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Summary
Bacteriophages encode anti-CRISPR (Acr) proteins that inactivate CRISPR-Cas bacterial immune techniques, permitting profitable invasion, replication, and prophage integration. Acr proteins inhibit CRISPR-Cas techniques utilizing all kinds of mechanisms. AcrIIA1 is encoded by quite a few phages and plasmids, binds particularly to the Cas9 HNH area, and was the primary Acr found to inhibit SpyCas9. Right here, we report the statement of AcrIIA1-induced degradation of SpyCas9 and SauCas9 in human cell tradition, the primary instance of Acr-induced degradation of CRISPR-Cas nucleases in human cells. AcrIIA1-induced degradation of SpyCas9 is abolished by mutations in AcrIIA1 that break a direct bodily interplay between the two proteins. Focused Cas9 protein degradation by AcrIIA1 may modulate Cas9 nuclease exercise in human therapies. The small measurement and specificity of AcrIIA1 may very well be utilized in a CRISPR-Cas proteolysis-targeting chimera (PROTAC), offering a instrument for creating protected and exact gene modifying purposes.
Quotation: Meacham Z, de Tacca LA, Bondy-Denomy J, Rabuka D, Schelle M (2023) Cas9 degradation in human cells utilizing phage anti-CRISPR proteins. PLoS Biol 21(12):
e3002431.
https://doi.org/10.1371/journal.pbio.3002431
Educational Editor: Franklin L. Nobrega, College of Southampton, UNITED KINGDOM
Obtained: March 23, 2023; Accepted: November 14, 2023; Printed: December 8, 2023
Copyright: © 2023 Meacham et al. That is an open entry article distributed underneath the phrases of the Artistic Commons Attribution License, which allows unrestricted use, distribution, and replica in any medium, supplied the unique creator and supply are credited.
Information Availability: All related knowledge are inside the paper and its Supporting Info recordsdata.
Funding: This work was supported by the Nationwide Institute of Basic Medical Sciences (R43GM145002) to DR. The funders had no function in research design, knowledge assortment and evaluation, choice to publish, or preparation of the manuscript.
Competing pursuits: I’ve learn the journal’s coverage and the authors of this manuscript have the next competing pursuits: D.R. and J.B.D. are founders of Acrigen Biosciences. D.R., M.S., Z.M., and L.T. are staff of Acrigen Biosciences.
Abbreviations:
CRISPR,
clustered frequently interspaced brief palindromic repeats; MGE,
cellular genetic ingredient; PAM,
protospacer adjoining motif; PROTAC,
proteolysis-targeting chimera
Introduction
CRISPR (clustered frequently interspaced brief palindromic repeats) arrays include fragments of DNA that micro organism use as protection towards invading nucleic acids [1,2]. RNA-guided CRISPR-associated (Cas) nucleases determine invaders by first binding to a brief protospacer adjoining motif (PAM) after which by Watson–Crick base-pairing, which results in nucleic acid cleavage [3]. Phages have developed CRISPR inhibitors that support in evasion of the CRISPR protection and improve the transmission of cellular genetic components (MGEs) [4]. Anti-CRISPR (Acr) proteins inactivate the CRISPR-Cas immune system of micro organism [4–7]. The primary instance of phage-encoded Acr proteins have been discovered to inhibit the Class 1 Kind I CRISPR-Cas techniques [8,9]. Shortly after this discovery, the primary antagonists of Class 2 Kind II CRISPR-Cas techniques, together with the clinically related SpyCas9, have been recognized in Listeria prophages [10,11]. AcrIIA1 was revealed to be widespread throughout Firmicutes prophages and MGEs and has even been used as a marker for the invention of latest Acr proteins [10]. AcrIIA1 is a broad-spectrum Cas9 inhibitor, able to inhibiting a number of Cas9 orthologs [12]. AcrIIA1 inhibits a number of Kind II-C Cas9 enzymes in addition to the extra frequent and therapeutically related Kind II-A nucleases, together with SauCas9 and SpyCas9 [12]. This broad inhibitory exercise is because of the capability of AcrIIA1 to bind with excessive affinity to the conserved HNH area of Cas9, particularly counting on the extremely conserved catalytic residue H840. This enables AcrIIA1 to inhibit extremely diverged Cas9 enzymes whereas different Acr proteins co-encoded with AcrIIA1 in Listeria phages, like AcrIIA4 and AcrIIA12, bind the extremely variable PAM-interacting area and inhibit a a lot smaller vary of Cas9 orthologs. Binding to a conserved area and the ensuing broad inhibition profile doubtless influenced the large phylogenetic distribution of AcrIIA1 [10].
Different broad-spectrum Cas9 inhibitors, like AcrIIC1, additionally bind the conserved HNH area [13]. AcrIIC1 features by trapping the DNA-bound Cas9 complicated. Surprisingly, AcrIIA1 was discovered to stimulate degradation of catalytically lively Cas9 protein in Listeria [12]. In Pseudomonas, AcrIIA1 inhibited Cas9 with out degrading the protein, suggesting that the degradation mechanism was particular to Listeria. Whereas AcrIIA1 was in a position to weakly inhibit Cas9 in human cells, it was unable to degrade Cas9 in vitro, main the authors to imagine that the degradation mechanism relied on bacterial-specific protein degradation equipment [12].
Right here, we show that AcrIIA1 induces Cas9 degradation in human cells. We present that AcrIIA1 stimulates the degradation of each SpyCas9 and SauCas9 however is unable to inhibit or stimulate the degradation of Kind V CRISPR-Cas12a. To our information, that is the primary demonstration of Acr-induced Cas9 degradation in eukaryotic cells. This discovery permits for the event of therapeutic gene modifying instruments like CRISPR-Cas9 proteolysis-targeting chimera (PROTAC) [14,15]. Acr-Cas9 PROTAC may very well be used to restrict publicity of human genomes to Cas9 modifying, decreasing the potential for off-target results and growing the protection of gene modifying therapies.
Outcomes and dialogue
AcrIIA1 inhibits Cas9 gene modifying in human cells
We transfected HEK293T human cells with a plasmid expressing SpyCas9 and a information concentrating on the hemoglobin beta (HBB) locus and a second plasmid expressing AcrIIA1 (Fig 1A). Much like earlier outcomes [10], AcrIIA1 encoded with native bacterial codons (AcrIIA1-bac) doesn’t absolutely inhibit SpyCas9 modifying. Nevertheless, expression of a human codon optimized model of the acrIIA1 gene (AcrIIA1-hum) absolutely inhibited SpyCas9 modifying. Enhancing at a recognized HBB off-target website (HBD) was additionally absolutely inhibited. Titration of AcrIIA1-bac plasmid confirmed a dose-dependent enhance in SpyCas9 modifying (Fig 1B). Western blot evaluation reveals a concomitant enhance in AcrIIA1 expression with growing plasmid quantity (Fig 2B). The AcrIIA1-hum assemble was in a position to inhibit SpyCas9 modifying at 0.5:1 plasmid weight:weight ratio. We additionally assessed AcrIIA1 inhibition of SpyCas9 in a second human cell line (Hep G2 liver cells) and noticed the same discount in modifying exercise (S1 Fig).
Fig 1. AcrIIA1 inhibits SpyCas9 modifying in human cells.
(A) Enhancing by SpyCas9 of the HBB gene and the carefully associated off-target website HBD. AcrIIA1-bac makes use of the native bacterial codons. AcrIIA1-hum is codon optimized for human expression (P < 0.001). HEK293T cells have been transiently transfected at a plasmid ratio of 1:2 SpyCas9:AcrIIA1 plasmid. (B) Dose-dependent inhibition of SpyCas9 modifying of HBB by AcrIIA1. “x” represents the fold w/w plasmid quantity of AcrIIA1 relative to SpyCas9. Whole plasmid DNA transfected in every situation was fixed. Bars signify the imply of organic replicates (dots). Underlying knowledge may be present in S1 Information.
AcrIIA1 induces Cas9 degradation in human cells
We subsequent sought to find out the mechanism of AcrIIA1 inhibition of Cas9 in human cells. We probed for the presence of SpyCas9 following expression in HEK293T cells utilizing an anti-SpyCas9 monoclonal antibody. We expressed SpyCas9 and information RNA alone or alongside varied Acr constructs in HEK293T cells. Surprisingly, SpyCas9 was not detected when expressed with AcrIIA1 in HEK293T (Fig 2A) or Hep G2 (S1 Fig) human cells. That is in distinction to co-expression of SpyCas9 with different Acr proteins together with AcrIIA4, a robust SpyCas9 inhibitor [10], or AcrVA1, a Cas12a Acr that doesn’t inhibit Cas9 [16]. Neither AcrIIA4 nor AcrVA1 affected SpyCas9 expression in HEK293T cells. This end result urged that AcrIIA1 is stimulating the degradation of Cas9 in human cells, much like the mechanism noticed in Listeria [12]. In distinction to AcrIIA1, AcrIIA4 is a potent SpyCas9 inhibitor that binds competitively to the PAM-interacting area of SpyCas9 and doesn’t set off Cas9 degradation [17,18]. The presence of SpyCas9 within the AcrIIA4 situation signifies that binding and inhibition of SpyCas9 is impartial of degradation.
Fig 2. AcrIIA1-dependent degradation of SpyCas9.
(A) Western blot displaying AcrIIA1-dependent lower in SpyCas9 protein stage in HEK293T cells. Expression of AcrIIA4 or AcrVA1 doesn’t present a lower in SpyCas9 protein. (B) Western blot displaying the dose-dependent lower in SpyCas9 protein with growing expression of AcrIIA1 in HEK293T cells. AcrIIA1 plasmid is dosed from 1× to 0.125× relative to SpyCas9 plasmid. (C) Western blots of anti-FLAG immunoprecipitations flattening FLAG-tagged SpyCas9 and probing for SpyCas9 and AcrIIA1. AcrIIA1-HA alone is absolutely eluted within the FT. SpyCas9-FLAG effectively binds to the anti-FLAG beads and is eluted (E1 and E2). Co-expression of SpyCas9-FLAG and AcrIIA1-HA (1:0.125 plasmid ratio) leads to decrease SpyCas9. AcrIIA1 binds and elutes (E1 and E2) together with the residual SpyCas9. I = enter, FT = movement by, W1 = wash 1, W2 = wash 2, E1 = elution 1, E2 = elution 2. (D) Enhancing by SpyCas9 on the HBB gene alone or together with AcrIIA1 or the AcrIIA1 double mutant T114A/F115A. Bars signify the imply of organic replicates (dots). Underlying knowledge may be present in S1 Information. (E) Western blot displaying the presence of SpyCas9 with the T114A/F115A AcrIIA1 double mutant.
We subsequent assessed the dose-dependence of AcrIIA1-induced Cas9 degradation. Plasmid encoding AcrIIA1 tagged with an HA epitope (S2 Fig) was titrated and transfected into HEK293T cells together with a plasmid expressing SpyCas9 and information RNA (Fig 2B). An anti-HA antibody reveals a rise in AcrIIA1 expression with growing plasmid focus. SpyCas9 protein focus is inversely correlated with AcrIIA1 expression, in keeping with AcrIIA1-induced degradation. The SpyCas9 protein focus additionally correlates with the modifying noticed in Fig 1B, with elevated modifying and SpyCas9 protein at 0.125-fold AcrIIA1 plasmid focus.
AcrIIA1 binds SpyCas9 in human cells
To find out if AcrIIA1 is instantly binding SpyCas9 in human cells, we co-immunoprecipitated AcrIIA1 and SpyCas9 (Fig 2C). Lysates from HEK293T cells transfected with plasmids encoding HA-tagged AcrIIA1 and FLAG-tagged SpyCas9 have been immunoprecipitated with magnetic beads conjugated to an anti-FLAG antibody to drag down the SpyCas9 protein. HEK293T cells have been transfected with SpyCas9-FLAG and AcrIIA1-HA plasmids at both a 1:1 ratio (S3 Fig) or 1:0.125 ratio (Fig 2C). SpyCas9 is barely detectable within the lysate at each AcrIIA1 ratios, although immunoprecipitation enriched for remaining Cas9. In each situations, AcrIIA1 co-elutes with SpyCas9, indicating direct binding between SpyCas9 and AcrIIA1 in human cells. To evaluate if AcrIIA1-induced SpyCas9 degradation results in truncation merchandise, we probed a western blot utilizing an anti-SpyCas9 antibody. We didn’t observe any apparent degradation merchandise when AcrIIA1 was added, solely a lower in general protein ranges (S4 Fig). We confirmed that AcrIIA1 is instantly concerned in SpyCas9 degradation by testing a mutated AcrIIA1 that abolishes direct SpyCas9 binding in vitro and SpyCas9 degradation in micro organism [12]. The AcrIIA1 T114A/F115A double mutant did not inhibit SpyCas9 modifying (Fig 2D) and didn’t result in degradation (Fig 2E).
AcrIIA1 induces degradation of Cas9 orthologs in human cells
Given its large inhibition spectrum noticed in micro organism, we examined AcrIIA1 for inhibition of SauCas9 in human cells. In micro organism, SauCas9 is inhibited to a lesser diploma than SpyCas9 by AcrIIA1 [12]. Equally, we noticed that AcrIIA1 is just in a position to modestly inhibit SauCas9 in human cells (Fig 3A). That is in distinction to the entire inhibition seen with SpyCas9 (Fig 1A). Like with SpyCas9, the inhibition is dose-dependent, with a decrease focus of AcrIIA1 plasmid leading to much less inhibition of SauCas9 modifying. Regardless of the modest inhibition of SauCas9 modifying by AcrIIA1, the Acr nonetheless effectively induces degradation of SauCas9 protein (Fig 3B). Certainly, on the 1:1 plasmid ratio, SauCas9 is barely detectable in HEK293T cell lysates. In contrast to with SpyCas9, SauCas9 protein ranges are absolutely restored on the 1:0.125 plasmid ratio, indicating that the AcrIIA1-induced degradation of SauCas9 is weaker than with SpyCas9. These outcomes point out that even extremely diverged Cas9 orthologue are prone to the degradation mechanism employed by the AcrIIA1 household. Additional testing of the extremely numerous AcrIIA1 household will likely be wanted to evaluate whether or not an AcrIIA1 homologs exist that would offer extra sturdy inhibition and degradation of SauCas9 and different orthologs.
Fig 3. AcrIIA1 induces degradation of SauCas9 however not Cas12a.
(A) Enhancing efficiencies for SauCas9 concentrating on Chrm2. AcrIIA1 solely modestly inhibits SauCas9 at a 1:1 plasmid ratio (AcrIIA1 (1x), P = 0.01). Bars signify the imply of organic replicates (dots). Underlying knowledge may be present in S1 Information. (B) Western blot of SauCas9 co-expressed with varied Acr proteins. Co-expression of AcrIIA1 induces degradation of SauCas9 equally to SpyCas9. AcrIIA4 and AcrVA1 don’t have an effect on SauCas9 protein concentrations. (C) Western blot of AsCas12a co-expressed with varied Acr proteins. AsCas12a protein focus isn’t affected by both AcrIIA1 or AcrIIA4.
In micro organism, AcrIIA1 was unable to inhibit CRISPR-Cas techniques past the Kind II Cas9 household. We examined if AcrIIA1 was in a position to degrade the Kind V nuclease AsCas12a, which lacks an HNH area and the catalytic residue AcrIIA1 is thought to work together with [12]. Co-expression of AsCas12a utilizing the identical promoter as SpyCas9 and AcrIIA1 from plasmids at a 1:1 ratio reveals no degradation of AsCas12a (Fig 3C). Probing for AcrIIA1 reveals that the protein is expressed, indicating that there isn’t a interplay between the Kind V nuclease and AcrIIA1. Taken collectively, these outcomes point out that AcrIIA1 broadly inhibits and induces the degradation of Cas9 nucleases in human cells and that this mechanism is particular to Kind II CRISPR-Cas techniques.
Dialogue
On this work, we present for the primary time that an anti-CRISPR protein is able to inducing the degradation of a CRISPR-Cas nuclease in human cells. Destabilization or degradation of a Cas protein by an Acr is an unusual mechanism. AcrIIA1 was beforehand proven to inhibit and induce degradation of Cas9 orthologs in Listeria [12]. Key binding residues have been elucidated on each the Acr and Cas9 protein, explaining the broad phylogenetic distribution of the AcrIIA1 household and breadth of Cas9 inhibition. Whereas the precise mechanism of AcrIIA1-induced Cas9 degradation stays unknown, the authors concluded that the degradation mechanism was more likely to be restricted to sure bacterial species the place Cas9 and AcrIIA1 are naturally discovered.
On this report, we present that AcrIIA1 induces degradation of SpyCas9 and SauCas9 by direct binding in human cells. This stunning statement may very well be used to develop a Cas9 PROTAC, which is able to managed Cas9 degradation, much like beforehand engineered auxin inducible degron fusions [15]. Altogether, the power of a single protein area (roughly 80 amino acid C-terminal area of AcrIIA1) to inhibit and degrade a number of Cas9 proteins in human cells means that this protein is both a protease or a Cas9 destabilizer. AcrIIA1 binds tightly to the Cas9 (D10A) nickase [12,19,20], generally utilized in base modifying purposes [21], suggesting that this gene modifying instrument is also degraded. The utility of irreversibly degrading (versus inhibiting) Cas9-based instruments may present a sturdy stand alone “Cas9 off-switch” or be paired with robust inhibitors of DNA binding (e.g., AcrIIA4), analogous to the method utilized by bacteriophages [22,23].
Supplies and strategies
Plasmid cloning
Three plasmids containing AcrIIA1 have been constructed expressing both: a local bacterial codon acrIIA1 (AcrIIA1-bac), a human codon-optimized acrIIA1 (AcrIIA1-hum), or AcrIIA1-hum with an HA-tag on the N-terminus (AcrIIA1-HA). These have been ordered as gene fragments from Twist Bioscience and cloned into Twist’s CMV expression vector utilizing HindIII and BamHI restriction websites. AcrVA1 and AcrIIA4 used as controls have been codon-optimized for human expression and ordered and cloned precisely as AcrIIA1.
The SpyCas9 plasmid was bought from Genscript with BbsI cloning websites for information addition. An HBB information was added to the SpyCas9 plasmid by the oligo anneal protocol supplied from Dr. Feng Zhang’s lab accessible on-line underneath “PX330 cloning protocol.” The oligos used to make the HBB goal are listed in S1 File.
The SauCas9 plasmid was bought from Genscript with BsaI cloning websites for information addition and the sequence is similar as PX601 from Dr. Feng Zhang’s lab. Information cloning protocol for the Chrm2 additionally follows “PX330 cloning protocol” and makes use of oligo anneal because the strategies. The oligos used for Chrm2 information listed in S1 File.
Sequencing
For sequencing of the endogenous areas assessed for modifying efficiencies, we used primers that annealed to every particular area. The off-target area proven in Fig 1 is positioned within the HBD locus and the sequence assessed is positioned on the Intergenic Place: chr2:116069276–116069298:+ and the sequence is GGGAACGTGGATGAAGCTGG (AGG) through which the daring letters signify mismatches to the information. Every area was amplified utilizing the primers listed in S1 File and checked on a 2% agarose gel for purity. They have been cleaned up utilizing a PCR clear up equipment from Zymo (CAT D4033) and submitted to Sanger sequencing utilizing the sequencing primer supplied above. TIDE evaluation was carried out following the printed methodology [24] and carried out in accordance with suggestions. All PCR and sequencing primers are listed in S1 File.
Human cell transfection
Cas9 and information plasmids and the Acr plasmids have been examined for exercise in HEK293T or Hep G2 cells following plasmid transfection utilizing Mirus Transit X2 reagent. Checks have been carried out in 96-well plates transfected with 100 ng of nuclease expression vector and ranging quantities of Acr vectors relying on the experiment following the Mirus Transit X2 transfection suggestions. Samples have been incubated for 72 h and harvested with Fast Extract (Lucigen). Genomic DNA was amplified and sequenced as described above.
Western blot and immunoprecipitations
We used NP40 lysis buffer (50 mM HEPES (pH 7.5), 150 mM KCl, 2 mM EDTA, 0.5% NP40, and 1 mM DTT). Earlier than use, we add 1 Roche full pill for 10 mL of buffer. Samples have been loaded utilizing SDS loading buffer (100 mM Tris-Cl (pH 6.8), 4% SDS, 0.2% bromophenol blue, 20% glycerol, 200 mM of DTT for 10 mL of water). Transfected HEK293T or Hep G2 cells have been lysed, and we carried out Bradford to normalize gel loading quantities. We used the iBind system to switch the gel earlier than blotting and iBlot 2 for blotting the western blots. For SpyCas9, we used a mouse monoclonal antibody from Cell Signaling Know-how (CAT 65832) at a 1:1,000 dilution. For SauCas9, we used a rabbit polyclonal antibody from Millipore Sigma (CAT AB356480) at 1:1,000 dilution. For AsCas12a, we used a rabbit polyclonal antibody from Cell Signaling Know-how (CAT 38150) at a 1:1,000 dilution. All AcrIIA1 detection was achieved utilizing HA-tagged AcrIIA1 and a rabbit monoclonal anti-HA antibody from R&D techniques (CAT MAB0601). For the anti-FLAG IP, we used anti-FLAG M2 Affinity Gel from Merck (CAT A2220) following producer’s directions. For HSP90, we used a polyclonal antibody raised in rabbit from Cell Signaling Know-how (CAT 4874) at a 1:1,000 dilution. For β-Actin, we used a rabbit polyclonal antibody from Cell Signaling Know-how (CAT 4967) at a 1:1,000 dilution. Western blot pictures are consultant of three impartial experiments.
Supporting data
S1 Fig. AcrIIA1 inhibits SpyCas9 in Hep G2 Human Cells.
(A) Enhancing by SpyCas9 of the HBB gene in Hep G2 human liver cells. Bars signify the imply of organic replicates (dots). Underlying knowledge may be present in S1 Information. (B) AcrIIA1 degrades SpyCas9 in Hep G2 cells. Western blot is consultant of triplicate.
https://doi.org/10.1371/journal.pbio.3002431.s001
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S2 Fig. AcrIIA1-HA inhibits equally to AcrIIA1-hum.
Enhancing by SpyCas9 of the HBB gene and the carefully associated off-target website HBD. AcrIIA1-bac makes use of the native bacterial codons. AcrIIA1-hum is codon optimized for human expression. AcrIIA1-HA is the AcrIIA1-hum with an HA tag. HEK293T cells have been transiently transfected at a plasmid ratio of 1:2 SpyCas9:AcrIIA1 plasmid. No distinction is noticed between AcrIIA1-hum and AcrIIA1-HA (P = 0.35). Bars signify the imply of organic replicates (dots). Underlying knowledge may be present in S1 Information.
https://doi.org/10.1371/journal.pbio.3002431.s002
(TIF)
S3 Fig. AcrIIA1 binds SpyCas9.
Western blot of anti-FLAG immunoprecipitations flattening FLAG-tagged SpyCas9 and probing for SpyCas9 and AcrIIA1. Co-expression of SpyCas9-FLAG and AcrIIA1-HA (1:1 plasmid ratio). AcrIIA1 binds and elutes (E1) together with the residual SpyCas9. I = enter, FT = movement by, W1 = wash 1, W2 = wash 2, E1 = elution 1, E2 = elution 2.
https://doi.org/10.1371/journal.pbio.3002431.s003
(TIF)
S4 Fig. No SpyCas9 degradation merchandise detected.
Western blot displaying AcrIIA1-dependent lower in SpyCas9 protein stage in HEK293T cell lysates in comparison with AcrVA1. No degradation merchandise are seen within the AcrIIA1 situation that aren’t current within the AcrVA1 lysate. SpyCas9 is detected utilizing a monoclonal anti-SpyCas9 antibody.
https://doi.org/10.1371/journal.pbio.3002431.s004
(TIF)
S1 Uncooked Photographs. Uncropped and minimally adjusted pictures for all related figures on this article.
Areas of curiosity utilized in figures are outlined in a purple field. Experimental method, antibody, detection technique, and gear are indicated for every picture. Sizes are proven akin to molecular weight markers for proteins. Lanes not used within the determine are marked with “X.”
https://doi.org/10.1371/journal.pbio.3002431.s005
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Acknowledgments
We need to thank the members of Acrigen Biosciences for his or her help and useful dialogue.
References
- 1.
Brouns SJJ, Jore MM, Lundgren M, Westra ER, Slijkhuis RJH, Snijders APL, et al. Small CRISPR RNAs information antiviral protection in prokaryotes. Science. 2008;321(5891):960–964. pmid:18703739 - 2.
Garneau JE, Dupuis MÈ, Villion M, Romero DA, Barrangou R, Boyaval P, et al. The CRISPR/cas bacterial immune system cleaves bacteriophage and plasmid DNA. Nature. 2010;468(7320). pmid:21048762 - 3.
Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, et al. CRISPR offers acquired resistance towards viruses in prokaryotes. Science. 2007;315(5819):1709–1712. pmid:17379808 - 4.
Sontheimer EJ, Davidson AR. Inhibition of CRISPR-Cas techniques by cellular genetic components. Curr Opin Microbiol. 2017;37:120–127. pmid:28668720 - 5.
Maxwell KL. Phages Struggle Again: Inactivation of the CRISPR-Cas Bacterial Immune System by Anti-CRISPR Proteins. PLoS Pathog. 2016;12(1):e1005282. pmid:26741979 - 6.
Maxwell KL. The Anti-CRISPR Story: A Battle for Survival. Mol Cell. 2017;68(1):8–14. pmid:28985512 - 7.
Borges AL, Davidson AR, Bondy-Denomy J. The Discovery, Mechanisms, and Evolutionary Impression of Anti-CRISPRs. Annu Rev Virol. 2017;4(1):37–59. pmid:28749735 - 8.
Bondy-Denomy J, Pawluk A, Maxwell KL, Davidson AR. Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system. Nature. 2013;493(7432):429–432. pmid:23242138 - 9.
Pawluk A, Bondy-Denomy J, Cheung VHW, Maxwell KL, Davidson AR. A brand new group of phage anti-CRISPR genes inhibits the sort I-E CRISPR-Cas system of pseudomonas aeruginosa. Hendrix R, editor. mBio. 2014;5(2). - 10.
Rauch BJ, Silvis MR, Hultquist JF, Waters CS, McGregor MJ, Krogan NJ, et al. Inhibition of CRISPR-Cas9 with Bacteriophage Proteins. Cell. 2017;168(1–2):150–158.e10. - 11.
Stanley SY, Borges AL, Chen KH, Swaney DL, Krogan NJ, Bondy-Denomy J, et al. Anti-CRISPR-Related Proteins Are Essential Repressors of Anti-CRISPR Transcription. Cell. 2019;178(6):1452–1464.e13. pmid:31474367 - 12.
Osuna BA, Karambelkar S, Mahendra C, Christie KA, Garcia B, Davidson AR, et al. Listeria Phages Induce Cas9 Degradation to Shield Lysogenic Genomes. Cell Host Microbe. 2020;28(1):31–40.e9. pmid:32325050 - 13.
Harrington LB, Doxzen KW, Ma E, Liu JJ, Knott GJ, Edraki A, et al. A Broad-Spectrum Inhibitor of CRISPR-Cas9. Cell. 2017;170(6):1224–1233.e15. pmid:28844692 - 14.
Sakamoto KM, Kim KB, Kumagai A, Mercurio F, Crews CM, Deshaies RJ. Protacs: Chimeric molecules that concentrate on proteins to the Skp1-Cullin-F field complicated for ubiquitination and degradation. Proc Natl Acad Sci U S A. 2001;98(15). - 15.
Kleinjan DA, Wardrope C, Nga Sou S, Rosser SJ. Drug-tunable multidimensional artificial gene management utilizing inducible degron-tagged dCas9 effectors. Nat Commun. 2017;8(1). pmid:29084946 - 16.
Marino ND, Zhang JY, Borges AL, Sousa AA, Leon LM, Rauch BJ, et al. Discovery of widespread sort I and kind V CRISPR-Cas inhibitors. Science. 2018;362(6411):240–242. pmid:30190308 - 17.
Kim I, Jeong M, Ka D, Han M, Kim NK, Bae E, et al. Answer construction and dynamics of anti-CRISPR AcrIIA4, the Cas9 inhibitor. Sci Rep. 2018;8(1):3883. pmid:29497118 - 18.
Yang H, Patel DJ. Inhibition Mechanism of an Anti-CRISPR Suppressor AcrIIA4 Concentrating on SpyCas9. Mol Cell. 2017;67(1):117–127.e5. pmid:28602637 - 19.
Jinek M, Chylinski Ok, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337(6096):816–821. pmid:22745249 - 20.
Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complicated mediates particular DNA cleavage for adaptive immunity in micro organism. Proc Natl Acad Sci U S A. 2012;109(39). pmid:22949671 - 21.
Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. Programmable modifying of a goal base in genomic DNA with out double-stranded DNA cleavage. Nature. 2016;533(7603):420–424. pmid:27096365 - 22.
Marino ND, Talaie A, Carion H, Zhang Y, Silas S, Li Y, et al. Translation-dependent downregulation of Cas12a mRNA by an anti-CRISPR protein. bioRxiv. 2022;2022.11.29.518452. - 23.
Osuna BA, Karambelkar S, Mahendra C, Sarbach A, Johnson MC, Kilcher S, et al. Vital Anti-CRISPR Locus Repression by a Bi-functional Cas9 Inhibitor. Cell Host Microbe. 2020;28(1):23–30.e5. pmid:32325051 - 24.
Brinkman EK, van Steensel B. Speedy Quantitative Analysis of CRISPR Genome Enhancing by TIDE and TIDER. Strategies Mol Biol. 2019;1961:29–44. pmid:30912038
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