide sequence and then a second polynucleotide sequence. [02491 In some embodiments, components of a tandem guide complex are provided separately and assembled with the contacting (i.e., at substantially the same time as the contacting). For example, in some embodiments, a tandem guide agent, a first variant Cas protein, and/or a second variant Cas protein are provided separately and assemble with the contacting to form a tandem guide complex. In some embodiments, one or more of a tandem guide agent, a first variant Cas protein, a second variant Cas protein a first polynucleotide sequence, and a second polynucleotide sequence are provided separately and the contacting assembles a complex comprising a tandem guide complex that is associated with the first and second polynucleotide sequences.

[0250] In some embodiments, one or more of the polynucleotide sequences for assembly (which include as referred to herein, a first polynucleotide sequence, a second polynucleotide sequence, an additional polynucleotide sequence, an assembly polynucleotide sequence, or pluralities of any thereof) comprise sticky ends. In some embodiments, one or more of the polynucleotide sequences to be assembled are restriction digested. In some embodiments, all of the polynucleotide sequences to be assembled are restriction digested prior to the contacting step. In some embodiments one or more of the polynucleotide sequences to be assembled are restriction digested prior to the contacting step (e.g., contacting with tandem guide complexes and/or polynucleotide scaffold).

[0251] In some embodiments, assembling comprises ligating the polynucleotide sequences to form an assembled polynucleotide. In some embodiments, a tandem guide complex promotes ligation efficiency by bringing the polynucleotide sequences to be assembled into close proximity of one another.

[0252] In some embodiments, assembling comprises annealing the two or more assembly polynucleotide sequences to each other followed by amplification to form the assembled polynucleotide. In some embodiments, amplification comprises polymerase chain reaction (PCR), rolling circle amplification (RCA), isothermal amplification, DNA polymerase-mediated extension, or a combination thereof. [02531 In some embodiments, provided methods further include detecting a sequence corresponding to an assembled polynucleotide(s). In some embodiments, detecting includes amplification of an assembled polynucleotide(s), such as by, e.g., PCR, RCA, isothermal amplification, or a combination thereof.

[0254] In some embodiments, the polynucleotide sequences for assembly and/or the assembled polynucleotides comprise or consist of DNA and/or RNA. In some embodiments, the polynucleotide sequences for assembly and/or the assembled polynucleotides comprise or consist of DNA. In some embodiments, the polynucleotide sequences for assembly and/or the assembled polynucleotides comprise or consist of RNA. In some embodiments, the polynucleotide sequences for assembly and/or the assembled polynucleotides comprise or consist of a combination of DNA and RNA.

[02551 In some embodiments, provided methods are multiplex methods. In some embodiments, the method further comprises: contacting the plurality of assembled polynucleotides with an additional plurality of tandem guide complexes and an additional plurality of polynucleotides, wherein each tandem guide complex of the additional plurality comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, wherein the first variant Cas protein of the additional tandem guide complex binds an assembled polynucleotide and the second variant Cas protein of the additional tandem guide complex binds a polynucleotide of the additional plurality, and assembling the additional polynucleotide and the assembled polynucleotide, thereby further extending the plurality of assembled polynucleotides. In some embodiments, the contacting and assembling steps are repeated, thereby iteratively extending the plurality of assembled polynucleotides.

[0256] In some embodiments, the contacting and assembling steps are repeated, thereby iteratively extending the assembled polynucleotide. In some embodiments, provided multiplex assembly methods at least 2 times, at least 3 times, at least 4 times, at least 5 times, at least 6 times, at least 7 times, at least 8 times, at least 9 times, at least 10 times, or more.

[0257] In some embodiments, the polynucleotide sequences for assembly are not limited to a particular length. In some embodiments, the polynucleotide sequences for assembly have a length within a range bounded by a lower limit and an upper limit, the upper limit being larger than the lower limit. In some embodiments, the lower limit is about 5 nucleotides, 10 nucleotides, 15 nucleotides, 20 nucleotides, 25 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 125 nucleotides, 150 nucleotides, 175 nucleotides, 200 nucleotides, 225 nucleotides, 250 nucleotides, 275 nucleotides, 300 nucleotides, 325 nucleotides, 350 nucleotides, 375 nucleotides, 400 nucleotides, 425 nucleotides, 450 nucleotides, 475 nucleotides, or 500 nucleotides. In some embodiments, the upper limit is about 20 nucleotides, 25 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 60 nucleotides, 70 nucleotides, 80 nucleotides, 90 nucleotides, 100 nucleotides, 125 nucleotides, 150 nucleotides, 175 nucleotides, 200 nucleotides, 225 nucleotides,

250 nucleotides, 275 nucleotides, 300 nucleotides, 325 nucleotides, 350 nucleotides, 375 nucleotides, 400 nucleotides, 425 nucleotides, 450 nucleotides, 475 nucleotides, 500 nucleotides,

550 nucleotides, 600 nucleotides, 650 nucleotides, 700 nucleotides, 750 nucleotides, 800 nucleotides, 850 nucleotides, 900 nucleotides, 950 nucleotides, 1000 nucleotides, 1100 nucleotides, 1200 nucleotides, 1300 nucleotides, 1400 nucleotides, 1500 nucleotides, 1600 nucleotides, 1700 nucleotides, 1800 nucleotides, 1900 nucleotides, 2000 nucleotides, 2500 nucleotides, 3000 nucleotides, 3500 nucleotides, 4000 nucleotides, 4500 nucleotides, or 5000 nucleotides.

[0258] In some embodiments, the polynucleotide sequences for assembly have a length of 5 to 5000 nucleotides. In some embodiments, the polynucleotide sequences for assembly have a length of 10 to 1000 nucleotides. In some embodiments, the polynucleotide sequences for assembly have a length of 25 to 500 nucleotides.

[0259] In general, the target sequences for the tgRNA(s) can be located at any position within each polynucleotide sequence for assembly. In some embodiments it may be advantageous to position the target sequence at least 5, 10, 15, 20 or 25 nucleotides away from the 5’ or 3’ terminus of the polynucleotide sequence, e.g., to avoid interfering with ligation enzymes. Array Fabrication

[0260] Traditional protein microarray fabrication requires synthesizing and isolating individual DNA sequences, protein expression and purification, and individual spotting at discrete positions on a microarray. The present disclosure provides compositions and methods that enable self-assembling protein microarray fabrication from mixed polynucleotide (e.g., DNA) libraries using libraries of tandem guide agents (tgRNAs).

[0261] In some embodiments, tandem guide agents (e.g., tgRNAs) and tandem guide complexes are used for assembly (e.g., self-assembly) of spatially arrayed protein-encoding polynucleotides fragments for protein microarray fabrication. For example, protein microarrays can be generated using a solid surface harboring target polynucleotides (e.g., DNA molecules). Appropriate DNA arrays are understood by one of skill in the art and can include commercially available DNA arrays or custom arrays.

[0262] In some embodiments, provided are single-batch mixed libraries of tandem guide agents (e.g., tgRNAs). In some embodiments, protein-encoding DNA fragments can incubated with the microarray surface, enabling localization of protein-encoding DNA molecules at positions of interest pre-programmed by the tandem guide agent sequences (e.g., targeting sequences within each unit or gRNA of a tgRNA). Subsequent in vitro translation enables proteins to be produced in situ.

[0263] In some embodiments, the present disclosure provides in vitro methods that comprise: obtaining or providing a surface associated with a first polynucleotide sequence, and contacting the first polynucleotide sequence with a tandem guide complex and a second polynucleotide sequence, wherein the second polynucleotide sequence encodes an agent of interest (e.g., a polypeptide or protein). In some embodiments, the second polynucleotide sequence is present in solution.

[0264] In some embodiments, a first polynucleotide is associated with a defined position on the surface. In some embodiments, the contacting brings the second polynucleotide sequence into proximity with the first polynucleotide sequence via the tandem guide agent and/or tandem guide complex. In this way, each the second polynucleotide is associated with a defined position on the surface, which is in close proximity to the first polynucleotide.

[0265] In some embodiments, the present disclosure provides in vitro methods that comprise: obtaining or providing a surface associated with a first plurality polynucleotide sequences, and contacting the surface with a plurality of tandem guide complexes and a second plurality of polynucleotide sequences, wherein each polynucleotide sequence of the second plurality encodes an agent of interest (e.g., a polypeptide or protein). In some embodiments, the second polynucleotide sequence is present in solution. In some embodiments, each polynucleotide of the first plurality of polynucleotides is associated with a defined position on the surface.

[0266] In some embodiments, each polynucleotide of the first plurality of polynucleotides is associated with a defined position on the surface. In some embodiments, the contacting brings the second plurality of polynucleotide sequences into proximity with the first plurality of polynucleotide sequences via the tandem guide agents and/or tandem guide complexes. In this way, each polynucleotide of the second plurality of polynucleotides is associated with a defined position on the surface.

[0267] In some embodiments, the number of polynucleotide sequences to be arrayed along a surface are not limited to a particular number. In some embodiments, the number of polynucleotide sequences on the array is within a range bounded by a lower limit and an upper limit, the upper limit being larger than the lower limit. In some embodiments, a plurality of sequence comprises at least 3 polynucleotides, at least 5 polynucleotides, at least 10 polynucleotides, at least 25 polynucleotides, at least 50 polynucleotides, at least 75 polynucleotides, at least 100 polynucleotides, at least 200 polynucleotides, at least 300 polynucleotides, at least 400 polynucleotides, at least 500 polynucleotides, at least 600 polynucleotides, at least 700 polynucleotides, at least 800 polynucleotides, at least 900 polynucleotides, at least 1000 polynucleotides, at least 2,000 polynucleotides, at least 3,000 polynucleotides, at least 4,000 polynucleotides, at least 5,000 polynucleotides, at least 6,000 polynucleotides, at least 7,000 polynucleotides, at least 8,000 polynucleotides, at least 9,000 polynucleotides, at least 10,000 polynucleotides, at least 20,000 polynucleotides, at least 30,000 polynucleotides, at least 40,000 polynucleotides, at least 50,000 polynucleotides, at least 60,000 polynucleotides, at least 70,000 polynucleotides, at least 80,000 polynucleotides, at least 90,000 polynucleotides, at least 100,000 polynucleotides, at least 200,000 polynucleotides, at least

300,000 polynucleotides, at least 400,000 polynucleotides, at least 500,000 polynucleotides, at least 600,000 polynucleotides, at least 700,000 polynucleotides, at least 800,000 polynucleotides, at least 900,000 polynucleotides, at least 1 ,000,000 polynucleotides, or more.

[0268] In some embodiments, the present disclosure provides in vitro methods for assembling an array of protein encoding sequences. In some embodiments, provided methods are multiplex methods. In some embodiments, provided methods use multiplex CASsembly technology described above to iteratively build a protein-encoding polynucleotide array with protein-encoding sequences at defined positions on a surface.

Self- Assembling Materials

[0269] In some embodiments, the present disclosure provides methods for selfassembling materials using tandem guide agents and tandem guide complexes as described above.

[0270] In some embodiments, the present disclosure provides in vitro methods comprising: obtaining or providing a first surface associated with a first polynucleotide and a second surface associated with a second polynucleotide, contacting the first surface and the second surface with a tandem guide complex, wherein the tandem guide complex comprises (i) a tandem guide agent, (ii) a first valiant Cas protein, and (iii) a second valiant Cas protein, and wherein the first variant Cas protein binds the first polynucleotide and the second variant Cas protein binds the second polynucleotide, thereby assembling the first and second surfaces.

[0271] In some embodiments, the present disclosure provides in vitro methods comprising: obtaining or providing a plurality of surfaces, wherein each surface is associated with a unique polynucleotide, and contacting the plurality of surfaces with a plurality of tandem guide complexes, wherein each tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, and wherein each tandem guide complex binds to two unique polynucleotides, each associated with a different surface among the plurality of surfaces, thereby bringing together two different surfaces into close proximity.

[0272] In some embodiments, the two different polynucleotides are each associated with a surface, and wherein the contacting brings the surfaces into close proximity to one another.

[0273] In some embodiments, the contacting brings the surfaces within a proximity of about 200 nm or less of one another. In some embodiments, the proximity of the surfaces is measured and/or characterized. In some embodiments, the proximity of the surfaces is measured and/or characterized using a proximity assay, such as, e.g., an AlphaLISA proximity assay (PerkinElmer, Waltham, MA). In some embodiments, the proximity of the surfaces is measured and/or characterized using microscopy.

V. Compositions

[0274] Among other things, the present disclosure provides compositions comprising one or more components of technologies described herein. In some embodiments, a composition comprises a tandem guide agent (e.g., tgRNA) or nucleic acid encoding the same.

[0275] In some embodiments, the present disclosure provides compositions comprising one or more variant Cas proteins (e.g., inactive variant, e.g., dCas, e.g., dCas9) and one or more tandem guide agents (e.g., tgRNA). For example, the present disclosure provides compositions comprising dCas polypeptides (e.g., dCas9) (and/or a nucleic acid encoding dCas polypeptides); and a tandem-guide agent (and/or a nucleic acid encoding the tandem guide agent).

[0276] The present disclosure provides compositions comprising a tandem guide complex (e.g., dCas-tgRNA complex) comprising: dCas polypeptides and a tandem-guide agent (e.g., a tgRNA). In some embodiments, the present disclosure provides a composition comprising a dCas-tgRNA complex (e.g., dCas9-tgRNA complex).

[0277] In some embodiments, the present disclosure provides compositions for selfassembling materials using tandem guide complexes. In some embodiments, the present disclosure provides self-assembling biomolecules and/or cells that have polynucleotides associated on their surfaces. In some embodiments, self-assembly is mediated through binding of tandem guide complexes of the present disclosure

[0278] In some embodiments, compositions, agents or systems of the present disclosure are prepared by any methods known to one of skill in the art. In some embodiments, compositions are prepared using any standard synthesis and/or purification system that will be known to one of skill in the ail. For example, in some embodiments as described herein, one or more methods may include techniques such as de novo gene synthesis, DNA fragment assembly, PCR, mutagenesis, Gibson assembly, molecular cloning, standard single-stranded DNA synthesis, PCR, molecular cloning, digestion by restriction enzymes, small RNA molecule synthesis, cloning into plasmids with U6 promoter for RNA transcription, etc.

VI. Nucleic Acid Constructs

[0279] Among other things, the present disclosure provides nucleic acid constructs that encode one or more components of a tandem guide complex. In some embodiments, nucleic acid constructs provided by and/or utilized in accordance with the present disclosure encode a tandem guide agent and/or a Cas protein, or a component thereof. In some embodiments, provided nucleic acid constructs are DNA constructs. In some embodiments, nucleic acid constructs (e.g., DNA constructs) provided by and/or utilized in accordance with the present disclosure are comprised in a vector.

[0280] Non-limiting examples of a vector include plasmid vectors, cosmid vectors, phage vectors such as lambda phage, viral vectors such as retroviral, adenoviral or baculoviral vectors, or artificial chromosome vectors such as bacterial artificial chromosomes (BAC), yeast artificial chromosomes (YAC), or Pl artificial chromosomes (PAC). In some embodiments, a vector is an expression vector. In some embodiments, a vector is a cloning vector. In general, a vector is a nucleic acid construct that can receive or otherwise become linked to a nucleic acid element of interest (e.g., a construct that is or encodes a payload, or that imparts a particular functionality, etc.) [0281] Expression vectors, which may be plasmid or viral or other vectors, typically include an expressible sequence of interest (e.g., a coding sequence) that is functionally linked with one or more control elements (e.g., promoters, enhancers, transcription terminators, etc.). Typically, such control elements are selected for expression in a system of interest. In some embodiments, a system is ex vivo (e.g., an in vitro transcription system); in some embodiments, a system is in vivo (e.g., a bacterial, yeast, plant, insect, fish, vertebrate, mammalian cell or tissue, etc.).

[0282] Cloning vectors are generally used to modify, engineer, and/or duplicate (e.g., by replication in vivo, for example in a simple system such as bacteria or yeast, or in vitro, such as by amplification such as polymerase chain reaction or other amplification process). In some embodiments, a cloning vector may lack expression signals.

[0283] In many embodiments, a vector may include replication elements such as primer binding site(s) and/or origin(s) of replication. In many embodiments, a vector may include insertion or modification sites such as, e.g., restriction endonuclease recognition sites.

[0284] In some embodiments, a vector is a viral vector (e.g., an AAV vector). In some embodiments, a vector is a non-viral vector. In some embodiments, a vector is a plasmid.

[0285] Those skilled in the art are aware of a variety of technologies useful for the production of recombinant polynucleotides (e.g., DNA or RNA) and/or polypeptides as described herein. For example, restriction digestion, reverse transcription, amplification (e.g., by polymerase chain reaction), Gibson assembly, etc., are well established and useful tools and technologies. Alternatively or additionally, certain nucleic acids may be prepared or assembled by chemical and/or enzymatic synthesis.

[0286] In some embodiments, an expression vector comprising a polynucleotide of the present disclosure is used to produce a RNA and/or protein in a host cell. In some embodiments, a host cell may be in vitro (e.g., a cell line) - for example a cell or cell line suitable for producing polynucleotides of the present disclosure and proteins and/or polypeptides encoded by said polynucleotides. [02871 A variety of methods are known in the art to introduce an expression vector into host cells. In some embodiments, a vector may be introduced into host cells using transfection. In some embodiments, transfection is completed, for example, using calcium phosphate transfection, lipofection, or polyethylenimine-mediated transfection. In some embodiments, a vector may be introduced into a host cell using transduction.

[0288] In some embodiments, transformed host cells arc cultured following introduction of a vector into a host cell to allow for expression of said recombinant polynucleotides and/or proteins. A skilled artisan would recognize that appropriate culture conditions for expression in host cells are well known in the art.

[0289] The disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the disclosure in any way.

EXAMPLES

Example 1: Production of an exemplary tandem guide complex

[0290] This example describes production of an exemplary tandem guide complex comprising an exemplary tandem guide agent (e.g., tgRNA) and exemplary variant Cas proteins that are inactive (e.g., dCas9). Specifically, DNA sequences encoding an exemplary tgRNA sequence were synthesized, transcribed to RNA, and complexed with dCas9.

[0291] DNA sequences encoding exemplary tgRNAs were ordered from a vendor. Tandem guide agents can be produced from DNA using in vitro transcription, specifically exemplary tgRNAs were transcribed using a commercially available kit, e.g., a T7 RiboMAX kit (Promega). Complexing with Cas proteins to form an exemplary tandem guide complex (e.g., dCas9-tgRNA) was performed either co-transcriptionally or post-transcriptionally.

[0292] A DNA sequence that encodes an exemplary tgRNA is provided below. aagatgatagTA

atagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcaaaaaaaaaaaA TAAAAGAA ACCCTCCGCATgtttcagagctatgctggaaacagcatagcaagttgaaataaggctagtccgttatcaacttgaaaaagtg gcaccgagtcggtgc (SEQ ID NO: 1).

[0293] Legend for SEQ ID NO: 1 from 5’ to 3’ : T7 PROMOTER. SPACER (TARGETING SEQUENCE 1), sgRNA scaffold 1, polyA (12 As) linker, SPACER (TARGETING SEQUENCE 2). sgRNA scaffold 2

[0294] For co-transcriptional complexing of an exemplary tandem guide agent (tgRNA) and an exemplary variant Cas protein (e.g., inactive variant, e.g., dCas9), 1 p.g of dCas9 is mixed into a 20 L T7 RiboMAX reaction including 50-500 ng template, incubated at 37 °C for 2 hours. This reaction mixture is then used directly for applications.

[0295] For post-transcriptional complexing of an exemplary tgRNA and exemplary variant Cas protein (e.g., inactive variant, e.g., dCas9), a 20 pL T7 RiboMAX reaction including 500 ng-1 p.g of DNA template encoding exemplary tgRNAs was first incubated at 37 °C.

Resulting tgRNA is then cleaned up using a commercially available kit, e.g., a MEGAclear spin column (Ambion) and tgRNA concentration is estimated by Nanodrop. tgRNA was then incubated at 98 °C for 2 minutes and then cooled at room temperature for 10 minutes to promote proper folding. Subsequently, 1 pg dCas9 was then mixed with tgRNA at a 2:1 molar ratio in 10 pL binding buffer including 20 mM Tris-HCl pH 7.5, 100 mM KC1, 5 mM MgCh, 5% glycerol, 0.05 mg/mL heparin, 1 mM DTT, and 0.005% Tween 20 (Boyle et al., (2017) PNAS 114(21):5461-5466), and incubated at room temperature for 10 minutes.

Example 2: Exemplary tandem guide complexes can bind to and co-localize two target polynucleotides in vitro

[0296] The present example demonstrates that exemplary tandem guide complexes can bind to and co-localize two distinct target polynucleotide (DNA) sequences in vitro.

Specifically, this example describes binding analyses of exemplary tandem guide complexes using an AlphaLISA proximity assay (Perkin Elmer Inc.), as depicted in FIGs. 2A, 2B, and 3 and an assay based on capture of a fluorescent DNA strand followed by flow cytometry readout, as depicted in FIG. 4.

2.1 - Proximity assay shows specific binding of a tandem guide complex to two target DNA sequences

[0297] This example describes use of an exemplary proximity assay to demonstrate that an exemplary tandem guide complex (tgRNA-dCas9) complex brings together two target DNA sequences.

[0298] A schematic of an exemplary proximity assay (AlphaLISA proximity assay (Perkin Elmer, Waltham, MA)) is provided in FIG. 2A. As depicted in FIG. 2A, binding of a tgRNA-dCas9 complex to each target DNA sequence (via the two unique spacer sequences that are complementary to each target DNA sequence), brings together the two separate DNA sequences into a proximity of 200 nm or less of one another. Proximity dependent fluorescence occurs via the excitation of donor beads at 680 nm inducing a 615 nm emission of acceptor beads. Thus, the ability of dCas9-tgRNA to bind and bring together both target DNA sequences is observed by fluorescence-based readout (the results for this particular configuration is shown in columns 7 and 8 and the zoomed-in panel of FIG. 2B, as described below).

[0299] Streptavidin-coated AlphaLISA donor and acceptor beads were used in combination with biotinylated target DNA sequences, enabling flexibility in which reaction components were immobilized on donor and acceptor beads. A first target DNA sequence was immobilized on donor beads and a second target DNA sequence was immobilized on acceptor beads. After allowing target DNA sequences to be immobilized on donor and acceptor beads, beads were blocked with biotinylated BSA to ensure other reaction components could not bind directly to the beads. The beads were then mixed together and fluorescence visualized using an Envision plate reader.

[0300] A consensus for exemplary target DNA sequences along with exemplary spacer sequences (including for a non-target control DNA sequence) are provided below.

[0301] Target DNA sequences from 5’ to 3’ (NGG PAM sequence is underlined): [0302] 5’-biotin- ggcgtatcacgaggcagaatttcagataaaaaaaatccttagctttcgctaaggatgatttctggaattctaaagatctttgac agctagctcagtcctaggtataatactagt[20bp spacer] tggctttctttcttatct (SEQ ID NO: 2)

[0303] Spacer Sequence 1 (shown as blue in FIG. 2A and FIG. 2B): CCGTACCTAGATACACTCAA (SEQ ID NO: 3)

[0304] Spacer Sequence 2 (shown as red in FIG. 2A and FIG. 2B): ataaaagaaaccctccgcat (SEQ ID NO: 4)

[0305] Spacer Sequence 3 (shown as black in FIG. 3): tgttagttgccccatatctt (SEQ ID NO: 5)

[0306] First, it was assessed whether a dCas9-tgRNA could bind to a single target DNA sequence complementary to a spacer sequence within the tgRNA. Target DNA or non-target DNA was bound to a donor bead and dCas9-tgRNA bound to an acceptor bead; results are depicted in the first six columns of FIG. 2B. Fluorescence emission was higher for samples with a target DNA sequence immobilized to a donor bead (columns 1 to 4) than samples with nontarget control DNA sequence immobilized to a donor bead (columns 5 and 6), indicating that dCas9-tgRNA bound to each target DNA sequence at a higher level than the non-target control DNA sequence.

[0307] Next, the ability of a dCas9-tgRNA to simultaneously bind to two target sequences was assessed. A first target DNA sequence was associated with an acceptor bead and second target DNA sequence or non-target control DNA sequence was associated with a donor bead; results are provided in columns 7 and 8 and the zoomed in right panel of FIG. 2B. Fluorescence emission was higher for a sample with donor and acceptor beads with target DNA sequences at a higher level than those where beads harbored a non-target control DNA sequence. This supports that dCas9-tgRNA bound to both target DNA sequences and brought the beads into close proximity.

[0308] Together, these results indicate that dCas9-tgRNA is able to bind both of the target DNA sequences complementary to the two spacer regions within the tgRNA, either individually or simultaneously. 2.2 - Proximity analysis of varying concentration of a tandem guide complex

[0309] This example describes use of an exemplary proximity assay (AlphaLISA proximity assay (Perkin Elmer, Waltham, MA)) assay to assess the impact of varying tgRNA concentrations in complex with dCas9. An AlphaLISA proximity assay was used with first and second target DNA sequences immobilized to donor and acceptor beads, respectively, as described above in Example 2.1 and depicted in FIG. 2A and column 7 of FIG. 2B, except that 0, 5 ng, 50 ng, 500 ng, or 5 pg of tgRNA were complexed with 1 pg of dCas9 (when present). Results are depicted in FIG. 3. As shown in the first four columns (from left to right), fluorescence emission increased with increasing concentration of tgRNA, until the 5 pg concentration of tgRNA, where the signal decreased. This decrease in signal is suggested to be a “hook effect”, which is a known phenomenon in AlphaLISA proximity assay experiments, in which assay signal decreases in the presence of too much analyte. tgRNA alone or dCas9 alone were unable to bind to both target DNA sequences.

[0310] Various negative controls were also performed: this assay was repeated using (i) tandem guide complexes with a tgRNA that includes a gRNA that binds to a non-target control DNA sequence (columns 5 to 8 of FIG. 3), (ii) tgRNA without dCas9 (columns 9 to 12 of FIG. 3), (iii) a tgRNA that includes a gRNA that binds to a non-target control DNA sequence without dCas9 (columns 13 to 16 of FIG. 3) and dCas9 without tgRNA (column 17 of FIG. 3). All of these conditions produced a similarly low signal.

2.3 - Flow cytometry analysis of tandem guide complex binding to target DNA

[0311] The present example describes an orthogonal assay for demonstrating that exemplary tandem guide complexes can bind to and co-localize two distinct target polynucleotide (DNA) sequences. Specifically, streptavidin-coated magnetic beads (M-270, ThermoFisher) and fluorescently labeled target DNA were used and fluorescence was detected by flow cytometry, as depicted in FIG. 4. [03121 A first target DNA sequence was initially immobilized on the beads, which were then further blocked with biotinylated BSA to occupy remaining streptavidin sites on the beads. Then, dCas9-tgRNA was incubated with the beads, followed by the fluorescently labeled second target DNA sequence. Fluorescent DNA immobilization was only observed when the used tgRNA contained spacers complementary to both the bead-immobilized DNA and the fluorescently labeled DNA, indicating that dCas9-tgRNA was able to bind both DNA sequences simultaneously.

[0313] Various negative controls were also performed: (i) a tandem guide complex with tgRNA with spacers complementary to the first target DNA sequence immobilized on the magnetic beads but not the fluorescent second target DNA sequence; (ii) a control dCas9 alone, and (iii) a control with no first target DNA sequence immobilized on the magnetic beads.

Example 3: Exemplary tandem guide complex facilitates assembly of target polynucleotides [0314] High-throughput multiplexed long DNA synthesis is essential for the production and subsequent characterization of complex protein libraries, including antibodies and other candidate therapeutic biologies. Long DNA sequences (greater than 200-300 bp) generally need to be assembled from multiple short oligonucleotide fragments (Hughes and Ellington, (2017) CSH Perspectives 9(l):a023812). However, the per-base cost of generating libraries of longer DNA sequences greatly exceeds that of the constituent oligonucleotides (Kosuri and Church, (2014) Nature Methods 11:499-507). Efforts are currently being made to perform robust multiplexed gene synthesis starting from complex, mixed oligonucleotide pools (Sidore et al., (2020) Nucleic Acids Research 48(16):e95), but these workflows often result in heterogeneous or undesired chimeric DNA products.

[0315] This example describes use of an exemplary tandem guide complex to facilitate DNA assembly of target DNA sequences. Specifically, this example demonstrates the programmable proximity ligation of two target DNA fragments that are bound by dCas9-tgRNA, as depicted in FIG. 5A and FIG. 5B. [03161 Specifically, in one example based on the approach depicted in FIG. 5A, dCas9- tgRNA was bound to two target DNA fragments in solution. After buffer exchange and removal of unbound DNA by 100MWCO spin column (Amicon), the mixture was subjected to ligation using T4 ligase (NEB). A tandem guide complex with off-target affinity was used as a negative control, an in-solution direct ligation without dCas9-tgRNA complex was used as a positive control. After ligation, PCR amplification was used to amplify assembled DNA fragments and samples run on an agarose gel. The expected size of the assembled (ligated) DNA product is approximately 420 bp. Results are depicted in FIG. 5C where L = ladder, 1 = dCas9 with on- target tgRNA, 2 = dCas9 with off-target tgRNA and 3 = in-solution direct ligation control.

[0317] A greater band intensity of an assembled DNA product was obtained in the presence of targeted dCas9-tgRNA complex compared to off-target dCas9-tgRNA complex, supporting that target DNA ligation was enhanced by the presence of dCas9-tgRNA complex.

[0318] The present disclosure encompasses a recognition that this DNA assembly can be scaled up and applied to the assembly of larger pieces of DNA in a multiplex fashion, for example using libraries of oligonucleotides and tgRNAs. An exemplary schematic of multiplexed DNA assembly using tandem guide complexes (e.g., dCas9-tgRNA) is provided in FIG. 6. The ligation control as described herein involved DNA ligation in the absence of CASsembly reagents.

Example 4; Exemplary pooled CASsembly of target polynucleotides using tandem guide complexes

[0319] This example describes the use of pooled exemplary tandem guide complexes (e.g., tgRNAs) to programmably assemble target polynucleotide (e.g., DNA) fragments, also referred to herein as a pooled CASsembly method. Specifically, a pool of 10 short (230 mer) exemplary DNA fragments were ligated in a tgRNA-dependent manner (FIG. 7A). In this example, four exemplary tgRNAs were synthesized, with spacers designed to bring together distinct pairs of DNA sequences (tgRNAs were designed to bring together DNA sequences harboring “barcodes” 1&2, 3&4, 5&6, and 7&8). Two negative control DNA sequences that did not correspond to any tgRNA spacers were also included. A listing of exemplary DNA sequences used is provided in Table 2 below.

[0320] Table 2 - Exemplary DNA sequences for pooled CASsembly

[0321] CASsembly was performed in solution as described above in Example 3, using DNA sequences with configurations as shown in FIG. 7B. The ligated DNA was PCR-amplified and the barcode pairings in the assembled product were identified by next- generation sequencing. Odd numbered DNA sequences were amplified with exemplary primers, primer 1A and primer 2; even numbered with primer IB and primer 2; assembled DNA sequences after CASsembly were amplified using primer 1A and primer IB.

[0322] Primer 1 A - ACTGCGCACATCTACGATTG (SEQ ID NO: 20)

[0323] Primer IB - CAATCATCGTTCGCTCGAC (SEQ ID NO: 21) [03241 Primer 2 - CTGTGTACTTTATCTGCTCG (SEQ ID NO: 22)

[0325] Results are depicted in FIG. 7C and FIG. 7D. Compared to a “ligation control” condition in which the digested DNA was directly ligated without CASsembly, negative control DNA barcodes not targeted by tgRNAs were 9-fold more abundant in the “ligation control” compared to CASsembly, indicating that tgRNA complementarity was necessary to bind DNA sequences to mediate CASsembly (FIG. 7C). After CASsembly, the percentage of reads with correct anticipated DNA pairs identified by NGS was 79%, compared to 26% for the “ligation control’ (FIG. 7D). For each DNA barcode tested, the percentage of anticipated correct pairings was higher by CASsembly compared to the “ligation control”, indicating that pooled tgRNAs are able to mediate multiplexed assembly of DNA sequences targeted by the two spacers within each tgRNA (FIG. 7D). Negative control DNA sequences were excluded from the analysis in FIG. 7D to avoid spurious inflation of correct pairings analysis by CASsembly compared to the “ligation control”. Thus, this application demonstrates successful pooled assembly of a plurality of target sequences to form a plurality of assembled polynucleotides. The “ligation control,” as described herein, involved bulk DNA ligation in the absence of CASsembly reagents.

Example 5: Use of tandem guide complexes to facilitate polynucleotide assembly along a scaffold

[0326] This example describes coordinated polynucleotide assembly (CASsembly) using a scaffold polynucleotide and exemplary tandem guide agents (tgRNAs) as described herein. Specifically, an additional scaffold polynucleotide (DNA) is used to coordinate positioning of constituent target DNA molecules for assembly, for example, as depicted in FIG. 8. Tandem guide agents (e.g., tgRNAs) suitable for such scaffolded CASsembly methods will have a first spacer that binds to a sequence on the DNA scaffold and a second spacer that binds to a sequence of a target DNA. A pool of such tandem guide agents (tgRNAs) each recognizing a different sequence along the DNA scaffold can be used to arrange target DNA molecules in a desired order. In this manner, a pool of tandem guide complexes facilitate simultaneous localization of a plurality of target polynucleotide sequences via self-assembly to known positions on a DNA scaffold. Target polynucleotide sequences assembled along the scaffold may then be ligated to form an assembled polynucleotide product. Additionally, target sequences may be restriction digested DNA sequences with sticky ends to further increase the specificity of ligation between each pair of DNA sequences. The present disclosure encompasses a recognition such scaffolded CASsembly methods may reduce the time and material preparation needed for assembling polynucleotide (e.g., DNA) sequences.

Example 6: Use of tandem guide complexes with nickase Cas proteins to facilitate polynucleotide assembly

[0327] This example describes use of tandem guide complexes with variant Cas protein(s) having nickase activity for polynucleotide assembly (CASsembly). Specifically, this example describes use of an exemplary nickase variant, H840A spCas9, for DNA assembly.

FIG. 9A depicts schematics of exemplary target sequences that have a PAM site (e.g., 5'-NGG- 3') and a cleavage site upstream of the PAM. FIG. 9B depicts a schematic of an exemplary tandem guide complex with a nickase Cas protein, e.g., H840A variant of spCas9 protein, (nickase-tgRNA). In FIG. 9C, two Cas9 nickase mutants (not shown for simplicity) are bound to a tgRNA; the two spacer sequences within the tgRNA enable targeting of two DNA sequences that are partially complementary to each other (an ~14 bp internal region).

[0328] Upon nickase-tgRNA binding to the two target DNA sequences, both DNA sequences are nicked 3 bp upstream of (5’ relative to) the PAM on the non-target strand (FIG. 9C). The resulting “flaps” are complementary and thus capable of annealing to each other. The annealed strands are then extended using a polymerase, synthesizing a new double stranded DNA sequence that includes the two target pieces of DNA. The present disclosure encompasses a recognition that libraries of nickase-tgRNAs can be designed to bring together multiple pieces of DNA for larger DNA sequence assembly. In some embodiments, this results in the removal of relevant PAM sequences upon assembly of the targeted DNA. A listing of exemplary DNA sequences used is provided in Table 3 below. [03291 Table 3 - Exemplary DNA sequences for nickase mediated CASsembly (with target sequences in target DNA sequences underlined and PAM in bold and spacers (or targeting sequences) in tgRNA underlined and linker in bold).

Example 7; Use of tandem guide complexes for production of protein arrays

[0330] This example describes the use of exemplary tandem guide complexes, dCas9- tgRNAs, to localize protein-encoding DNA sequences, to positions of interest on a solid surface. The present disclosure encompasses a recognition that tandem guide agents may reduce the time and material preparation needed for producing protein microarrays. FIG. 10 depicts a schematic describing the use of tandem guide complexes, e.g., dCas9-tgRNAs, to facilitate production of protein microarrays. A microarray comprising a solid surface with target DNA molecules immobilized, e.g., within micro wells of a multiwall plate is prepared or obtained from commercial sources. A protein-encoding barcoded plasmid DNA library will also be prepared or obtained from commercial sources. dCas9-tgRNA libraries, capable of binding to both specific immobilized DNA sequences within the microarray and the specific barcodes of the plasmids of interest can also be generated. dCas9-tgRNA library, protein-encoding barcoded plasmid DNA library and DNA microarray will be pooled and incubated. dCas9-tgRNAs of the library will facilitate localization of each plasmid via self-assembly to a known position without requiring laboring intensive spotting individual plasmids or proteins on the microarray surface. Then, introduction of in vitro transcription/translation mixture onto the array will enable in situ expression of each protein of interest. The arrays can then be used for subsequent multiplexed protein assays. In some embodiments, a protein array will use target DNA sequences located within microwells to limit protein product diffusion.

[0331] Thus, each microwell will express a protein of interest that is ready for direct analysis. Moreover, the present disclosure recognizes that such DNA microarrays may be reused by stripping the DNA microarray of dCas9-tgRNA and plasmid components.

Example 8: Use of tandem guide complexes to facilitate self-assembly of materials [0332] This example describes the use of exemplary tandem guide complexes, e.g., dCas9-tgRNAs, to generate compositions capable of self-assembly of cells and materials (e.g., micro- and nanomaterials, e.g., biomaterials). The present disclosure encompasses a recognition that tandem guide complexes as described herein can be used for assembly of various surfaces functionalized with target DNAs. For example, as depicted in FIG. 11, a first surface functionalized with a first target DNA and a second surface functionalized with a second target DNA can be brought together when a tandem guide complex binds to both the first and second target DNAs. Various cell types with target DNA sequences attached to their surfaces, or multiple solid surfaces decorated with target DNAs can be self-assembled. In some exemplary embodiments, a first and second surface can be a cell surface and the binding of a tandem guide complex to the first and second target DNAs can bring the cell surfaces together, FIG. 11, left panel.

Example 9: Use of tandem guide complexes to facilitate polynucleotide assembly

[0333] This example demonstrates that tandem guide complexes, e.g., dCas9-tgRNAs, can bind target DNA at any position within a sequence, except at an end needed for ligation, to assemble polynucleotides (CASsembly). The present disclosure encompasses a recognition that one or more tandem guide complex(es), as described herein, can bind one or more target DNA at one or more location(s), except at any end sequence used for ligation, to assemble polynucleotides. A library of 3,120 oligonucleotides (oligos) 230 mer in length were designed to encode 20 base pair (bp) target regions (for tgRNAs) that moved incrementally by 1 bp in each strand. Target sequences were positioned along both sense and antisense strands. 156 oligos each encoding the incremental 1 bp shift in target region were used per barcode on each DNA strand (312 total oligos per barcode), as depicted in FIG. 12A.

[0334] Twenty base pairs on the 5’ and 3’ ends were used for PCR amplification, and a Bsal restriction enzyme site was included on the 3’ end for fragment digestion/eventual CASsembly -based ligation. All bases in the DNA sequences were randomized except the target region, PAM, Bsal site, and PCR amplification sequences. Randomization did not produce additional PAMs (i.e., no polyG or polyC sequences). Ten different tgRNA binding sites and five tgRNAs, each targeting two distinct DNA barcodes, were used in assembly experiments.

[0335] DNA sequences were assembled via CASsembly with dCas9-tgRNA complexes binding target sequences at any target position except within approximately 10 bp of the Bsal site. FIG. 12B depicts a graphical overview of the above described work flow. Correct pairing of each of the 312 oligos associated with one target region with the 312 oligos associated with a second target region are depicted in FIG. 12C. The correct pairing control consists of Bsal- digested DNA pieces ligated directly in the absence of CASsembly. Paired-end Illumina sequencing was used to identify DNA sequences joined together by CASsembly. [03361 In some embodiments, dCas9-tgRNA bound DNA is “shielded” from DNA ligase when a dCas9-tgRNA is bound too close to the sticky end. This “shielding” effect was not observed in a ligation control consisting of Bsal-digested DNA pieces ligated directly in the absence of CASsembly.

EQUIVALENTS

[0337] It is to be understood that while the disclosure has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:

1. An in vitro method comprising: obtaining or providing a tandem guide complex, wherein the tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, and contacting the tandem guide complex with a first polynucleotide sequence and a second polynucleotide sequence, wherein the tandem guide complex is capable of binding to the first polynucleotide sequence and the second polynucleotide sequence.

2. A method comprising: contacting a first polynucleotide sequence and a second polynucleotide sequence with a tandem guide complex, wherein the tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, wherein the first variant Cas protein binds the first polynucleotide sequence and the second variant Cas protein binds the second polynucleotide sequence, and assembling the first polynucleotide sequence and the second polynucleotide sequence to form an assembled polynucleotide that comprises the first polynucleotide sequence and the second polynucleotide sequence.

3. The method of claim 1 or 2, wherein the contacting brings the first polynucleotide sequence and the second polynucleotide sequence within a proximity of about 200 nm or less of one another.

4. The method of any one of claims 1 to 3, wherein the first polynucleotide sequence and the second polynucleotide sequence arc each present in solution.

5. The method of any one of claims 1 to 3, wherein the first polynucleotide sequence and the second polynucleotide sequence are each associated with a surface.

6. A method comprising: obtaining or providing a surface associated with a first polynucleotide sequence, contacting the first polynucleotide sequence with a tandem guide complex and a second polynucleotide sequence, wherein the tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, and wherein the first variant Cas protein binds the first polynucleotide sequence and the second variant Cas protein binds the second polynucleotide sequence, and assembling the first polynucleotide sequence and the second polynucleotide sequence to form an assembled polynucleotide that comprises the first polynucleotide sequence and the second polynucleotide sequence.

7. The method of claim 6, wherein the second polynucleotide sequence and/or the tandem guide complex are present in solution.

8. The method of any one of claims 2 to 7, wherein the contacting brings the first and second polynucleotide sequences into close proximity to one another.

9. The method of claim 1 or 8, wherein the first polynucleotide sequence and the second polynucleotide sequence are brought within a proximity of about 200 nm or less.

10. The method of any one of claims 1 to 9, wherein contacting the first polynucleotide sequence and the second polynucleotide sequence with the tandem guide complex occurs simultaneously.

11. The method of any one of claims 1 to 9, wherein the contacting comprises contacting the first polynucleotide sequence and the tandem guide complex and subsequently contacting the second polynucleotide sequence, such that the tandem guide complex binds to both the first polynucleotide sequence and the second polynucleotide sequence.

12. The method of any one of claims 1 to 11, wherein the first polynucleotide sequence and/or the second polynucleotide sequence are restriction digested prior to the contacting step.

13. The method of any one of claims 2 to 12, wherein the assembling comprises ligating the first polynucleotide sequence and the second polynucleotide sequence to form the assembled polynucleotide.

14. The method of any one of claims 2 to 12, wherein the assembling comprises annealing the first polynucleotide sequence and the second polynucleotide sequence to each other followed by amplification to form the assembled polynucleotide.

15. The method of claim 14, wherein the amplification comprises polymerase chain reaction (PCR), rolling circle amplification (RCA), isothermal amplification, DNA polymerase-mediated extension, or a combination thereof.

16. The method any one of claims 2 to 15, further comprising detection of a sequence corresponding to the assembled polynucleotide.

17. The method of any one of claims 2 to 16, wherein the first polynucleotide sequence, the second polynucleotide sequence and/or the assembled polynucleotide comprise or consist of DNA.

18. The method of any one of claims 2 to 17, wherein the method further comprises: contacting the assembled polynucleotide with an additional tandem guide complex and an additional polynucleotide sequence, wherein the additional tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, wherein the first variant Cas protein of the additional tandem guide complex binds the assembled polynucleotide and the second variant Cas protein of second tandem guide complex binds the additional polynucleotide sequence, and assembling the additional polynucleotide sequence and the assembled polynucleotide, thereby further extending the assembled polynucleotide.

19. The method of claim 18, wherein the contacting and assembling steps are repeated, thereby iteratively extending the assembled polynucleotide.

20. A method comprising: obtaining or providing two or more tandem guide complexes, wherein each tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, contacting the two or more tandem guide complexes with (i) a polynucleotide scaffold comprising two or more scaffold sequences that are positioned adjacently in series along the polynucleotide scaffold, and (ii) two or more assembly polynucleotide sequences, wherein the first variant Cas protein of each tandem guide complex binds a scaffold sequence and the second variant Cas protein of each tandem guide complex binds an assembly polynucleotide sequence, wherein the contacting brings the two or more assembly polynucleotide sequences into close proximity to one another, and assembling the two or more assembly polynucleotide sequences to form an assembled polynucleotide.

21. The method of claim 20, wherein the two or more assembly polynucleotide sequences and the polynucleotide scaffold are present in solution.

22. The method of any one of claims 20 or 21, wherein contacting the two or more tandem guide complexes with the polynucleotide scaffold and the two or more assembly polynucleotide sequences occurs simultaneously.

23. The method of any one of claims 20 to 22, wherein one or more of the assembly polynucleotide sequences are restriction digested prior to the contacting step.

24. The method of any one of claims 20 to 23, wherein the assembling comprises ligating the two or more assembly polynucleotide sequences to form the assembled polynucleotide.

25. The method of any one of claims 20 to 24, wherein the assembling comprises annealing the two or more assembly polynucleotide sequences to each other followed by amplification to form the assembled polynucleotide.

26. The method of claim 25, wherein the amplification comprises polymerase chain reaction (PCR), rolling circle amplification (RCA), isothermal amplification, DNA polymerase-mediated extension, or a combination thereof.

27. The method any one of claims 20 to 26, further comprising detection of a sequence corresponding to the assembled polynucleotide.

28. An in vitro method comprising: obtaining or providing a plurality of tandem guide complexes, wherein each tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, and contacting the plurality of tandem guide complexes with one or more compositions that comprise a plurality of polynucleotides, wherein each tandem guide complex is capable of binding to two different polynucleotides among the plurality of polynucleotides.

29. The method of claim 28, wherein each polynucleotide of the plurality of polynucleotides comprises a unique barcode.

30. A method comprising: contacting a plurality of tandem guide complexes with one or more compositions that comprise a plurality of polynucleotides, wherein each tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, and wherein each tandem guide complex binds to two different polynucleotides among the plurality of polynucleotides, and assembling the two different polynucleotides bound by each tandem guide complex, thereby forming a plurality of assembled polynucleotides.

31. The method of any one of claims 28 to 30, wherein the two different polynucleotides are each associated with a surface, and wherein the contacting brings the surfaces into close proximity to one another.

32. The method of any one of claims 28 to 30, wherein the two different polynucleotides are each present in solution.

33. The method of any one of claims 28 to 32, wherein the plurality of polynucleotides are restriction digested prior to the contacting step.

34. A method comprising: obtaining or providing a surface associated with a first plurality of polynucleotides, contacting the first plurality of polynucleotides with a plurality of tandem guide complexes and a second plurality of polynucleotides, wherein each tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, and wherein the first variant Cas protein binds a polynucleotide of the first plurality of polynucleotides and the second variant Cas protein binds a polynucleotide of the second plurality of polynucleotides, and assembling the polynucleotides bound by each tandem guide complex, thereby forming a plurality of assembled polynucleotides.

35. The method of claim 34, wherein the second plurality of polynucleotides and/or the plurality of tandem guide complexes are present in solution.

36. The method of claim 34 or 35, wherein the plurality of assembled polynucleotides are each associated with the surface.

37. The method of any one of claims 34 to 36, wherein each polynucleotide of the first plurality of polynucleotides is associated with a defined position on the surface.

38. The method of claim 37, wherein each assembled polynucleotide of the plurality of assembled polynucleotides is associated with a defined position on the surface.

39. The method of any one of claims 34 to 38, wherein contacting the first plurality of polynucleotides with a plurality of tandem guide complexes and a second plurality of polynucleotides occurs simultaneously.

40. The method of any one of claims 34 to 38, wherein the contacting comprises contacting the first plurality of polynucleotides and the plurality of tandem guide complexes and subsequently contacting the second plurality of polynucleotides.

41 . The method of any one of claims 34 to 40, wherein the second plurality of polynucleotides arc restriction digested prior to the contacting step.

42. The method of any one of claims 30 to 41, wherein the assembling comprises ligating the polynucleotides bound by each tandem guide complex to form the plurality of assembled polynucleotides.

43. The method of any one of claims 30 to 42, wherein the assembling comprises annealing polynucleotides bound by each tandem guide complex to each other followed by amplification to form the plurality of assembled polynucleotides.

44. The method of claim 43, wherein the amplification comprises polymerase chain reaction (PCR), rolling circle amplification (RCA), isothermal amplification, DNA polymerase-mediated extension, or a combination thereof.

45. The method any one of claims 30 to 44, further comprising detection of sequences corresponding to the plurality of assembled polynucleotides.

46. The method of any one of claims 30 to 45, wherein the plurality of polynucleotides and/or the plurality of assembled polynucleotides comprise or consist of DNA.

47. The method of any one of claims 30 to 46, wherein the method further comprises: contacting the plurality of assembled polynucleotides with an additional plurality of tandem guide complexes and an additional plurality of polynucleotides, wherein each tandem guide complex of the additional plurality comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, wherein the first variant Cas protein of the additional tandem guide complex binds an assembled polynucleotide and the second variant Cas protein of the additional tandem guide complex binds a polynucleotide of the additional plurality, and assembling the additional polynucleotide and the assembled polynucleotide, thereby further extending the plurality of assembled polynucleotides.

48. The method of claim 47, wherein the contacting and assembling steps are repeated, thereby iteratively extending the plurality of assembled polynucleotides.

49. A method comprising: obtaining or providing a first surface associated with a first polynucleotide and a second surface associated with a second polynucleotide, contacting the first surface and the second surface with a tandem guide complex, wherein the tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, and wherein the first variant Cas protein binds the first polynucleotide and the second variant Cas protein binds the second polynucleotide, thereby assembling the first and second surfaces.

50. A method comprising: obtaining or providing a plurality of surfaces, wherein each surface is associated with a unique polynucleotide, and contacting the plurality of surfaces with a plurality of tandem guide complexes, wherein each tandem guide complex comprises (i) a tandem guide agent, (ii) a first variant Cas protein, and (iii) a second variant Cas protein, and wherein each tandem guide complex binds to two unique polynucleotides, each associated with a different surface among the plurality of surfaces, thereby bringing together two different surfaces into close proximity.

51. The method of any one of claims 1 to 50, wherein the tandem guide agent or each tandem guide agent of the plurality comprises: a first unit comprising a first spacer (or a first targeting sequence) and a first scaffold, and a second unit comprising a second spacer (or a second targeting sequence) and a second scaffold.

52. The method of claim 51, wherein the tandem guide agent further comprises a linker between the first unit and the second unit.

53. The method of claim 51 or 52, wherein:

(i) the first unit comprises in 5’ to 3’ order: the first spacer and the first scaffold and/or the second unit comprises in 5’ to 3’ order: the second spacer and the second scaffold;

(ii) the first unit comprises in 3’ to 5’ order: the first spacer and the first scaffold and/or the second unit comprises in 3’ to 5’ order: the second spacer and the second scaffold;

(iii) the first unit comprises in 5’ to 3’ order: the first spacer and the first scaffold and/or the second unit comprises in 3’ to 5’ order: the second spacer and the second scaffold; or

(iv) the first unit comprises in 3’ to 5’ order: the first spacer and the first scaffold and/or the second unit comprises in 5’ to 3’ order: the second spacer and the second scaffold.

54. The method of any one of claims 51 to 53, wherein the first unit comprises a sgRNA and/or the second unit comprises a sgRNA.

55. The method of any one of claims 51 to 54, wherein the first scaffold and second scaffold bind to the same variant Cas protein.

56. The method of any one of claims 51 to 54, wherein the first scaffold and second scaffold bind to different variant Cas proteins.

57. The method of any one of claims 1 to 56, wherein the first variant Cas protein and/or second variant Cas protein is a variant Class 2 Cas protein.

58. The method of any one of claims 1 to 57, wherein the first variant Cas protein and/or second variant Cas protein is a variant Type II, Type V or Type VI Cas protein.

59. The method of any one of claims 1 to 58, wherein the first variant Cas protein and/or second variant Cas protein is a variant Cas9 protein, a valiant Cas 12 protein, a variant Cas 13 protein, and/or a variant Cas 14 protein.

60. The method of any one of claims 1 to 59, wherein the first variant Cas protein and/or second variant Cas protein is inactive.

61. The method of any one of claims 1 to 59, wherein the first variant Cas protein and/or second variant Cas protein is a nickase.

62. The method of any one of claims 1 to 61, wherein the first variant Cas protein and/or second variant Cas protein is PAM-less.

63. The method of any one of claims 52 to 62, wherein the linker is a polynucleotide linker, a non-polynucleotide covalent linker, or a non-covalent linker.

64. The method of any one of claims 52 to 63, wherein the linker is a poly-adenosine linker.

65. The method of claim 64, wherein the poly-adenosine linker comprises 5 to 50 adenosine residues.

66. The method of claim 64, wherein the poly-adenosine linker comprises 10 to 15 adenosine residues.

67. A tandem guide agent for use in a method of any one of claims 1 to 50.

68. A tandem guide agent, comprising: a first unit comprising a first spacer (or a first targeting sequence) and a first scaffold, and a second unit comprising a second spacer (or a second targeting sequence) and a second scaffold.

69. The tandem guide agent of claim 67 or 68, wherein the tandem guide agent further comprises a linker between the first unit and the second unit.

70. The tandem guide agent of any one of claims 67 to 69, wherein:

71. The tandem guide agent of any one of claims 67 to 70, wherein the first unit comprises a sgRNA and/or the second unit comprises a sgRNA.

72. The tandem guide agent of any one of claims 67 to 71, wherein the first scaffold and second scaffold bind to the same variant Cas protein.

73. The tandem guide agent of any one of claims 67 to 71, wherein the first scaffold and second scaffold bind to different variant Cas proteins.

74. The tandem guide agent of any one of claims 67 to 73, wherein the first variant Cas protein and/or second variant Cas protein is a variant Class 2 Cas protein.

75. The tandem guide agent of any one of claims 67 to 74, wherein the first variant Cas protein and/or second variant Cas protein is a variant Type 11, Type V or Type VI Cas protein.

76. The tandem guide agent of any one of claims 67 to 75, wherein the first variant Cas protein and/or second variant Cas protein is a variant Cas9 protein, a variant Cas 12 protein, a variant Cas 13 protein, and/or a variant Cas 14 protein.

77. The tandem guide agent of any one of claims 67 to 76, wherein the first variant Cas protein and/or second variant Cas protein is inactive.

78. The tandem guide agent of any one of claims 67 to 76, wherein the first variant Cas protein and/or second variant Cas protein is a nickase.

79. The tandem guide agent of any one of claims 67 to 78, wherein the first variant Cas protein and/or second variant Cas protein is PAM-less.

80. The tandem guide agent of any one of claims 69 to 79, wherein the linker is a polynucleotide linker, a non-polynucleotide covalent linker, or a non-covalent linker.

81. The tandem guide agent of any one of claims 69 to 79, wherein the linker is a polyadenosine linker.

82. The tandem guide agent of claim 81, wherein the poly-adenosine linker comprises 5 to 50 adenosine residues.

83. The tandem guide agent of claim 81, wherein the poly-adenosine linker comprises 10 to 15 adenosine residues.

84. A tandem guide complex comprising: (i) a tandem guide agent of any one of claims 67 to 83, (ii) a first variant Cas protein, and (iii) a second variant Cas protein.

85. The tandem guide complex of claim 84, wherein the first variant Cas protein and/or second variant Cas protein is a variant Class 2 Cas protein.

86. The tandem guide complex of claim 84 or 85, wherein the first variant Cas protein and/or second variant Cas protein is a variant Type II, Type V or Type VI Cas protein.

87. The tandem guide complex of any one of claims 84 to 86, wherein the first variant Cas protein and/or second variant Cas protein is a variant Cas9 protein, a variant Casl2 protein, a variant Casl3 protein, and/or a variant Casl4 protein.

88. The tandem guide complex of any one of claims 84 to 87, wherein the first variant Cas protein and/or second variant Cas protein is inactive.

89. The tandem guide complex of any one of claims 84 to 87, wherein the first variant Cas protein and/or second variant Cas protein is a nickase.

90. The tandem guide complex of any one of claims 84 to 89, wherein the first variant Cas protein and/or second variant Cas protein is PAM-less.

91. A composition comprising a tandem guide agent of any one of claims 67 to 83 or a tandem guide complex of any one of claims 84 to 90.

92. A composition comprising one or more surfaces associated with a polynucleotide, and one or more tandem guide complexes of any one of claims 84 to 90.

93. A composition comprising: two or more surfaces each associated with a polynucleotide, and one or more tandem guide complexes of any one of claims 84 to 90, wherein the one or more tandem guide complexes bind to a polynucleotide on each of the two or more surfaces, thereby self-assembling.

94. The composition of claim 92 or 93, wherein the surfaces comprise a planar surface, a bead, a microbead, and/or a cell surface.