Materials and Methods

Potent Cas9 Inhibition in Bacterial and Human Cells by AcrIIC4 and AcrIIC5 Anti-CRISPR Proteins

MATERIALS AND METHODSBioinformatics analysis for anti-CRISPR identification. Putative anti-CRISPR genes were identified using the guilt-by-association bioinformatic method described previously (15). Briefly, BLASTp searches were conducted using aca2 ({"type":"entrez-protein","attrs":{"text":"WP028357637.1","term_id":"654906280","term_text":"WP_028357637.1"}}WP_028357637.1) from B. oedipodis DSM 13743 ({"type":"entrez-nucleotide","attrs":{"text":"NZ_KK211205.1","term_id":"654906589","term_text":"NZ_KK211205.1"}}NZ_KK211205.1), and orthologs of aca2 that had a small, uncharacterized hypothetical ORF immediately upstream were curated manually. The search yielded two high-confidence putative type II-C Acrs in strains of H. parainfluenzae 146_HPAR 254_56103_2121718_4319843 (accession {"type":"entrez-nucleotide","attrs":{"text":"NZ_JVSL01000013.1","term_id":"896444009","term_text":"NZ_JVSL01000013.1"}}NZ_JVSL01000013.1) and S. muelleri ATCC 29453 (accession {"type":"entrez-nucleotide","attrs":{"text":"NZ_CP019448.1","term_id":"1353421432","term_text":"NZ_CP019448.1"}}NZ_CP019448.1).Characterization of HpaCas9 and SmuCas9. CRISPRfinder ( was used to identify the CRISPR locus of H. parainfluenzae. The spacers targeting the phage sequences were blasted via CRISPRTarget ( to predict the PAM present on the 3′ sequences. DNA and protein sequences of HpaCas9 and SmuCas9 orthologs are provided in Table S1 in the supplemental material.Plasmid construction. Plasmids used in this study are described in Table S4.TABLE S4Strains and plasmids used in this study. Download Table S4, PDF file, 0.04 MB.Copyright © 2018 Lee et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.Cas9/sgRNA and anti-CRISPR vector for bacterial expression, protein purification, and in vitro transcription. The pMCSG7-NmeCas9 expression vector and the sgRNA for in vitro transcription are as previously described (15). To make the HpaCas9 expression vector pEJS-MCSG7-HpaCas9, genomic DNA sequence from H. parainfluenzae DSM 8987 was obtained from DSMZ and cloned into the pMCSG7-NmeCas9 expression plasmid, replacing the NmeCas9 sequence using Gibson Assembly (NEB). The GeoCas9-expressing plasmid (expressing the GeoCas9 ortholog from G. stearothermophilus strain ATCC 7953) was obtained from Addgene (catalog no. 87700) and similarly cloned into the pMCSG7 vector. To make GeoCas9 from G. stearothermophilus strain L300, a gBlock (IDT) containing the PID was used to replace the PID of GeoCas9 from G. stearothermophilus strain ATCC 7953. For construction of sgRNA scaffolds for HpaCas9 and GeoCas9, the tracrRNA was predicted by crRNA repeat complementarity as well as homology to the NmeCas9 tracrRNA. These sgRNA scaffolds were ordered as gBlocks (IDT) along with overhangs to clone into pLKO.1 plasmid (15, 44) using Gibson Assembly (NEB). The CjeCas9 sgRNA plasmid was used as previously reported (20, 45). All sgRNA scaffolds were used as the templates to create in vitro-transcribed sgRNAs.DNA sequences encoding candidate anti-CRISPR proteins were synthesized and cloned into a pUC57 mini (AmpR) vector with an N. meningitidis 8013 Cas9 promoter sequence for bacterial work, as done previously for other anti-CRISPRs (15). For anti-CRISPR protein purification, the Acr insert was amplified and inserted into the pMCSG7 backbone by Gibson Assembly (NEB), resulting in pMCSG7-Acr. Table S1 contains the DNA and protein sequences of the anti-CRISPRs tested in this study.Cas9/sgRNA and Acr vectors for mammalian expression. For editing of genomic dual target sites by both SpyCas9 and NmeCas9, we used Cas9 and cognate sgRNA expression vectors that were described previously (15). To generate the Acr expression vector, the Acr ORF was amplified from pUC57-Acr and inserted into XhoI-digested pCSDest2 by Gibson Assembly (NEB).Vectors for fluorescence microscopy. pHAGE-TO-DEST dSpyCas9-(mCherry)3 and dNmeCas9-(sfGFP)3 plasmids (38) were purchased from Addgene (catalog no. 64108 and 64109, respectively) and used directly for no-sgRNA control experiments. dNmeCas9-(sfGFP)3 and dSpyCas9-(mCherry)3 all-in-one plasmids have been described previously (15). To make Acr plasmids, we amplified an mTagBFP2 cassette and incorporated it into pCSDest2 vectors expressing the respective Acr by Gibson Assembly (NEB).Expression and purification of Acr and Cas9 proteins. The expression and purification of Acrs and Cas9s were performed as described previously (7, 15). 6×His-tagged anti-CRISPRs and Cas9s were expressed in E. coli strain BL21 Rosetta(DE3). Cells were grown in LB or 2× YT medium at 37°C to an optical density (OD600) of 0.6 in a shaking incubator. At this stage the bacterial cultures were cooled to 18°C, and protein expression was induced by adding 1 mM IPTG. Bacterial cultures were grown overnight at 18°C (∼16 h), after which cells were harvested and resuspended in lysis buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 5 mM imidazole, 1 mM DTT) supplemented with 1 mg/ml lysozyme and protease inhibitor cocktail (Sigma). Cells were lysed by sonication, and the supernatant was then clarified by centrifugation at 18,000 rpm for 30 min. The supernatant was incubated with preequilibrated Ni-NTA agarose (Qiagen) for 1 h. The resin was then washed twice with wash buffer (50 mM Tris-HCl pH 7.5, 500 mM NaCl, 25 mM imidazole, 1 mM DTT). The proteins were eluted in elution buffer containing 300 mM imidazole. For Acr proteins, the 6×His tag was removed by incubation with His-tagged tobacco etch virus (TEV) protease overnight at 4°C followed by a second round of Ni-NTA purification to isolate successfully cleaved, untagged anti-CRISPRs (by collecting the unbound fraction). Cas9s were further purified using cation exchange chromatography using a Sepharose HiTrap column (GE Life Sciences). Size exclusion chromatography was used to purify NmeCas9 further in 20 mM HEPES-KOH (pH 7.5), 300 mM KCl and 1 mM TCEP.In vitro DNA cleavage. For the in vitro DNA cleavage experiments with NmeCas9 (Fig. 1B and Fig. S1), NmeCas9 sgRNA targeting NTS4B was generated by in vitro T7 transcription (NEB). NmeCas9 (150 nM) was incubated with purified, recombinant anti-CRISPR protein (0 to 5 µM) in cleavage buffer (20 mM HEPES-KOH pH 7.5, 150 mM KCl, 1 mM DTT) for 10 min. Next, sgRNA (1:1, 150 nM) was added and the mixture was incubated for another 15 min. Plasmid containing the target protospacer NTS4B was linearized by ScaI digestion. Linearized plasmid was added to the Cas9/sgRNA complex at 3 nM final concentration. The reaction mixtures were incubated at 37°C for 60 min, treated with 1 U proteinase K (NEB) at 50°C for 10 min, and visualized after electrophoresis in a 1% agarose/1× TAE gel.Phage immunity assay. Plasmids expressing Cas9 targeting E. coli phage Mu were cotransformed into E. coli strain BB101 with plasmids expressing the anti-CRISPRs (20). Cells carrying both plasmids were grown in lysogeny broth (LB) supplemented with streptomycin (50 µg/ml) and chloramphenicol (34 µg/ml). Anti-CRISPR gene expression was induced using 0.01 mM IPTG for three hours. A lawn of 200 μl of cells in top agar was applied to LB agar plates supplemented with streptomycin, chloramphenicol, 200 ng/ml anhydrotetracycline (aTc), 0.2% arabinose ± 200 ng/ml aTc, and 10 mM MgSO4. Tenfold serial dilutions of phage Mu were spotted on top of the lawn, and the plates were incubated overnight at 37°C. To confirm the expression levels of the anti-CRISPR proteins in this assay, 500-µl aliquots of cells applied to the top agar were pelleted by centrifugation, resuspended in 100 µl of SDS-PAGE loading buffer, and run on a 15% Tris-Tricine gel, and the resulting protein gel was visualized by Coomassie blue (Bio-Rad).Cas9-Acr copurification. Cas9 proteins were expressed from plasmid pMCSG7 with an N-terminal 6×His affinity tag in E. coli Rosetta cells. Untagged Acrs were coexpressed in the same cells from plasmid pCDF1b. Cells were grown in LB to an OD600 of 0.8, and protein production was induced with 2 mM IPTG overnight at 16°C. Cells were collected by centrifugation, resuspended in binding buffer (20 mM Tris, pH 7.5, 250 mM NaCl, 5 mM imidazole), and lysed by sonication, and cellular debris was removed by centrifugation. The cleared lysates were applied to Ni-NTA columns, washed with binding buffer supplemented with 30 mM imidazole, and eluted with 300 mM imidazole. Protein complexes were analyzed by SDS-PAGE followed by Coomassie staining.PAM determination. A library of a protospacer with randomized PAM sequences was generated using overlapping PCRs, with the forward primer containing the 10-nt randomized sequence flanking the protospacer. The library was subjected to in vitro cleavage by purified recombinant HpaCas9 or SmuCas9 proteins as well as in vitro-transcribed sgRNAs. Briefly, 300 nM Cas9:sgRNA complex was used to cleave 300 nM target fragment in 1× reaction buffer (NEBuffer 3.1) at 37°C for 60 min. The reaction mixture was then treated with 1 U proteinase K (NEB) at 50°C for 10 min and run on a 4% agarose gel with 1× TAE. The segment of a gel where the cleavage products were expected to be was purified and subjected to library preparation as described previously (46). The library was sequenced using the Illumina NextSeq500 sequencing platform and analyzed with custom scripts.Electrophoretic mobility shift assay (EMSA). NmeCas9 (1 µM) was incubated with 1 µM sgRNA in 1× binding buffer (20 mM Tris-HCl pH 7.5, 150 mM KCl, 2 mM EDTA, 1 mM DTT, 5% glycerol, 50 µg/ml heparin, 0.01% Tween 20, 100 μg/ml BSA) for 20 min at room temperature to form the RNP complex. Acrs were added to a final concentration of 10 µM and incubated for an additional 20 min. Finally, the FAM-tagged NTS4B protospacer oligonucleotide was added to the mixture and incubated at 37°C for 1 h. The mixture was loaded onto a native 6% acrylamide gel, and the FAM-tagged DNA was visualized using a Typhoon imager.sgRNA EMSA. NmeCas9 (1.5 µM) and anti-CRISPR (20 µM) proteins were preincubated in 1× binding buffer for 10 min, and then sgRNA (0.15 µM) was added to the reaction mixture for an additional 10 min. The complexes were resolved on a 6% polyacrylamide native gel, stained by SYBR Gold (ThermoFisher), and visualized with a Typhoon imager.Mammalian genome editing. Plasmids for mammalian expression of NmeCas9, SpyCas9, their respective sgRNAs, and the anti-CRISPR proteins are listed in Table S4. Plasmid transfections, collection of genomic DNA, and T7E1 digestions were as described previously (15).Genome editing by Cas9 ribonucleoprotein (RNP) delivery. RNP delivery of NmeCas9 was performed using a Neon electroporation system following the manufacturer’s instructions (ThermoFisher). Briefly, in a 10 μl reaction volume, 15 pmol of NmeCas9 and 150 pmol of anti-CRISPR protein were mixed in buffer R and incubated at room temperature for 20 min. Then, 20 pmol of T7 in vitro-transcribed sgRNA was added to the Cas9-Acr complex and incubated at room temperature for 30 min. Approximately 50,000 to 100,000 cells were mixed with the RNP-Acr-sgRNA complex, electroporated (Neon nucleofection system), and then plated in 24-well plates. Genomic DNA was extracted 48 h post-nucleofection using a DNeasy Blood and Tissue kit (Qiagen) according to the manufacturer’s protocol. Quantification of editing (% of amplicons exhibiting lesions) was done using TIDE analysis (34). PCR products spanning the target site were amplified using 2× Q5 master mix (NEB) and column-purified (Zymo). Purified amplicons were sent for Sanger sequencing (Genewiz), and trace files were analyzed by TIDE.Fluorescence microscopy of dNmeCas9. Experimental procedures were as described previously (15). Briefly, U2OS cells were cotransfected with all-in-one plasmids (150 ng of each dNmeCas9 and dSpyCas9 plasmid), additional sgRNA-expressing plasmid, and 100 ng of anti-CRISPR/mTagBFP2 plasmid using PolyFect (Qiagen) according to the manufacturer’s instructions. After 24 h of incubation, live cells were imaged with a Leica DMi8 microscope equipped with a Hamamatsu camera (C11440-22CU), a 63× oil lens objective, and Microsystems software (LASX). Further imaging processing was done with Fiji-ImageJ. For quantification, only cells that exhibited mTagBFP2 and sfGFP fluorescence as well as dSpyCas9-(mCherry)3 telomeric foci were assessed for the presence or absence of colocalizing dNmeCas9-(sfGFP)3 telomeric foci.Fluorescence polarization. For fluorescence polarization assays, preformed RNP complex of NmeCas9 and sgRNA was added to 1× binding buffer (20 mM Tris-HCl pH 7.5, 150 mM KCl, 5 mM EDTA, 5 mM MgCl2, 1 mM DTT, 5% vol/vol glycerol, 50 μg/ml heparin, 0.01% Tween 20, and 100 μg/ml BSA) and incubated for 30 min followed by the addition of 10 μM Acrs. This mixture was incubated for 30 min followed by the addition of 8 nM FAM-tagged NTS4B protospacer (34 bp containing only 8-bp PAM duplex). After an incubation of 30 min the polarization measurements were made on Victor3 multilabel plate counters (Perkin Elmer). To calculate fraction-bound values, data were normalized by setting the lowest anisotropy to 0 and highest to 1. The curve fitting was performed in GraphPad Prism using the following equation: Y=(|DNA|+|RNP|+Kd)-(|DNA|+|RNP|+Kd)2-(4×|DNA|×|RNP|)2×|DNA|Coimmunoprecipitation. Plasmids expressing NmeCas9 and each anti-CRISPR protein were cotransfected into HEK293T cells. After 48 h, cell lysates were collected and bound to M2 FLAG magnetic beads (Sigma) overnight at 4°C. The beads were washed 5 times before elution by boiling with sample buffer (125 mM Tris-HCl, pH 6.8, with 4% SDS, 20% vol/vol glycerol, and 0.004% bromphenol blue). Subsequent Western blotting was performed as described below.Western blots. For estimating anti-CRISPR protein levels in cells, plasmids encoding each Acr were transiently transfected into HEK293T cells using Polyfect (Qiagen). After 72 h, cell lysate was collected with lysis buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1× protease inhibitor cocktail Sigma). Lysates were boiled with the sample buffer supplemented with 5% 2-mercaptoethanol at 95°C for 5 min before running on 15% SDS-PAGE gels (Bio-Rad). Proteins were then transferred onto a PVDF membrane on a semidry transfer blot using the manufacturer’s instructions (Bio-Rad). Membranes were blocked in 5% dry milk, incubated with 1:5,000 primary antibodies (anti-FLAG raised in rabbit; Sigma) overnight, washed three times with TBST (25 mM Tris-HCl pH 7.6, 125 mM NaCl, 1% Tween 20) for 5 min, and then incubated with HRP-conjugated secondary antibodies for detection with X-ray film (Kodak). For the NmeCas9 stability experiment, 150 ng of Cas9-expressing and 150 ng of sgRNA-expressing plasmids were transiently transfected with an additional 100 ng of Acr-expressing plasmid. For the no-sgRNA control, 150 ng of empty vector was used. Cell lysates were collected and run on a 6% SDS-PAGE gel as described above. After transfer and blocking steps, membranes were incubated with 1:5,000 anti-HA (mouse; Sigma) antibodies overnight and washed with TBST three times for 5 min before incubation with secondary antibodies (ThermoFisher) for 1 h. As a loading control, 1:5,000 anti-GAPDH (rabbit; Abcam) primary antibodies and HRP-conjugated secondary antibodies against rabbit (Bio-Rad) were used.Targeted deep sequencing analysis. Targeted deep sequencing analyses were done as previously described (44). Briefly, we used a two-step PCR amplification approach to produce DNA fragments for each on-target and off-target site. In the first step, we used locus-specific primers bearing universal overhangs with ends complementary to the TruSeq adaptor sequences (Data Set S1). DNA was amplified with High Fidelity 2× PCR Master Mix (NEB) using appropriate annealing temperatures for the on-target (NTS1C) and off-target (NTS1C-OT1) sites. In the second step, the purified PCR pool was amplified with a universal forward primer and an indexed reverse primer to reconstitute the TruSeq adaptors. Full-size products (∼250 bp in length) were extracted using AMPure beads (Beckman Coulter). The purified library was deep sequenced using a paired-end 150 bp MiSeq run. Raw deep sequencing data and the results of statistical tests are reported in Data Set S1.DATA SET S1Raw deep sequencing data and the results of statistical tests. Download Data Set S1, XLSX file, 0.03 MB.Copyright © 2018 Lee et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.Data availability. Raw data files are available upon reasonable request. High-throughput sequencing data are available at the NCBI Sequence Read Archive (accession no. PRJNA505886).FIG S6AcrIIC4Hpa or AcrIIC5Smu inhibit NmeCas9 before DNA binding. (A) Binding of NmeCas9 to partially duplexed DNA measured by fluorescence polarization assays with or without the indicated Acrs. The graph shows the average values (±SD) of three replicates. The curve was fitted to the equation shown in Materials and Methods, and the resulting KD values (nM) for AcrIIC5Smu, AcrIIC4Hpa, AcrIIC1Nme, and “No Acr” were 450.7 ± 47.6, 749.6 ± 157.7, 82.4 ± 6.5, and 85.9 ± 3.9, respectively. (B) Quantitation of dNmeCas9-(sfGFP)3 telomeric foci, as judged by colocalization with dSpyCas9-(mCherry)3 telomeric foci, in cells that express no anti-CRISPR, negative control anti-CRISPR (AcrE2), positive-control AcrIIC3Nme, AcrIIC4Hpa, or AcrIIC5Smu. Foci were scored blinded, i.e., without the experimenter knowing the sample identities. n, the number of cells that were scored under each condition over three biological replicates. Download FIG S6, PDF file, 1.0 MB.Copyright © 2018 Lee et al.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

Article TitlePotent Cas9 Inhibition in Bacterial and Human Cells by AcrIIC4 and AcrIIC5 Anti-CRISPR Proteins


In their natural settings, CRISPR-Cas systems play crucial roles in bacterial and archaeal adaptive immunity to protect against phages and other mobile genetic elements, and they are also widely used as genome engineering technologies. Previously we discovered bacteriophage-encoded Cas9-specific anti-CRISPR (Acr) proteins that serve as countermeasures against host bacterial immunity by inactivating their CRISPR-Cas systems (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829–1838.e9, 2016, We hypothesized that the evolutionary advantages conferred by anti-CRISPRs would drive the widespread occurrence of these proteins in nature (K. L. Maxwell, Mol Cell 68:8–14, 2017,; A. Pawluk, A. R. Davidson, and K. L. Maxwell, Nat Rev Microbiol 16:12–17, 2018,; E. J. Sontheimer and A. R. Davidson, Curr Opin Microbiol 37:120–127, 2017, We have identified new anti-CRISPRs using the same bioinformatic approach that successfully identified previous Acr proteins (A. Pawluk, N. Amrani, Y. Zhang, B. Garcia, et al., Cell 167:1829–1838.e9, 2016, againstNeisseria meningitidisCas9 (NmeCas9). In this work, we report two novel anti-CRISPR families in strains ofHaemophilus parainfluenzaeandSimonsiella muelleri, both of which harbor type II-C CRISPR-Cas systems (A. Mir, A. Edraki, J. Lee, and E. J. Sontheimer, ACS Chem Biol 13:357–365, 2018, We characterize the type II-C Cas9 orthologs fromH. parainfluenzaeandS. muelleri, show that the newly identified Acrs are able to inhibit these systems, and define important features of their inhibitory mechanisms. TheS. muelleriAcr is the most potent NmeCas9 inhibitor identified to date. Although inhibition of NmeCas9 by anti-CRISPRs fromH. parainfluenzaeandS. muellerireveals cross-species inhibitory activity, more distantly related type II-C Cas9s are not inhibited by these proteins. The specificities of anti-CRISPRs and divergent Cas9s appear to reflect coevolution of their strategies to combat or evade each other. Finally, we validate these new anti-CRISPR proteins as potent off-switches for Cas9 genome engineering applications.

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