CN116514935B

CN116514935B - Inhibitors AcrIIIA of RNA editing system 1IPLA35And applications thereof

Info

Publication number: CN116514935B
Application number: CN202310197452.2A
Authority: CN
Inventors: 林平; 蒋建新; 吴敏
Original assignee: Western Chongqing Science City Germplasm Creation Science Center; Chinese Peoples Liberation Army Army Specialized Medical Center
Current assignee: Western Chongqing Science City Germplasm Creation Science Center; Chinese Peoples Liberation Army Army Specialized Medical Center
Priority date: 2021-03-04
Filing date: 2021-03-04
Publication date: 2024-04-30
Anticipated expiration: 2041-03-04
Also published as: CN112961226B; CN116514935A; CN116514934A; CN112961226A; CN116514934B

Abstract

The invention belongs to the technical field of RNA editing, and particularly relates to a III-A type CRISPR-Cas system inhibitor AcrIIIA1 _IPLA35 and application thereof, wherein a AcrIIIA1 homologous protein AcrIIIA1 _IPLA35 screened by the invention is derived from StaphylococcusvirusIPLA phage virus, acrIIIA1 _IPLA35 is identified to inhibit III-A type CRISPR-Cas gene editing activity, acrIIIA1 _IPLA35 in bacteria controls 'closing' of III-A type CRISPR-Cas gene editing capacity, and the variety of the III-A type CRISPR-Cas system inhibitor is enriched. AcrIIIA1 _IPLA35 inhibitor can control III-A type CRISPR-CasRNA editing efficiency in time or space, and improve safety and practicability of III-A type CRISPR-Cas editing technology in fields of biological treatment, biotechnology, agriculture and the like.

Description

Inhibitors AcrIIIA of RNA editing system IPLA35 and application thereof

The application relates to a Chinese patent application with the application date of 2021, 3-month and 4-date, the application number of 202110241224.1 and the name of 'III-A CRISPR-Cas system inhibitor AcrIIIA and application thereof'.

Technical Field

The invention belongs to the technical field of RNA editing, and particularly relates to a III-A type CRISPR-Cas system inhibitor AcrIIIA- _IPLA35 and application thereof.

Background

One of the most exciting findings of microbiology in the last decade is that, like eukaryotes, bacteria also have an acquired immune system, breaking the long-felt theorem that "acquired immunity is unique to eukaryotes. During foreign invasion, bacteria have evolved a new unique immune defense system, the CRISPR-Cas system (clustered regularly interspaced short palindromic repeats and related Cas proteins). CRISPR-Cas systems protect bacteria and archaea from invasion by foreign phages, viruses and plasmids by capturing integrated foreign nucleic acid fragments and under the combined action of Cas proteins and CRISPR RNAS (crrnas). CRISPR-Cas systems have been developed for gene editing, applied in the fields of bioscience, medical diagnostics, crop breeding, and the like. However, since Cas protein is continuously activated after editing of the target gene is completed, nonspecific cleavage at the whole gene level may be caused, thereby causing unknown consequences. Therefore, the editing activity of the CRISPR-Cas is reasonably controlled, the off-target effect is reduced, and the CRISPR-Cas system is a scientific problem which is urgently needed to be solved when the CRISPR-Cas system is applied to gene editing, biological treatment and the like.

Disclosure of Invention

Accordingly, one of the purposes of the present invention is to provide a protein AcrIIIA, _IPLA35, which is homologous to the III-A CRISPR-Cas system inhibitor AcrIIIA, another purpose of the present invention is to provide a reagent or composition containing AcrIIIA1 protein AcrIIIA, _IPLA35, and a third purpose of the present invention is to provide an application of the reagent or composition in preparing a medicament for inhibiting RNA editing activity of the III-A CRISPR-Cas system. In order to achieve the above purpose, the present invention provides the following technical solutions:

1. and a III-A CRISPR-Cas system inhibitor AcrIIIA, wherein the amino acid sequence of the AcrIIIA is SEQ ID NO. 4.

As one of the preferred technical schemes, the gene sequence of AcrIIIA is a sequence site targeted and identified by a III-A CRISPR-Cas system in S.argnteus 3688STDY6125118 bacteria.

As one of the preferred embodiments, acrIIIA has at least 70% sequence identity with the sequence of the AcrIIIA homologous protein and has the same biological function as AcrIIIA.

As one of the preferable technical schemes, the amino acid sequence of the homologous protein is SEQ ID NO.6, and the homologous protein is AcrIIIA1 _IPLA35.

As one of the preferred embodiments, the AcrIIIA or the AcrIIIA homologous protein inhibits the activity of the CRISPR-Cas system of type III-a to cleave RNA.

2. An agent or composition comprising AcrIIIA and/or AcrIIIA1 homologous protein.

3. Use of the agent or composition in the preparation of a medicament for inhibiting the RNA editing activity of a CRISPR-Cas system of type III-a.

The invention has the beneficial effects that:

The invention screens out type III anti-CRISPR (Acr) inhibitor AcrIIIA which inhibits the III-A CRISPR-Cas gene editing activity from Staphylococcus argenteus-3688-STDY 6125118 bacterial gene sequences, and AcrIIIA controls the 'closing' of the III-A CRISPR-Cas gene editing ability in bacteria and mammalian cells. In addition, the invention screens AcrIIIA homologous proteins, has the same inhibition effect, and enriches the variety of III-A CRISPR-Cas system inhibitors. AcrIIIA1 inhibitors and homologous proteins thereof can control RNA editing efficiency of the III-A type CRISPR-Cas system in time or space, and improve safety and practicability of the III-A type CRISPR-Cas editing technology in fields of biological treatment, biotechnology, agriculture and the like.

Drawings

FIG. 1 shows bioinformatic screening and TXTL identification of AcrIIIA gene. A is Staphylococcus argenteus3688STDY6125118 containing the site of recognition of the crRNA itself and a candidate AcrIIIA gene schematic diagram; b is a schematic diagram of a transmission-translation (TXTL) reaction system; C. d is GFP fluorescence detection candidate AcrIIIA gene inhibits the activity of III-A CRISPR-Cas system to cut RNA.

Figure 2 is a diagram showing that AcrIIIA and AcrIIIA2 inhibit the activity of type III-a CRISPR-Cas systems to cleave RNA in bacteria. A is the design of a plaque experiment for an MS2 RNA phage infected host; b is plaque experiment to verify that candidate gene orf1-23 inhibits III-A CRISPR-Cas system against MS2 RNA phage infection host; the AcrIIIA and AcrIIIA genes inhibit the activity of the type III-a CRISPR-Cas system to cleave RNA.

FIG. 3 is a graph depicting the activity of AcrIIIAs gene inhibition III-A CRISPR-Cas system editing technology in mammalian HEK 293T. A is a schematic diagram for establishing and detecting AcrIIIAs gene inhibition III-A CRISPR-Cas system RNA cutting activity in HEK293T cells; b is qRT-PCR detection IAV virus RNA expression level evaluation AcrIIIA and AcrIIIA gene inhibition III-A CRISPR-Cas system activity efficiency; c is an IAV titer evaluation AcrIIIA and AcrIIIA gene to inhibit the active efficiency of type III-A CRISPR-Ca systems.

FIG. 4 is a phylogenetic tree analysis of AcrIIIA and homologous proteins.

FIG. 5 is a phylogenetic tree analysis of AcrIIIA and homologous proteins.

FIG. 6 is a diagram showing the activity of AcrIIIA and AcrIIIA homologous proteins to inhibit RNA cleavage by type III-A CRISPR-Cas system in bacteria; a is the amino acid sequence alignment of AcrIIIA homologous proteins; b is the amino acid sequence alignment of AcrIIIA homologous proteins; c is a plaque assay that verifies AcrIIIA and AcrIIIA homology genes inhibit the activity of type III-A CRISPR-Cas systems to cleave RNA.

Detailed Description

Other advantages and effects of the present invention will become apparent to those skilled in the art from the following disclosure, which describes the embodiments of the present invention with reference to specific examples. The invention may be practiced or carried out in other embodiments that depart from the specific details, and the details of the present description may be modified or varied from the spirit and scope of the present invention.

Example 1

AcrIIIA1 identification and cloning of AcrIIIA2

(1) Candidate AcrIIIAs gene for screening and analyzing biological information

Based on the presence of RNA sequences in the transcriptome of bacteria that can be recognized by type III-a CRISPR-Cas targeting, it is suggested that inhibitors that prevent RNA editing activity of type III-a CRISPR-Cas systems may be included in the bacterial genome itself. According to this principle, the sequence site recognized by type III-A CRISPR-Cas targeting was obtained by Self-TARGETING SPACE SEARCH Platform (SSTS) analysis in Staphylococcus argenteus3688STDY6125118 bacterial gene sequences (https:// www.ncbi.nlm.nih.gov/, accession number: NZ-FQLY 01000002.1) containing type III-A CRISPR-Cas system (A in FIG. 1), suggesting that inhibitors inhibiting type III-A CRISPR-Cas cleavage RNA activity may be present in S.argnteus 3688STDY 6125118. Since the Acr gene is typically clustered with the Acr-associated (Aca) gene containing a conserved helix-helix (HTH) domain. On this basis, first, 6 prophage regions (prophage regions) were identified in S.argnteus 3688STDY6125118 by PHAGE SEARCH Tool (PHAST), encoding 204 open reading frames (open READING FRAMES: ORFs); next, the domain of these 204ORFs was analyzed using Pfam (protein families database) to identify the Aca gene containing the HTH domain; finally, 23 genes of unknown function (orf 1-23) were obtained as candidate genes for AcrIIIAs for the next verification under the conditions of proximity to the Aca gene and identical transcription direction.

(2) Cloning of candidate Gene AcrIIIAs and construction of recombinant expression vector thereof

The full-length sequence of orf1-23 candidate gene is synthesized by GeneUniversal company, gene fragments are recovered by double enzyme digestion of Nco I and Xho I, simultaneously pET28a vector is digested by both of Nco I and Xho I, vector skeleton is recovered, the recovered gene digested fragments and pET28a skeleton fragments are connected and transformed, so that recombinant plasmids pET28a-orf1 to pET28a-orf23 are obtained, and correct recombinant plasmid transformation of escherichia coli is confirmed by sequencing.

GFPpre-crRNA sequence: (SEQ ID NO: 1)

5'-GATATAAACCTAATTACCTCGAGAGGGGACGGAAACGGACACGCTGAACTTGT GGCCGTTTACGTCGCCGTCGATATAAACCTAATTACCTCGAGAGGGGACGGAAACCTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGATATAAACCTAATTACCTCGAGAGGGGACGGAAACGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTGATATAAACCTAATTACCTCGAGAGGGGACGGAAAC-3'

GFP pre-crRNA sequence was synthesized by GeneUniversal and linked to pBlueScript II SK (+) by double cleavage with Kpn I and Xba I to obtain pBlueScript II SK (+) -GFPpre-crRNA (T7-pre-CRRNAFRAGMENTS) recombinant plasmid.

(3) Cell-free Transcription-translation System (TXTL) System selection of AcrIIIAs Gene

TXTL reaction A reaction system was composed of deGFP reporter plasmid (P70 a-deGFP, arbor bioscience), type III-A CRISPR-Cas expression plasmid (T7-pCas/Csm), GFP pre-crRNA (T7-pre-CRRNA FRAGMENTS), T7rnap expression plasmid (pTXTL-P70 a-T7rnap HP, arbor bioscience), and pET28a-orf1 to pET28a-orf23 recombinant plasmids according to the conditions recommended by the manufacturer (FIG. 1B). All plasmids were extracted with QIAGEN PLASMIDMINI KIT (QIAGEN) to give ultrapure plasmids, and the plasmids were purified by AMPure XPbeads (Beckman Coulter).

Each 12. Mu.L TXTL of reaction contains 9. Mu. LTXTLmaster mix,0.1nM of pTXTL-P70a-T7rnap HP,0.125nM of P70 a-deGFP, 1nM of T7-pCas/Csm,2nM of pBlueScript II SK (+) -GFPpre-crRNA,2nM of pET28a-orf1 to pET28a-orf23, and 5. Mu.M of IPTG. GFP fluorescence was measured with a BioTek SYNERGY HT Multi-Mode Microplate Reader at 29℃for 20 hours. At the same time, GFP fluorescence photographs were taken with IVIS XRII system (Perkinelmer). The algorithm for inhibiting type III-a CRISPR-Cas cleavage activity is as follows:

GFP _20h represents the GFP fluorescence value at 20h when crRNA targets GFP, GFP _min represents the minimum GFP fluorescence value during detection, GFPev _,20h represents the GFP fluorescence value in the absence of Acr, GFP _NT,20h represents the GFP fluorescence value when crRNA does not target GFP, and GFP _NT,min represents the minimum GFP fluorescence value during detection.

TXTL the results show that orf10 and orf18 are capable of inhibiting type III-a CRISPR-Cas mediated RNA cleavage capacity in vitro, designated AcrIIIA and AcrIIIA, respectively (C, D in fig. 1).

Example 2

AcrIIIA1 and AcrIIIA inhibit III-A CRISPR-Cas RNA cleavage Activity in bacteria

It was verified in bacteria whether AcrIIIA and AcrIIIA2 inhibited the activity of type III-a CRISPR-Cas. First, PCRISPR MS recombinant plasmids were constructed to obtain crRNA expression vectors for recognition of rep RNA of MS2RNA phages. The pre-crRNA sequence was synthesized by GeneUniversal, as shown in FIG. 2A, and the pre-crRNA was linked to pBluescript II sk (+) 1 vector via T4 DNA ligase to obtain recombinant plasmid PCRISPR MS. Next, pET28a-AcrIIIA1, pET28a-AcrIIIA2 or pET28a and T7-pCas/Csm plasmid and PCRISPR MS plasmid were transformed into competent cells to obtain E.coli c-3000/T7-pCas/Csm/PCRISPR MS2/pET28a, E.coli c-3000/T7-pCas/Csm/PCRISPR MS2/pET28a-AcrIIIA 1and E.coli c-3000/T7-pCas/Csm/PCRISPR MS2/pET28a-AcrIIIA2 strains for plaque formation experiments of MS2RNA phage invasion (FIG. A). The recombinant strain obtained was cultured overnight at 37℃at 220rpm/min, inoculated onto LB agar (0.3%) solid medium containing 1mM IPTG, infected with MS2RNA phage diluted 10-fold in gradient, cultured overnight, observed for plaque formation and photographed.

The results are shown in figure 2 at B, C, where AcrIIIA and AcrIIIA inhibit the ability of type III-a CRISPR-Cas to mediate RNA cleavage, resulting in MS2 RNA phage infection of the host and growth proliferation, resulting in host lysis, ultimately forming plaques. It was shown that AcrIIIA and AcrIIIA can prevent type III-a CRISPR-Cas RNA editing activity in bacteria.

Example 3

AcrIIIA1 and AcrIIIA inhibit the editing activity of type III-a CRISPR-Cas RNAs in mammalian cells

To verify whether AcrIIIA1 and AcrIIIA2 inhibited type III-a CRISPR-Cas RNA editing activity in HEK293T cells, the effect of AcrIIIA and AcrIIIA2 on the efficiency of type III-a CRISPR-Cas cleaving influenza viruses (Influenza A virus, IAV) was tested in HEK293T cells. IAV virus was detected RNA and virus titer (a in fig. 3) after transfection of HEK293T cells with IAV-targeted CRISPR-Cas complex type III-a with AcrIIIA or AcrIIIA proteins using Lipofectamine ^TM CRISPRMAX^TM Transfection Reagent (Thermo FISHER SCIENCTIFIC) (moi=0.01 and 0.5,multiplicity ofinfection). The process is as follows:

1) AcrIIIA Ni column affinity chromatography purification of AcrIIIA, acrIIIA2 and type III-a CRISPR-Cas proteins:

E.coli BL21 competent cells are transformed by the constructed pET28a-His-AcrIIIA1, pET28a-His-AcrIIIA1 and T7-pCas/Csm/His-Csm2 plasmids to obtain recombinant plasmid bacteria, the recombinant plasmid bacteria are collected by centrifugation at 7000rpm for 10 minutes after expansion culture, the recombinant plasmid bacteria are washed 3 times by PBS, the liquid nitrogen is repeatedly frozen and thawed 3 times, the recombinant plasmid bacteria are crushed by ultrasonic waves for 30 minutes, supernatant is collected by centrifugation at 16000rpm for 30 minutes, and the supernatant is filtered by a 0.45 mu m filter membrane; loading the supernatant after suction filtration into a Ni affinity column equilibrated with PBS, respectively washing the column rapidly with 30mL of PBS solution and 20mM of imidazole to wash unbound proteins, respectively washing the column slowly with 10mL of PBS containing 100mM, 500mM and 1M of imidazole and collecting eluate, collecting a tube approximately every 2mL, subjecting the collected eluate to SDS-PAGE electrophoresis detection to discard eluate containing hybrid proteins, dialyzing with PBS solution for 36h, changing the dialysate for every 8h, and finally performing SDS-PAGE electrophoresis detection, wherein the result shows that single bands of AcrIIIA1 and AcrIIIA proteins and III-A type CRISPR-Cas complex proteins are obtained through separation and purification; and its concentration was measured with BCA protein concentration measurement kit.

2) IAV guide RNA sequence Synthesis

5’-ACGGAAACGUAAUGAAGGAUCUUAUUUCUUCGGAGACAAU-3’(SEQ ID NO:2)；

5’-ACGGAAACGGUCGGUUGCUCACAAGUCCUGCCUGCCUGCC-3’(SEQ ID NO:3)；

3) Transfection

Referring to Lipofectamine CRISPRMAX ^TM Transfection Reagent (Thermo FISHER SCIENCTIFIC) transfection reagent instructions, IAV virus (moi=0.01 and 0.5,multiplicity ofinfection) was infected after HEK293T cells were transfected with 3000ng of CRISPR-Cas complex protein type III-a, 1200ng IAV gRNAs,2000ng AcrIIIA1 or AcrIIIA protein, and RNA and virus titer of IAV virus were detected after 24 h.

As shown in figure 3B, C, acrIIIA and AcrIIIA prevent type III-a CRISPR-Cas mediated RNA editing activity in HEK293T cells, acrIIIA and AcrIIIA can effectively control "turn off" of type III-a CRISPR-Cas gene editing. This would help improve the safety of type III-a CRISPR-Cas mediated RNA editing techniques in clinical therapies and application studies.

Example 4

Homology protein analysis of AcrIIIA1 and AcrIIIA2

The protein sequences in all NCBI are subjected to blastp comparison by utilizing the protein sequences of AcrIIIA, and proteins with e-value less than 10 ^-3 and homology ratio more than 70% are selected as homologous proteins of AcrIIIA 1. Identifying that the homologous proteins obtained AcrIIIA1 include WP_001552317.1,YP_009196757.1,YP_002332371.1,EWJ86503.1,EHO90800.1,KMR53579.1,EUQ10906.1,SCU38681.1,EWA35716.1,EWR63129.1,EWK80326.1,AXJ28344.1,SHD87588.1,KFA43737.1,AWQ90359.1,EVD55746.1,EUR30676.1,SGS29864.1,EWH71077.1,COE55786.1,ARM68199.1,WP_095376943.1,KFB80258.1,EVG06959.1,EJE28889.1,EVV21927.1,CZQ83597.1 and wp_037544580.1; wherein ARM68199.1 (AcrIIIA 1 _IME1367)、YP_009196757.1(AcrIIIA1_23MRA) and YP_002332371.1 (AcrIIIA 1 _IPLA35) are homologous proteins from phage viruses. Meanwhile, phylogenetic tree analysis was performed using MEGA7 to make AcrIIIA a1 and its homologous protein phylogenetic tree (fig. 4).

The protein sequences in all NCBI are subjected to blastp alignment by utilizing the protein sequences of AcrIIIA, and proteins with e-value less than 10 ^-3 and homology ratio more than 70% are selected. Identification of homologous proteins to obtain AcrIIIA2 included:

WP_072465245.1,WP_072539211.1,WP_031868661.1,WP_064131496.1,WP_117232106.1,WP_001077670.1,WP_001573838.1,WP_106104614.1,WP_053005550.1,WP_001077638.1,WP_070059026.1,WP_015978251.1,WP_103259238.1,WP_103252633.1,WP_103147425.1,WP_072458005.1,WP_031921279.1,WP_129934257.1,WP_101766656.1,WP_145340959.1,WP_070848524.1,WP_053031406.1,WP_105967223.1,WP_107399202.1,WP_119504741.1,WP_050331550.1,PTF96982.1,WP_075778679.1,WP_002468630.1,WP_021298890.1,WP_145449609.1,WP_002501272.1,WP_141489545.1,WP_002469096.1,WP_049391373.1,WP_115343401.1,WP_049387433.1,WP_002469451.1,WP_064587828.1,WP_002439153.1,PTG35301.1,WP_015365401.1,WP_031765295.1,WP_017804551.1,WP_024273300.1,WP_029625613.1,WP_072599561.1,WP_048527588.1,YP_006382263.1,WP_001837400.1,WP_000896616.1,YP_003857099.1,WP_110179714.1,WP_111762068.1,WP_050961184.1,WP_042856227.1,WP_032099440.1,WP_094969788.1,WP_093514686.1,WP_072527761.1,WP_070859732.1,WP_049401137.1,WP_046467714.1,WP_115287758.1,SUM72483.1,WP_072492559.1,RCV80954.1,WP_114288318.1,WP_032604936.1,WP_002495917.1,WP_002502889.1,WP_124263453.1,WP_069996864.1,WP_135789161.1,EGS40332.1,WP_060556001.1,WP_031764150.1,WP_070481548.1,WP_046597470.1,AGZ24991.1,WP_099816467.1,WP_002475547.1,EHM65174.1,EJE02307.1,WP_129531134.1,WP_087437151.1,WP_070664144.1;

Wherein YP_006382263.1 (AcrIIIA 2 _TEM123) and YP_003857099.1 (AcrIIIA 2 _SAP26) are homologous proteins from phage viruses. Meanwhile, phylogenetic tree analysis was performed using MEGA7 to make AcrIIIA a2 and its homologous protein phylogenetic tree (fig. 5).

To examine whether the phage-derived AcrIIIA and AcrIIIA homologous proteins in table 1 inhibit the activity of type III-a CRISPR-Cas, a plaque formation experiment of MS2 RNA phage invasion was performed as in example 2. As shown in figure 6A, B, C, the homologous proteins of AcrIIIA1 (AcrIIIA _IME1367、AcrIIIA1_23MRA and AcrIIIA1 _IPLA35) and AcrIIIA2 (AcrIIIA 2 _TEM123 and AcrIIIA2 _SAP26) inhibited the type III-a CRISPR-Cas mediated RNA cleavage ability, resulting in MS2 RNA phage infection and growth proliferation, resulting in host lysis, and eventually plaque formation. It was shown that AcrIIIA and AcrIIIA homologous proteins in phage virus sources can also prevent type III-a CRISPR-Cas RNA editing activity.

Tables 1AcrIIIA, acrIIIA and amino acid sequence listing of homologous proteins

Finally, the above embodiments are only intended to be examples. While the invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents substituted for elements thereof without departing from the spirit and scope of the invention, and it is intended to be encompassed by the scope of the claims.

Claims

Use of an acriiia1 homologous protein, an agent or composition comprising said AcrIIIA a homologous protein, said homologous protein having the amino acid sequence SEQ ID No. 6, for the preparation of an inhibitor for inhibiting RNA editing activity of a CRISPR-Cas system of type III-a.