CRISPR Handbook Enabling Genome Editing and Transforming Life Science Research www.GenScript.com GenScript USA Inc. 860 Centennial Ave. Piscataway, NJ 08854 USA Phone: 1-732-885-9188 Toll-Free: 1-877-436-7274 Fax: 1-732-885-5878 Table of Contents CRISPR Reagents and Services from GenScript gRNA sequence databases Validated gRNA sequences for efficient, specific targeing of WTCas9 or SAM CRISPR Plasmids Validated all-in-one, dual, non-viral and viral vectors for Cas9 & gRNA constructs CRISPR gRNA Libraries Validated GeCKO and SAM libraries for genome-scale loss- or gain-of-funcion screens SAM Transcripion Acivators A S M Validated SAM guide RNA sequences and efficient leniviral vectors for robust transcripional acivaion of endogenous genes or lncRNA CRISPR KO/KI mammalian cell lines CRISPR genome ediing service generates KO or KI mammalian cell lines Microbial genome ediing Efficient bacterial genome ediing using λ Red – CRISPR/Cas technology This handbook describes CRISPR/Cas9 genome ediing and other research applicaions for CRISPR technology. The CRISPR Genome Ediing Revoluion Discovery of CRISPR in bacterial immune system Evoluion of Genome Ediing technology Advantages of CRISPR genome ediing Improving the specificity of CRISPR genome ediing Improving gRNA and Cas9 delivery efficacy Expanding the applicability of CRISPR genome ediing Regulaing Cas9 expression 2 4 7 8 10 11 12 Puing CRISPR into Pracice: Workflows and Case Studies Design guide RNA and generate expression constructs Deliver CRISPR reagents to target cells Check for intended KO/KI and off-target effects Case studies 13 14 16 17 Expanding the Research Applicaions for CRISPR Genome-wide screens using CRISPR libraries Adaping CRISPR for transcripional regulaion Epigeneic Modificaions Stem Cell Differeniaion Therapeuics 21 22 24 25 25 Future of CRISPR 28 References 29 www.genscript.com 1 Figure 1: Mechanism of CRISPR-mediated immunity in bacteria viral DNA Discovery of CRISPR in bacterial immune systems Microbes have adapted many strategies to evade infecion by viruses and phages, from blocking virus adsorpion to prevening DNA inserion. Over the past 10 years, a new bacterial immune system has been discovered, employing a novel technique to prevent infecion. This immune system allows bacteria to both prevent foreign DNA from being inserted into the genome, and also target the invasive DNA for destrucion (Horvath et al., 2010). This system was first brought to light in 1987. Nakata and colleagues were studying the iap enzyme when they discovered curious repeat and non-repeat sequences downstream of the iap gene (Ishino et al., 1987). Just 5 years later, these repeat arrays would become referred to as CRISPR, or Clustered Regular Interspaced Short Palindromic Repeats (Jansen et al., 2002); however, their funcion was sill a mystery. In 2005, Mojica and colleagues revealed that these sequences, or “spacers”, actually contained DNA from bacteriophages (Mojica et al., 2005). Shortly ater this discovery, Boloin et al also observed the presence of cas genes, which encode for a DNA endonuclease, in close proximity to CRISPR structures, strongly suggesing that foreign DNA degradaion may be a primary funcion of CRISPR/Cas (Boloin et al., 2005). The specificity of this system for foreign DNA was further elucidated a few years later with the discovery of conserved moifs 2 www.genscript.com Cas1/Cas2 DNA is cleaved and new spacer unit is inserted } repeat + spacer TracrRNA Cas complex crRNA-tracrRNACas complex + immunity The ability to manipulate DNA has been a significant breakthrough in the scienific community – making it possible to beter understand the relaionship between the genome and its funcions. From inhibiing gene funcion to altering its expression, genome ediing can provide tremendous insight into the basis of disease and idenificaion of new targets for medical intervenion (Hsu et al., 2014). For this to become a reality, researchers need the ability to make specific, targeted changes to the genome, a simple principle that has been challenging in pracice. Over the last 20 years, advances in genome ediing technologies have overcome many of these challenges, allowing researchers to more precisely manipulate genomes in cell lines and animal models to more accurately model disease pathologies. Of these advances, one of the most exciing has been CRISPR/Cas, a system adapted from the bacterial immune system that is efficient, rapid, and easy to use (Doudna et al., 2014). In this handbook, we will discuss how CRISPR technology has fueled a genome ediing revoluion, as well as how it has been adapted for other biological applicaions and how it is expected to transform medicine. within the genome. Just upstream of the “protospacers,” or target genomic sequences on the foreign DNA, are conserved moifs called protospacer adjacent moifs (PAM). These moifs are preferenial targets for the Cas endonucleases (Horvath et al., 2008, Deveau et al., 2008), and allow the system to discern between self- and non-self DNA (Mali et al., 2013). Together, by the end of the immunization The CRISPR Genome Ediing Revoluion RNA Pol III viral DNA cleavage viral DNA early 2000s, the significance of the CRISPR as a defense strategy in bacteria was coming to light. By 2010, three CRISPR systems had been idenified in bacteria: Type I, II and III. Type II CRISPR interference, because of its relaive simplicity, would eventually become the system adapted for genome ediing in mammalian cells (Sapranauskas et al., 2011) (Figure 1). CRISPR-based immunity is composed of two main phases: immunizaion and immunity. In the immunizaion phase, Cas proteins (Cas1/Cas2) form a complex that cleaves the foreign, viral DNA (Jiang et al., 2015). This foreign DNA is then incorporated into the bacterial CRISPR loci as repeat-spacer units. In the immunity phase, following re-infecion, the repeat spacer units are transcribed to form pre-CRISPR RNA (pre-crRNA). The Cas9 endonuclease and trans-acivaing crRNA (tracrRNA, which helps guide Cas9 to crRNA) then bind to the pre-crRNA. A mature crRNA-Cas9-tracrRNA complex is formed following cleavage by RNA polymerase. This crRNA-Cas9-tracrRNA complex is essenial to target and destroy the foreign DNA. www.genscript.com 3 Evoluion of Genome Ediing technology Prior to the dawn of “genome ediing” in the early 2000s, studying gene funcion was primarily limited to transgenesis. The concept of gene ediing began in the late 1980s: in 1989, homologous recombinaion (HR) was used to target specific which recognizes 34-bp loci called loxP (Sauer et al., 1998). Recombinaion at these sites leads to knock-out of desired genes, which has been paricularly useful for the development of transgenic mouse models. While easier to control than HR, the Cre-lox system was less efficient as the geneic distance increased between loxP sites (Zheng et al., 2000). Figure 2: Advancements in genome ediing Figure 3: DNA repair by targeted genome ediing 1989 1998 HR-mediated targeing Zinc-finger nucleases (ZFNs) 2009 First study describing genome ediing via HR in mouse ES cells (Capecchi et al). Discovery of zinc-finger proteins that can target specific DNA sequences (Beerli et al). DNA binding proteins discovered in Xanthomonas bacteria (Boch et al). Transcripion-like effector nucleases (TALENs) 1992 2000 2013 Cre-lox Bacterial CRISPR/Cas The Cre-lox ediing technology was successfully used for site-specific recombinaion in mice (Orban et al). CRISPR/Cas genome ediing The CRISPR defense system is first idenified in prokaryotes (Mojica et al). First demonstraion that the CRISPR/Cas system can be used for mammalian cell genome ediing (Mali et al, Cong et al). genes in mouse embryonic stem cells to generate knock-in (KI) and knock-out (KO) cells (Capecchi et al., 1989) (Figure 2). Since HR occurs rather infrequently in mammalian cells, the recombinaion frequency was low (1 in every 3×104 cells); however, this work provided new ideas for how genes can be targeted and altered in specific ways. As the need for relevant animal disease models rose, so did the need for sophisicated and more efficient genome ediing tools. The Cre-lox technology became one of the most effecive gene ediing tools in the early 1990s, allowing scienists to control gene expression both spaially and temporally (Utomo et al., 1999, Orban et al., 1992). Cre-lox uses a site-specific DNA endonuclease Cre, 4 www.genscript.com Double-Strand Break Donor DNA Non-homologous end joining (NHEJ) Homology directed repair (HDR) Insertion/deletion mutations (indels) Precise alteration/correction Knock-out Knock-in Since HR alone rarely results in gene integraion in mammalian cells, the introducion of double strand breaks (DSB) into the genome can increase recombinaion significantly (Choulika et al., 1995). DSB resoluion occurs by either HDR or error-prone nonhomologous end joining (NHEJ) (Figure 3). If there is no donor DNA present, resoluion will occur by NHEJ, resuling in inserion/deleions (indels) that will ulimately knock-out gene funcion. Alternaively, if donor DNA sequences are available, the DSBs will be repaired by HDR, resuling in gene knock-in (Bibikova et al., 2002). Combined, these strategies represented new and more effecive approaches for modifying the eukaryoic genome (Hsu et al., 2014). www.genscript.com 5 CRISPR aside, the most effecive genome ediing techniques employing DSB-mediated repair have been zinc-finger (ZF) domains (Beerli et al., 1998) and transcripion acivator-like effectors (TALEs) (Moscou and Bogdanove, 2009; Boch et al., 2009). Both of these systems use DNA binding proteins with nuclease acivity that bind to DNA and create site-specific DSBs. While effecive, both of these methods require extensive experise in protein engineering, which has been a botleneck for many research labs’ use of this technology (Perez-Pinera et al., 2012). Figure 4: CRISPR/Cas system for genome ediing in mammalian cells CMV Human codon opimized Cas9 SV40 Target The adaptaion of CRISPR for mammalian cells has revoluionized genome ediing – not only for its accuracy but also for its ease of use in any lab regardless of molecular biology experise. Unlike ZF and TALE nucleases, CRISPR/Cas does not require protein engineering for every gene being targeted. The CRISPR system only requires a few simple DNA constructs to encode the gRNA and Cas9, and, if knock-in is being performed, the donor template for HR. In addiion, muliple genes can be edited simultaneously. The table below summarizes the key differences and advantages between the most common DSB-mediated genome ediing technologies. TK pA Table 1: Key differences between TALENs, ZFNs, and CRISPR/Cas + U6 Advantages of CRISPR genome ediing gRNA scaffold TTTTTT TALEN (transcripion acivator-like effector nucleases) ZFN (zinc finger nucleases) CRISPR/Cas (gRNA-Cas9): DNA Target Protein: DNA Protein: DNA Construct Proteins containing DNA-binding domains that recognize specific DNA sequences down to the base pair Zinc finger DNA binding moifs in a ββα configuraion, the α-helix recognizes 3 bp segments in DNA Cas 9 PAM Target DNA Target Sequence gRNA The use of CRISPR/Cas as a gene ediing tool began in 2013, with the observaion that type II CRISPR systems from S. Thermophilus and S. Pyogenes (SpCas) could be engineered to edit mammalian genomes (Mali et al., 2013, Cong et al., 2013). To further adapt the system for mammalian cells, a two-vector system was opimized (Mali et al., 2013). The two major components include (1) a Cas9 endonuclease and (2) the crRNA-tracrRNA complex; when co-expressed, they form a complex that is recruited to the target DNA sequence. The crRNA and tracrRNA can be combined to form a chimeric guide RNA (gRNA) with the same funcion – to guide Cas9 to target gene sequences (Jinek et al., 2012). These components can then be delivered to mammalian cells via transfecion or leniviral transducion. 6 www.genscript.com Design feasibility References Easy: - all-in-one gRNA-Cas9 vector system - muligene ediing is feasible Difficult: -Need a customized protein for each gene sequence -Low delivery efficiency Moscou and Bogdanove, 2009 Boch et al., 2009 Gaj et al., 2013 20nt crRNA (CRISPR RNA) fused to a tracrRNA and Cas9 endonuclease that recognize specific sequences to the base pair Beerli et al., 1998 Perez-Pinera et al., 2012 Gaj et al., 2013 Mali et al., 2013 Cong et al., 2013 Jiang et al., 2015 www.genscript.com 7 Improving the specificity of CRISPR genome ediing Several systemaic efforts have been undertaken to empirically determine the rules governing gRNA efficiency and specificity. One study looked at all possible targetable sites iling across 6 mouse and 3 human genes -- 1,841 sgRNAs in total – and quanified their ability to create null alleles as assayed by anibody staining and flow cytometry. The results were used to construct a predicive model of sgRNA acivity to improve sgRNA design for gene ediing and geneic screens. (Doench et al. 2014) The gRNA design tool, which returns a score predicing the acivity of any sgRNA based on empirical rules determined by this study, is freely available at htp://www.broadinsitute.org/rnai/public/analysis-tools/sgrna-design. Another more recent study measured sgRNA acivity across ~1,400 genomic loci, across muliple human cell types, using two Cas9 orthologs with different PAMs (S. Pyogenes and S. Thermophilus), to uncover parameters that govern gRNA efficiency based not only on the nucleoide sequences but also on epigeneic status (Chari et al., 2015). These results power an interacive web tool that can idenify putaive CRISPR/Cas9 sites) and assign a predicted acivity, freely available at htp://crispr.med.harvard.edu/sgRNAScorer. Although it is rare for a 20 bp gRNA sequence to have 100% homology at muliple sites throughout the genome, sgRNA-Cas9 complexes are tolerant of several mismatches in their targets. Cas9 binds to many locaions throughout the genome that display several mismatches to the guide (Kusco et al., 2014), but the enzyme only creates DSBs at a small subset of those locaions. Sill, DSBs have been observed at sites containing five or more mismatched nucleoides relaive to the guide RNA sequence (Tsai et al., 2015). Therefore, there has been a major effort to develop modified CRISPR/Cas9 systems with improved specificity. One strategy for improving gRNA-Cas9 targeing specificity is to require a pair of guides that target very nearby regions. Feng Zhang’s laboratory at the Broad Insitute and Keith Joung’s laboratory at Harvard/MGH both developed systems that implement this strategy in slightly different ways. The Zhang lab observed that an aspartate-to-alanine (D10A) mutaion in the RuvC catalyic domain of Cas9 causes it to create single strand breaks (nicks) instead of double strand breaks. Targeing this nickase mutant (Cas9n) to two loci within close proximity, but occurring on opposite strands of the genomic DNA, causes Cas9n to effecively nick rather than cleave DNA to yield single-stranded breaks. Appropriately offset sgRNA pairs can guide Cas9n to simultaneously nick both strands of the target locus to mediate a DSB, thus effecively increasing the specificity of target recogniion. Although each gRNA might have off-target binding sites throughout the genome, the Cas9n would cause only single strand breaks (SSB) at those locaions; SSBs are preferenially repaired through HDR rather than NHEJ, 8 www.genscript.com which can potenially decrease the frequency of unwanted indel mutaions from off-target DSBs. Figure 5. Increasing specificity through paired guides: Nickase or RFN Double-Nicking Cas9n D10A mutant sgRNA 1 N-bp sgRNA offset Cas9n Target 2 5 3 3 5 Target 1 sgRNA 2 5 overhang RNA-guided Fokl nuclease (RFN) d Cas9 gRNA 1 Fok 1 5 3 3 5 Fok 1 gRNA 2 Another strategy to improve specificity has focused on the gRNA itself. Although 20 bp regions were iniially used, it was observed that mismatches were tolerated most oten in the 3’ end of the gRNA, and some wondered if these final nucleoides were necessary. Researchers in the Joung lab found that gRNAs with 17 or 18 nucleoides of complementarity funcioned as efficiently as (or, in some cases, more efficiently than) 20 bp sequences to introduce mutaions by means of NHEJ or HDR at on-target sites, and they showed reduced mutagenic effects at closely matched off-target sites (Fu et al., 2014). These truncated gRNAs (tru-gRNAs) can be used with WT SpCas9 or in combinaion with the RNA-Fok1 nuclease described above (Wyvekens et al., 2015). Off-target binding of Cas9 throughout the genome has been observed to be concentraion-dependent (Wu et al., 2014) This finding spurred invesigaions of whether the frequency of off-target cleavage events could be reduced by delivering a short-lived Cas9 protein rather than plasmid that would drive expression of Cas9 for a longer period of ime than was strictly necessary. A purified Cas9 protein can be complexed to its guide RNA in vitro to form a ribonucleoprotein (RNP), which will cleave chromosomal DNA almost immediately ater delivery and then be degraded rapidly in cells, reducing off-target effects. RNPs can be efficiently delivered to hard-to-transfect cells such as human fibroblasts and pluripotent stem cells. Another advantage is that RNP delivery may be less stressful for cells than plasmid transfecion (Kim et al., 2014). www.genscript.com 9 Improving gRNA & Cas9 delivery efficiency Some of the most widely-used model systems for biomedical research are primary mammalian cell cultures or hard-to-transfect cell lines in which transfecion efficiency via lipofecion or electroporaion can be quite low. Leniviral vectors are preferred for these cell types (Figure 6). Figure 6 : Opimized Leniviral Vectors for CRISPR genome ediing in mammalian cells Opion 1: An all-in-one vector, pLeniCRISPRv2, enables CRISPR ediing in any cell type of interest without generaing stable Cas9-expressing cell line first. AmpR O • Create transgenic animal lines that express Cas9, either consituively or in an inducible manner, and then to deliver only the guide RNAs and any necessary inducer at the ime of the experiment (Plat et al., 2014). • Develop a split-Cas9 system using split-inteins (Truong et al., 2015). • Use smaller Cas9 orthologues from other species, such as Staphylococcus aureus (SaCas9), which are small enough to be packaged along with a single guide RNA expression cassete into a single AAV vector (Ran et al., 2015) HIV-1 ψ HIV-1 RRE HIV-1 cPPT LTR ri CRISPR/Cas9 system components can be delivered in vivo using modified viral vectors or any number of non-viral drug delivery systems. Modified recombinant adeno-associated virus (rAAV) paricles are a preferred vehicle for in vivo gene delivery, but the size of the SpCas9 gene (> 4 kb) exceeds the typical cargo limit of AAV vectors. Soluions that have been developed to date include: Bleo R U6 promoter G(N)20 gRNA gRNA scaffold LTR Expanding the applicability of CRISPR genome ediing EFS promoter pLentiCRISPR v2 13kb E WPR One limitaion of the first CRISPR genome ediing protocol was the constraint on genomic sequences that could be targeted. The SpCas9 enzyme requires the presence of the PAM sequence "NGG" at the end of the ~20-mer. Guide RNA expression was typically driven by the U6 human pol III promoter due to its efficiency at iniiaing transcripion, which iniiates transcripion from a guanosine (G) nucleoide. Therefore, U6-driven gRNAs used with SpCas9 needed to be selected from genomic sequences that fit the patern GN19NGG – which might occur infrequently in a gene of interest. Pu ro R DYK s Ca P2A 9 Opion 2: A two-vector system; sequenial transducion with, and selecion for, pLeni-Cas9-Blast followed by pLeniGuide-Puro, shows 10-fold higher efficiency compared to pLeniCRISPRv2. AmpR O ri G(N)20 gRNA gRNA scaffold HIV-1 cPPT LTR U6 promoter HIV-1 ψ EF1a HIV-1 RRE HIV-1 RRE pro mo te r Pu Bleo R HIV-1 ψ roR EFS promoter pLentiGuide-Puro 8.3kb WPR WPRE LTR pLentiCas9-Blast 12.8kb LTR HIV-1 cPPT i Or B P2A LT R E sd R DYK s Ca 9 One strategy to expand the possibiliies for CRISPR-mediated genome ediing was to drive gRNA expression from a different promoter. The H1 promoter can iniiate transcripion from A or G; therefore, H1-driven gRNAs can also target sequences of the form AN19NGG, which occur 15% more frequently than GN19NGG within the human genome. This small change in the gRNA expression cassete more than doubles the number of targetable sites within the genomes of humans and other eukaryotes. Another strategy has been to search for ways to relax the restricion on the PAM sequence, as SpCas9’s requirement for NGG presents a ight constraint. One approach to this has been to use protein engineering techniques to create novel engineered Cas mutants that recognize alternaive PAM sequences (Kleinsiver et Am p R 10 www.genscript.com www.genscript.com 11 al., 2015). Through a painstaking process that used structural informaion, bacterial selecion-based directed evoluion, and combinatorial design, researchers developed several mutant Cas9 that could recognize alternaive PAMs. Engineered Cas9 nucleases can cleave at PAM sites consising of NGA and NGCG, which allows targeing 50% more sites than can be reached with the NGG PAM alone. Addiionally, there is data showing that these newly engineered Cas9s have lower off-target acivity compared to wt SpCas9. Puing CRISPR into Pracice: Workflows and Case Studies With CRISPR genome ediing, modified clonal cell lines can be derived within 2–3 weeks staring from the guide RNA design stage; transgenic animal strains can be created in a single generaion; and clinically relevant animal models of disease can be rapidly created through introducing somaic mutaions in vivo. To jump-start your CRISPR experiments, the workflow and references below may help. Regulaing Cas9 expression In order to make Cas9 acive only at specific imes or in specific issues, several research groups have engineered CRISPR/Cas9 systems that are inducible or condiional. For example, spaial and temporal control of genome ediing can be accomplished using a photoacivatable Cas9 (paCas9) that was created by spliing Cas9 into two fragments each fused to a photoinducible dimerizaion domain; upon blue light irradiaion, paCas9 dimerizes and becomes acive, creaing targeted genome edits via NHEJ or HDR only while the opical simulus is present (Nihongaki et al., 2015). Tissue-specific genome ediing can be accomplished by using issue-specific promoters to drive Cas9 expression. Many mouse strains have been developed that stably express Cre recombinase under the control of issue-specific specific promoters (cre-driver mice); these can easily be crossed with mice harboring a CRE-driven Cas9 cassete to enable issue-specific genome ediing upon delivery of guide RNA (Plat et al. 2014). Heritable issue-specific Cas9 expression has also been achieved in diverse species other than mice, including zebrafish (Ablain et al., 2015; Yin et al., 2015), sea squirt Ciona intesinalis (Stolfi et al., 2014), and drosophila (Xue et al. 2014). Tissue-specific promoters are also useful for constraining Cas9 acivity ater in vivo delivery via AAVs, which can infect many different cell types (Cheng et al. 2014). Design guide RNA and generate expression constructs To perform CRISPR/Cas9-mediated gene ediing, the first step is to select the nuclease you will use (e.g. WT SpCas9, Paired-nickase with Cas9D10A, etc) and then to design, or select from a pre-exising database, the guide RNA sequences appropriate for your nuclease. Gene sequence analysis: It is advisable to sequence the region of interest within the host genome of the cell line or animal model you are using, rather than assuming that it will perfectly match the NCBI ref seq for your species/strain. GenScript offers custom gRNA design services for any target in any species, as well as searchable online databases of validated gRNAs for human and mouse Designing gRNA for single DSB-induced gene KO: Designing gRNAs against early exons tends to disrupt expression, reducing the chance of having truncated forms of the protein expressed. Alternaively, targeing a funcional site can generate a loss-of-funcion mutant. For genes with muliple splice variants, care should be taken to ensure that a consituive exon is targeted if the goal is to knock out all splice variants. Designing guides for paired nickase: Guide RNA for use with Cas9n should be designed to target opposite strands of the genomic DNA with an offset of 0-20 bp from the 5’ ends of the gRNA (i.e. a 40-60bp offset between PAM sequences). Designing constructs for knock-in: As a general rule, WT Cas9 is more efficient at mediaing homologous recombinaion than Cas9 nickase; although using a paired nickase strategy can reduce the risk for off-target acivity, the efficiency of HDR mediated by Cas9 nickase is highly dependent on the cell type (Ran et al., 2013). To introduce a specific change within the genome, for example a point mutaion that will cause a specific amino acid subsituion in the protein product, it is 12 www.genscript.com www.genscript.com 13 necessary to supply a donor template that GenScript offers custom gRNA can be used for HDR ater Cas9 creates a DSB. constructs built from vectors HDR templates may be delivered in plasmids developed in Feng Zhang’s or as single-stranded oligos (ssODN). To assist laboratory, offered through a in detecing successful HDR and quanifying license with the Broad Insitute. knock-in efficiency, donor templates are oten designed to include several synonymous mutaions so that sequencing can easily disinguish between the donor and the wild-type sequences. To prevent the cleavage of donor templates or of the genomic DNA ater successful HDR, the donor template should be designed with mutaions in the PAM sequence. Making gRNA and Cas9 Constructs Once you have designed your gRNA, you need to synthesize them and clone them into your vector of choice. The plasmid vector you choose will depend upon your host and delivery method (Table 2). Deliver CRISPR reagents to target cells CRISPR/Cas9 technology for precise genome ediing has already proven successful in many cell lines and species, including C. elegans (Friedland et al., 2013; Waaijers et al., 2013), Xenopus tropicalis (Guo et al., 2014), plants (Jiang et al., 2013), and even monkeys (Niu et al., 2014). Although the basic components are the same regardless of the target organism, the delivery method varies widely, and choosing the most appropriate vector for your host is criical for success. In vivo genome ediing: As with prior methods for creaing transgenic animal strains, CRISPR/Cas9 system components can be delivered to germ line cells to create heritable mutaions; stable, homozygous mutaions at muliple loci can be achieved in a single generaion in mice (Wang et al., 2013). CRISPR genome ediing can also be used to generate precise mutaions in somaic issues of adult animals, and to modify muliple genes at once in the same cells (Cong et al., 2013, Mali et al., 2013). This is especially valuable for creaing clinically relevant in vivo cancer models, because human tumors oten contain a combinaion of gain-of-funcion mutaions in oncogenes and loss-of-funcion mutaions in tumor suppressor genes (Plat et al., 2015). In addiion, CRISPR can be used to generate chromosomal rearrangements seen in human cancers, such as the EML4-ALK inversion observed in human non-small cell lung cancer. Viral-mediated delivery of CRISPR/Cas9 system to somaic cells in the lung of adult mice yielded a new clinically faithful mouse model of Eml4-Alk human lung cancer and presents a new paradigm for accurately modeling human cancers in mice (Maddalo et al., 2014). Table 2: gRNA & Cas9 Delivery Methods used for different hosts Host 14 www.genscript.com Reference Mammalian cells Cong et al., 2013, Mali et al., 2013 Schumann et al., 2015 Shalem et al., 2014 Microbial organisms - Transformaion of plasmids into competent cells Jiang et al., 2015 Pyne et al., 2015 Plants - Agrobacterium mediated transformaion of sgRNA and Cas9 vector Gao et al., 2014, Zhou et al., 2014 Mouse: heritable mutaions - Direct injecion into embryos - Electroporaion into zygotes Wang et al., 2014 Qin et al., 2015 Mouse: mutaions to adult somaic issue Direct injecion of AAV into issue - of interest Cheng et al., 2014 Maddalo et al., 2014 Yeast - Electroporaion of plasmids and galactose inducion of Cas9 DiCarlo et al., 2013 In vitro genome ediing: For easy-to-transfect cell lines, plasmids encoding gRNA and Cas9 can be delivered with high efficiency via lipofecion. CRISPR plasmids typically contain selecion markers such as genes conferring anibioic resistance, or fluorescent proteins for easy visualizaion or FACS. For difficult-to-transfect cell lines or primary cells, leniviral vectors are preferred. gRNA may be delivered either via an all-in-one plasmid that also encodes the Cas9 nuclease, or a separate plasmid that can be delivered into cells already expressing Cas9. Alternaively, gRNA may be introduced via a PCR-generated U6-sgRNA expression cassetes expression. Cleavage efficiency is typically lower than when gRNA is expressed from a plasmid; however, PCR-generated cassetes may be used for rapid comparison of sgRNA efficiencies so that the most opimal sgRNA, in terms of both efficiency and specificity, can be idenified before subsequent cloning into pSpCas9 (Ran et al., 2013). Delivery Method - Lipofecion-based transfecion of DNA plasmids - Electroporaion of DNA plasmids or RNP - Leniviral transducion of DNA plasmids www.genscript.com 15 Figure 7: Knock-out targeing strategy for K-Ras K-ras locus Exon1 16 www.genscript.com Exon3 Exon4 Exon5 Exon6 Exon7 Exon8 gRNA and Cas9 complex K-ras exon 4 is targeted for double stranded DNA break (DSB) DSB initiates non-homologous end joining (NHEJ) K-ras knock-out How to ensure that off-target Cas9 acivity won’t confound your experiments: Indel on Exon 4 Primers for sequencing gRNA and Cas9 complex Figure 8: Sanger sequencing (A) and western blot (B) results for HCT116 KRAS -/B K- ra s -/ - A 6 • For each guide RNA you use, isolate muliple, independent clonal cell populaions or founder individuals. The likelihood off-target DSBs occur in the same place in independent clones is very low. • Use at least two independent gRNA sequences in parallel to derive disinct clones or founder individuals. Models created through genome ediing with disinct guideRNA that share an on-target locus but do not share off-target loci are an excellent way to create independent replicates. • Although few labs have the resources to do staisically powerful whole genome sequencing verificaion protocols such as gUIDEseq, it is relaively easy to select the few predicted off-target sequences for each gRNA you use and then sequence around those loci to ensure that off-target indels have not been introduced. If you use most or all of these ips in combinaion, you can have confidence that your experiments will reveal true genotype/phenotype relaionships. Exon2 HCT116 6 Whole genome sequencing is oten not pracical for low frequency events. In addiion, targeted sequencing only of computaionally predicted off-target sites introduces a strong observaional bias. Therefore, researchers in Keith Joung’s lab developed a technique called Genome-wide Unbiased Idenificaion of DSBs Enabled by sequencing (GUIDE-seq) to beter quanify off-target acivity of Cas9 throughout the genome (Tsai et al., 2015). GUIDE-seq introduces a tag any ime a DSB occurs, and then sequences around the tags to determine all off-target cleavage locaions. They found surprising results, including that the majority of cleavage sites idenified by GUIDE-seq were not of GUIDE-seq OT sites were not predicted by any algorithm, because they contain up to 6 mismatched nucleoides and in many cases include non-canonical PAMs. To knock-out the K-Ras locus, gRNA and Cas9 vectors were encapsulated into a virus. In this case, exon 4 was targeted by the gRNA-Cas9 complex to generate a DSB. In the absence of donor DNA, the DSB was repaired by NHEJ to create an indel. Sanger sequencing (Figure 8A) and a western blot (Figure 8B) were used to confirm successful knock-out of the KRAS gene. T1 1 To determine off-target effects, you may sequence around regions that are predicted to be likely sites for off-target cleavage based on sequence similarity to the on-target site, paricularly in the “seed” region. A more rigorous measure of off-target cleavage can be performed using whole-genome sequencing. Using GenScript’s GenCRISPRTM cell line services, any gene can be targeted in any mammalian cell. All clones are target sequence validated and a detailed report on clone generaion is provided. HC In some cases, such as in populaions of primary cells, you may simply want to show that you achieved high KO or KI efficiency, without isolaing clones for confirmaion. Genome ediing efficiency is typically determined via Surveyor assay (T7E1 assay) or assayed with next-generaion sequencing (NGS). Many unique inserions and deleions will likely be observed. The KRAS gene encodes for a protein called K-Ras, which is an important regulator of cell division. This gene, when mutated, can cause cells to become cancerous. In this case study, the K-Ras locus was knocked-out in the human colon cancer cell line, HCT116 (Figure 7). T1 1 To idenify successful cases of CRISPR-mediated KO, the target site should be sequence to confirm a frame-shit mutaion has occurred. You should also confirm that the mRNA and protein are significantly depleted or absent, such as by qPCR and Western blot on genome-edited samples versus unedited (parental) controls. Case Study 1: Generaing K-Ras knock-out cell lines using CRISPR genome ediing HC Check for intended KO / KI and off-target effects K-ras HCT116 KRAS -/- beta acin www.genscript.com 17 Case Study 2: Using CRISPR to generate GLP-1R knock-in cell lines Glucagon-like pepide 1 receptor (GLP-1R) is expressed in pancreaic cells and when simulated increases insulin synthesis and release (Drucker et al., 1987). Consequently, it is a common target for the development of therapeuics for diabetes. In this study, a knock-in cell line was generated using GLP-1R donor DNA (containing the gene of interest and a puromycin selectable marker) and HEK 293T cells. The AAVS1 locus was targeted as the knock-in region (Figure 9). The cells were co-transfected with the donor DNA, Cas9 and gRNA, and posiive clones were selected from the cell pools by Sanger sequencing and PCR. Figure 9: Integraion of GLP-1R into HEK 293T cells gRNA and Cas9 complex AAVS1 locus DNA break stimulates homologous recombination Donor Vector Homologous Puro GLP-1R Homologous Integration of donor into AAVS1locus Gene edited locus Case Study 3: Microbial Genome Ediing Microbial genome ediing has many GenScript’s Microbial Genome applicaions in both pharma and industry – Ediing service uses λ Red – from studying gene funcion to the CRISPR/Cas ediing technology. producion of recombinant proteins for drug This technique is the most discovery and development. CRISPR/Cas can precise, efficient, and cost also be used to generate knock-in and effecive recombineering knock-outs in microbes, such as E. coli. Since method on the market! HR frequency is generally lower in microbes than mammalian cells, CRISPR/Cas can be combined with other recombinaion techniques to improve gene ediing efficiency (Jiang et al., 2015). In this example, λ Red recombineering, one of the most effecive recombinaion techniques in bacteria, is combined with CRISPR/Cas for efficient, seamless genome ediing in E. coli. In this case study, λ Red – CRISPR/Cas is used to knock-out cadA in the BL21 E. coli strain. The CasA protein is a component of lysine decarboxylase, an enzyme that helps bacteria survive in acidic environments (Lee et al., 2007). Ater the reacion, Sanger sequencing and colony PCR screening was used to confirm knock-out was successful (Figure 11). Figure 11: Seamless knock-out of cadA in BL21 E. coli Puro-GLP-1R Sanger sequencing of BL21 ΔcadA Primers for sequencing Figure 10: Immunocytochemistry (let) and western blot (right) analysis of GLP-1R clones Negaive Control GLP-1R clone Ani-GLP-1R Phase contrast Cell line: NC GLP-1R clone 80 kD – 60 kD – 50 kD – 40 kD – GLP-1R www.genscript.com Seq primer R cadA 30 kD – 20 kD – Ater 2 weeks of maintenance under puromycin selecion, surviving cells were isolated and PCR analyzed for the Puro-GFP insert, which indicated GLP-1R was successfully inserted into the AAVS1 locus. Along with the Sanger sequencing results, immunocytochemistry and western blot analysis confirmed that the transfecion was successful (Figure 10). 18 Seq primer F 1 2 3 4 5 6 7 8 M 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 (bp) 3000 2000 1500 1000 750 500 250 100 KO strain www.genscript.com 19 CRISPR genome ediing powers novel findings across disciplines CRISPR/Cas genome ediing has been used to accelerate research in many different arenas of basic life science and biomedical research. Table 3: Research applicaions for CRISPR/Cas9 genome ediing Neuroscience Cancer Biology CRISPR/Cas idenifies novel tumor suppressor genes and new animal models for brain tumors. Mutaions to tumor suppressor genes are oten causes for cancer progression, and developing animal models for these transformaions is a very ime-intensive. To address this, Zimermann et al (2015), used CRISPR/Cas to somaically induce loss-of-funcion (LOF) mutaions in genes in the Sonic Hedgehog (Shh) signaling pathway: in previous studies, the authors found that SHH regulates proliferaion of neural cells in the brain that can lead to malignant brain tumors. The results of this study confirmed that CRISPR/Cas could successfully induce these LOF mutaions for the development of new, relevant brain tumor models. Vaccines/ Virology T cell engineering with CRISPR/Cas reveals a new therapeuic strategy for HIV. While successful T cell ediing has historically been challenging, Schumann et al (2015) reported that the CRISPR/Cas ediing tool can be used to successfully knock-out CXCR4, a co-receptor that HIV uses to infect cells. Using this technology, the authors reported that approximately 40% of CD4+ T-cells are CXCR4- following transfecion with Cas9: gRNA ribonucleases. Plant Biology Immunology 20 A novel rat model for muscular dystrophy reveals new treatment targets. Muscular Dystrophy is a condiion associated with a loss of the protein Dystrophin, which is deadly when it affects the cardiac muscle. The lack of appropriate animal modes has made therapeuic discovery challenging; however, in a recent study by Nakamura et al (2014), CRISPR/Cas was used to knock-out the Dystrophin gene (Dmd) in rats. These mutaions were heritable, thus presening a new animal model to study new therapeuic targets for muscular dystrophy. Successful adaptaion of the CRISPR/Cas ediing system in rice. Targeted mutagenesis has many implicaions for developing new traits in plants; however, mutaion frequencies have varied significantly between species and delivery in plants can be paricularly difficult. In an effort to opimize the process in rice, Mikami et al (2015) tested the efficiency of muliple gRNA and Cas9 vectors in rice calli. From this study they idenified two Cas9 vectors, MMCas9 and FFCas9, as being the most effecive for rice plants. Knock-out nasal airway epithelial cells reveal a new pro-inflammatory funcion of the MUC18 gene. Genome ediing in primary cell lines has been a persistent challenge; however, Chu et al (2015) demonstrated that CRISPR/Cas could be used to knockout Muc18, a gene known to promote tumor metastasis, to beter understand its funcion. In this study, the group showed that MUC18 KO has a pro-inflammatory role in the airway epithelium following exposure to viral and bacterial simuli. www.genscript.com Expanding the Research Applicaions for CRISPR CRISPR/Cas9 technology has been adapted for many research applicaions other than genome ediing, such as: • in situ funcional assays in mouse tumor models (Malina et al., 2013), • targeing funcional long noncoding RNAs (lncRNA) or ribonucleoprotein (RNP) complexes to specific genomic loci (Shechner et al., 2015) • Studying genome architecture and long-distance gene-enhancer interacions by disruping megabase-scale topological chromain domains (Lupiáñez et al., 2015) Genome-wide screens using CRISPR libraries In addiion to targeing a single gene or a few specific genes at a ime, CRISPR has been adapted for genome-wide screening to discover genes whose inhibiion or aberrant acivaion can drive phenotypes implicated in disease, development, or other biological processes. GenScript offers amplified, NGS validated GeCKO and SAM libraries to accelerate your genome-wide screening efforts. Genome-scale CRISPR knock-out libraries (GeCKO v2) libraries for mouse and human genomes enable rapid screening for loss-of-funcion mutaions, as described by Sanjana et al (2014). GeCKO libraries are a mixed pool of CRISPR guide RNAs that target every gene and miRNA in the genome. Each gRNA is cloned into a leniviral vector opimized to produce high-iter virus for efficient leniviral transducion of primary cells or cultured cell lines. Either a single-vector or dual-vector system may be used (see Figure 6 on page 11). A cell populaion should be transduced with the GeCKO library pool at a low MOI ensuring no more than one gRNA enters any given cell. Ater transducion, deep sequencing with NGS should be performed to assess gRNA representaion in the cell pool before beginning a screening protocol. At the end of the screen, ater a second round of NGS, data analysis should be performed to idenify the guides that were lost or enriched over the course of the screen. In order to idenify true posiive hits from a GeCKO library screen, you should idenify genes for which muliple guides were enriched. A detailed GeCKO screening protocol may be found on the Genome Engineering website. GeCKO libraries were designed to contain 6 single guide RNA (sgRNA) molecules targeing each gene within the human or mouse genome, as well as 4 sgRNA targeing each miRNA, and 1000 control (non-targeing) sgRNAs. The gRNA sequences are distributed over three or four consituively expressed exons for each gene and were selected to minimize off-target genome modificaion. www.genscript.com 21 Each library was divided into two sublibraries, A and B, containing 3 unique sgRNA for each gene; only library A contains 4 sgRNA targeing each of 1,864 miRNAs; both A and B contain the same 1,000 nontargeing control sgRNAs. The use of a single sublibrary maintains comprehensive genome-scale coverage but reduces the number of cells required to perform a screen, which is useful when cell numbers are limiing (for example, with primary cells or in vivo screens); alternaively, larger screens can be performed by combining both sublibraries. The GeCKO library can be used in place of RNAi libraries for loss-of-funcion screening for any phenotype of interest, for example, to idenify genes whose loss of funcion enables drug resistance in cancer cells (see box on page 23). As a complimentary approach, a CRISPR-based gene acivaion library can be used in place of a cDNA overexpression library for gain-of-funcion screening, as described below. Adaping CRISPR for Transcripional Regulaion Several research groups have harnessed the specificity and easy re-programmability of the CRISPR/Cas9 system to create programmable transcripion factors that can acivate or repress transcripion of any desired coding region within a genome (Gilbert et al., 2013; Bikard et al., 2013; Cheng et al., 2013; Perez-Pinera et al., 2013). These systems use a nucleolyically inacive Cas9 protein (typically denoted as “dead” or dCas9) in order to target the Cas9-gRNA complex to the right posiion in the genome without cleaving or altering genomic DNA. They fuse the Cas9 to a well-characterized transcripion-regulaing domain, and then design guide RNA to direct the complex to just upstream of the transcripion start site. Several light-inducible CRISPR-based transcripion factors have been designed to allow precise spaial and temporal control of endogenous gene acivaion (Polstein et al., 2015; Nihongaki et al., 2015). MS2 RNA aptmers dCas9 One CRISPR-based transcripional acivator that sgRNA has been used not only to target single genes VP64 but also for genome-wide gain-of-funcion screening is the CRISPR/Cas9 Synergisic MS2 Acivaion Mediator (SAM) system developed in p65 HSF1 the laboratory of Feng Zhang at the Broad Insitute. SAM enables robust transcripional acivaion of endogenous genes targeted by guide RNA that binds within 200 bp upstream of the transcripion start site. SAM can be used to acivate transcripion of a single gene or up to 10 assembled SAM complex 22 www.genscript.com genes at once in the same cell. They can also be used to interrogate the funcion of long intergenic non-coding RNA (lincRNA) transcripts in addiion to genes. Stable expression of SAM components via leniviral transducion generates cell lines show stable and robust transcripional acivaion, even of genes that are normally transcripionally silent. These cell lines can be ideal research tools to characterize the funcion of specific candidate genes or groups of genes. SAM can also be used for discovery research to idenify the genes that drive phenotypes of interest in any disease model or developmental/differeniaion process by using a genome-wide SAM gRNA library for gain-of-funcion screening (Konermann et al., 2015). The screening process is similar to the GeCKO library screening experimental protocol described above, but the library is designed to acivate transcripion rather than edit the genome. The human genome-wide SAM library contains 3 guide RNA targeing within 200 bp upstream of each of 23,430 coding gene isoform with a unique transcripion start site in the human reference genome, for a total of 70,290 guides. This mixed pool of SAM guide RNAs is delivered along with the other SAM components using leniviral vectors. CRISPR libraries yield insights into Cancer Biology An oncogenic mutaion observed in melanoma cells, BRAF(V600E), makes cells suscepible to therapeuic treatment with BRAF inhibitors. However, some melanoma cells are able to develop resistance to these drugs over ime. Genome-wide CRISPR libraries were used to idenify genes whose upor down-regulaion within melanoma cells could confer resistance to BRAF inhibiing drugs (Shalem et al., 2014; Konermann et al., 2015) Both GeCKO and SAM libraries were used to screen A375 (BRAF(V600E)) melanoma cells, by transducing a cell pool with the library and performing NGS to quanify sgRNA representaion before and ater a 14-day drug treatment. Ater treatment, most gRNA were substanially reduced, while a small set were highly enriched. The gene expression signature based on the top screening hits correlated with markers of BRAF inhibitor resistance in cell lines and paient-derived samples, enhancing confidence in the clinical relevance of these results. Genes for which several unique gRNA were enriched were considered top hits; these included genes previously known to confer resistance, such as EGFR and other genes in the ERK pathway, as well as numerous novel candidate genes, which can be subsequently validated using individual sgRNA and cDNA overexpression. www.genscript.com 23 Epigeneic Modificaions Stem Cell Differeniaion Epigeneic modificaions to genomic DNA and to the histone proteins that help organize chromosomes are increasingly shown to play criical roles in biological processes. Epigeneic marks such as methylaion or acetylaion at specific genomic loci or histone residues can be inherited or acquired, and can influence gene expression. The enzymes that regulate epigeneic state can be targeted via CRISPR genome ediing or order to generate genomewide perturbaions in epigeneic state. This was seen, for example, ater CRISPR-mediated KO of all three acive DNA methyltransferases (DNMTs), individually or in combinaion, in human embryonic stem cells (ESCs), allowing researchers to characterize viable, pluripotent cell lines with disinct effects on the DNA methylaion landscape (Liao et al., 2015). CRISPR technology can be used to guide stem cell differeniaion for both basic research and therapeuics. Stem cell differeniaion typically requires the robust acivaion of specific genes – typically transcripion factors that control broad programs of downstream target gene expression – in specific combinaions and sequences, over the course of several weeks or months. A catalyically inacive Cas9 nuclease that is fused to transacivaion domains can be used as a programmable transcripion acivator to acivate genes required for differeniaion. For example, targeted acivaion of the endogenous Myod1 gene locus has been shown to yield stable and sustained reprogramming of mouse embryonic fibroblasts into skeletal myocytes (Chakraborty et al., 2014) for the repair of skeletal muscle issue. Researchers increasingly need methods for introducing epigeneic modificaions only at desired genomic loci in order to model diseases and test hypotheses regarding potenial therapeuic strategies. For example, specific epigeneic alteraions are oten necessary or sufficient to drive transformaion of normal cells into cancerous cells, and play roles in later steps of carcinogenesis; therefore, the enzymes that regulate epigeneic modificaions to DNA or histone proteins are candidate targets for cancer therapy (reviewed by Yao et al., 2015). Induced pluripotent stem cells (iPSCs) have also become popular choices for stem cell therapy since they can be derived from paient-specific cells, overcoming ethical issues associated with embryonic stem cells. Similar to embryonic stem cells, iPSCs must be pre-differeniated prior to implantaion to avoid teratoma formaion; however, differeniaion efficiency coninues to be a botleneck. Recent reports indicate that CRISPR may be an essenial tool to improve differeniaion, and has been used to derive a variety of cell types including muscle cells for the treatment of muscular dystrophy (Loperfido et al., 2015) and hematopoieic stem cells for the treatment of sickle cell anemia (Song et al., 2015). Recently, there have been muliple studies invesigaing the use of CRISPR to correct deleterious mutaions associated with geneic diseases. For instance, the inherited blood disease β-Thalassemia is caused by deleions to the β-globin (HBB) gene, and by generaing iPSCs with this mutaion corrected could be a potenial treatment opion (Xu et al., 2015). Together these results demonstrate that CRISPR/Cas can improve the efficiency of not only gene targeing, but also directed differeniaion. CRISPR technology allows a catalyically inacive Cas9 to serve as a precisely targeted DNA-binding domain; when fused to epigeneic enzymes such as DNA methylases, histone acetyltransferases or deacetylases (HATs or HDACs), the complex can alter the epigeneic state in a precise way at a single precise locaion, or at several specific locaions simultaneously. For example, a CRISPR-Cas9-based acetyltransferase consising of dCas9 fused to the catalyic core of the human acetyltransferase p300 was shown to acetylate histone H3 lysine 27 specifically at its target sites and to robustly acivate transcripion of target genes (Hilton et al., 2015). Similar to the capabiliies of the SAM complex for transcripion acivaion, Cas9 epigeneic effectors (epiCas9s) could also be used for genome-wide screening to discover novel relaionships between DNA methylaion or chromain states and phenotypes such as cellular differeniaion or disease progression (Hsu et al., 2014). 24 www.genscript.com Therapeuics Both well-established pharmaceuical companies and new start-up biotech companies are racing to create CRISPR-based therapeuics. Compared to other strategies for gene therapy, CRISPR genome ediing is thought to be faster, less expensive, and potenially far safer. CRISPR-based therapeuics are already in development for treaing blood cancers by modifying paients’ T cells; eliminaing disease-causing viruses in paients; and correcing single nucleoide mutaions that cause many inherited diseases such as sickle-cell anemia. www.genscript.com 25 CRISPR genome ediing is especially promising for diseases that can be tackled by modifying cells that can easily be removed from a paient, genome-edited, screened to ensure no off-target genome modificaions, and then infused back into the same paient. Autologous cell therapies that use genome ediing to correct a mutaion in the paient’s own cells could be far safer than current therapies that use transplants from healthy donors. For example, combining CRISPR-mediated genome engineering with autologous T-cell therapies holds great promise for many diseases including cancer, HIV, primary immune deficiencies, and autoimmune diseases. It has already been demonstrated that primary human CD4+ T cells can be genome-edited with high efficiency and specificity using Cas9 protein in complex with guide RNA (Cas9 RNPs) (Schumann et al., 2015). Fusing GFP to Cas9 allows FACS-based enrichment of transfected T-cells (Meissner et al., 2014), and other improvements to CRISPR-based T-cell therapy protocols are doubtless underway. While there are many examples of in vitro or animal studies in which CRISPR-mediated gene knockout corrects a disease phenotype, significant challenges nonetheless remain to translate these into safe, efficacious therapies for human paients. In order to address safety concerns prior to bringing CRISPR technology in to the clinic, a great deal of atenion has already been paid to developing nonviral vectors such as lipid- or polymer-based nanocarriers, and several are already in clinical trials (Li et al., 2015). Non-viral CRISPR-mediated gene therapy may bypass some of the risks of prior viral-based gene therapy strategies, including the risk that a viral vector might recombine in vivo and become replicaion-competent; the risk that randomly integraing viruses will induce inserional mutagenesis, inaccurate gene dosage; the risk that geneic modificaions could be made at unintended genomic loci or in unintended issues; or the chance that the gene therapy will simply be ineffecive due to immune responses directed against the viral vector. However, even non-viral Cas9 delivery may not completely avoid unwanted immune responses; a study delivering SpCas9 in vivo in mouse liver detected Cas9-specific humoral immune responses, highlighing the need for cauion in future translaional studies, and reinforcing the idea that ex vivo genome modificaion of autologous cells may be a safer route than in vivo delivery of Cas9 (Wang et al., 2015). 26 www.genscript.com Table 4: Lead Prospects for CRISPR-based Therapeuics Cancer CRISPR-mediated knockout of NANOG and NANOGP8 decreases the in vivo tumorigenic potenial of DU145 prostate cancer cell lines as well as in vitro phenotypes associated with malignancy such as sphere formaion, anchorage-independent growth, migraion capability, and drug resistance, suggesing that CRISPR-mediated gene knockout may be a viable addiion to the therapeuic arsenal for prostate cancer paients (Kawamura et al., 2015). Cardiovascular Disease CRISPR/Cas9-mediated gene therapies could be used to correct inherited or acquired mutaions that underlie cardiac disease, or to introduce therapeuic genes such as SERCA2a, S100A1, and adenylate cyclase 6 (Rincon et al., 2015) HIV HIV has been effecively eliminated in some paients via gene therapy to delete CCR5, which could be accomplished more efficiently in the future using CRISPR technology. In addiion, CRISPR could be used in stem cell-based gene therapies to treat chronic HIV infecion; hematopoieic stem/progenitor cells have been engineered to express a chimeric anigen receptor (CAR), so that they differeniate into funcional cytotoxic T lymphocytes and natural killer cells that are resistant to HIV infecion and suppress HIV replicaion (Zhen et al., 2015). Viral Diseases CRISPR genome ediing may be used to prevent, control, or cure viral diseases by targeing viral genes essenial for replicaion or virulence. For example, persistent infecion with HPV strains that cause genital warts, which have a high rate of recurrence ater treatment, could be tackled through CRISPR-mediated inacivaion of viral E 7 gene, as has already been demonstrated in transformed kerainocytes in vitro (Liu et al. 2015). CRISPR could also be used to target human genes that could enhance host immune responses against the virus. Immunodeficiences Immunodeficiencies such as SCID are typically treated by allogeneic hematopoieic stem cell (HSC) transplantaion, which carry a significant risk of incompaibility between donor and paient (Ot de Bruin et al., 2015). Geneic Diseases CRISPR genome ediing could enable treatments for a number of geneic diseases, such as Chronic granulomatous disease (CGD) (Flynn et al., 2015) and replacing dysfuncional proteins in photoreceptor cells to restore sight in paients with a geneic reinal disease. www.genscript.com 27 Future of CRISPR References CRISPR/Cas has revoluionized genome ediing for its ease of use and broad applicability to mammalian cells, microbes, and animal models. Not only does CRISPR have the potenial to enhance our ability to analyze and understand gene funcion, but this new tool can also reform the medical industry. Accessible genome ediing techniques can be used to correct geneic mutaions that are responsible for inherited disorders or diseases, and also for large-scale producion and screening of new drugs (Doudna et al., 2014). In addiion, the ability of CRISPR/Cas to both acivate and repress gene funcion in both coding and non-coding regions of the genome expands its potenial even further. The references cited in this handbook are not intended to be exhausive. We apologize for omiing, due to space constraints, many important contribuions from other researchers. Considering how recently the CRISPR system has been applied to mammalian and microbial gene ediing, there is sill room for improvement. As the mechanism for how Cas9 binds to DNA is revealed, more effecive Cas9-gRNA constructs can be designed (Sternberg and Doudna, 2015). Along the same vein, delivery of Cas9 into mammalian cells coninues to be a botleneck for some cell types. Designing smaller Cas9 variants that can be transfected into cells more easily will expand its applicaions and uses. Regardless of these improvements, the significant role that CRISPR/Cas plays in the biological sciences is apparent. CRISPR/Cas gene ediing remains the easiest and most exciing technology in genome engineering. There is no doubt that this is just the beginning of a revoluionary technology that can be used by generaions of scienists to come. Ablain J et al. A CRISPR/Cas9 vector system for issue-specific gene disrupion in zebrafish. Dev Cell. 2015 Mar 23;32(6):756. Boloin A et al. Clustered regularly spaced short palindromic repeats (CRISPRs) have spacers of extrachromosomal origin. Microbiology. 2005; 151: 2551. Brouns et al. Small CRISPR RNAs guide aniviral defense in prokaryotes. Science. 2008; 321 (5891): 960. Capecchi M. Altering the genome by homologous recombinaion. Science. 1989: 244; 1288. Chakraborty S et al. A CRISPR/Cas9-based system for reprogramming cell lineage specificaion. Stem Cell Rep. 2014 Dec 9;3(6):940. Chari R et al. Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach. Nat Methods. 2015 Jul 13. Want to keep up with advances in CRISPR technology? Visit www.genscript.com/crispr.html to learn about the most recent CRISPR-based tools and research findings 28 www.genscript.com Chu VT et al. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene ediing in mammalian cells. Nat Biotechnol. 2015 May;33(5):543. Cong L et al. Muliplex genome engineering using CRISPR/Cas systems. Science. 2013; 339: 819. Deveau H et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J. Bacteriol. 2008: 190; 1390. DiCarlo J. et al. Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic Acids Res. 2013 Apr;41(7):4336-43. Doench JG et al. Raional design of highly acive sgRNAs for CRISPR-Cas9-mediated gene inacivaion. Nat Biotechnol. 2014 Dec;32(12):1262. Drucker DJ et al. Glucagon-like pepide 1 simulates insulin gene expression and increases cyclic AMP levels in a rat islet cell line. PNAS. 1987; 84 (10): 3434. Choulika A et al. Introducion of homologous recombinaion in mammalian chromosomes by using the I-SceI system of Saccharomyces cerevisiae. Mol. Cell. Biol. 1995; 15: 1968. Friedland AE. et al. Heritable genome ediing in C. elegans via a CRISPR-Cas9 system. Nat Methods. 2013 Aug;10(8):741. Cheng R et al. Efficient gene ediing in adult mouse livers via adenoviral delivery of CRISPR/Cas9. FEBS Let. 2014 Nov 3;588(21):3954. Fu Y et al. Targeted genome ediing in human cells using CRISPR/Cas nucleases and truncated guide RNAs. Methods Enzymol. 2014;546:21. Chu HW et al. CRISPR-Cas9-mediated gene knockout in primary human airway epithelial cells reveals a proinflammatory role for MUC18. Gene ther. June 2015. Gao J et al. CRISPR/Cas9-mediated targeted mutagenesis in Nicoiana tabacum. Plant Mol Biol. 2015 Jan; 87(1-2): 99. www.genscript.com 29 Guilinger JP et al. Fusion of catalyically inacive Cas9 to FokI nuclease improves the specificity of genome modificaion. Nat Biotechnol. 2014;32(6):577. Guo X et al. Efficient RNA/Cas9-mediated genome ediing in Xenopus tropicalis. Development. 2014 Feb;141(3):707. Hermann M et al. Binary recombinase systems for high-resoluion condiional mutagenesis. Nucleic Acids Res. 2014 Apr;42(6):3894. Hilton IB et al. Epigenome ediing by a CRISPR-Cas9-based acetyltransferase acivates genes from promoters and enhancers. Nat Biotechnol. 2015 May;33(5):510. Horvath P and Barrangou R. CRISPR/Cas, the immune system of bacteria and archaea. Science. 2010; 327: 167. Horvath P et al. Diversity, acivity, and evoluion of CRISPR loci in Streptococcus thermophilus. J. Bacteriol. 2008: 190: 1401. Hsu PD et al. DNA targeing specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 2013; 31: 827. Hsu PD et al. Development and Applicaions of CRISPR-Cas9 for Genome Engineering. Cell. 2014 Jun 157:1262. Ishino Y et al. 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One step generaion of mice carrying mutaions in muliple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013 May 9;153(4):910. 32 www.genscript.com To request a hard copy of this handbook or learn more about GenScript’s CRISPR services, visit www.genscript.com/CRISPR.html CRISPR/Cas9 FAQs CRISPR Research Applicaions CRISPR Webinars CRISPR References Experimental Protocols Case Studies: KO/KI cell lines Legal Statement of GenCRISPR Services and Products (Updated on July 28, 2015): GenCRISPR™ services and products are covered under US 8,697,359, US 8,771,945, US 8,795,965, US 8,865,406, US 8,871,445, US 8,889,356, US 8,889,418, US 8,895,308, US 8,906,616 and foreign equivalents and licensed from Broad Insitute, Inc. Cambridge, Massachusets. 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