Materials and Methods

Anti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for Genome Editing

STAR★METHODSCONTACT FOR REAGENT AND MATERIALS AVAILABILYITYPlease direct any requests for further information or reagents generated in this study to the lead contact, Alan R. Davidson (ac.otnorotu@nosdivad.nala). All unique/stable reagents generated in this study are available from the Lead Contact without restriction.EXPERIMENTAL MODEL AND SUBJECT DETAILSEscherichia coli BB101 (DE3) E. coli BB101 (DE3) cells were used for protein expression for in vivo phage targeting experiments, in vitro studies, and RNP expression and purification. Cells were cultured in Luria broth (LB) at 37°C. To ensure plasmid maintenance, LB media was supplemented with chloramphenicol (34 μg/mL) and streptomycin (34 μg/mL) for the co-expression of the plasmid expressing Cas9s and the Anti-CRISPR proteins for the phage targeting experiments. For RNP expression and purification in the presence of Anti-CRISPR proteins, ampicillin (100 μg/mL) and streptomycin (34 μg/mL) were used for plasmid maintenance.Phage E. coli Mu phage was propagated at 30°C. Mu phage production was induced by increasing the temperature to 45°C for 15 minutes and transferred to 37°C until cell lysis occurred. Mu phage was stored at 4°C.Cell lines HEK293T and Traffic Light Reporter (TLR) cell line (TLR-MCV1, unpublished) were cultured in Dulbecco’s modified Eagle’s minimum essential medium (DMEM; Life Technologies) supplemented with 10% (vol/vol) FBS (Sigma) and 1% Penicillin/Streptomycin.Neuro-2a cells were cultured at 37°C, 5% CO2in DMEM + 10% FBS + 1% Penicillin/Streptomycin.METHOD DETAILSPhylogenetic analysis of Cas9 proteins The Neisseria meningitidis Cas9 sequence from strain 8013 (accession number {"type":"entrez-protein","attrs":{"text":"PHP22510","term_id":"1269814630"}}PHP22510) was used to query the NCBI complete bacterial genome database (four psiBLAST iterations, max target sequences 30,000). Protein sequences corresponding to unique accession numbers (e-value < 0.1) were collected. A non-redundant set of proteins was compiled by filtering out proteins with > 90% sequence identity. The resulting sequences were aligned using MAFFT (Katoh et al., 2002) and Clustal Omega (Sievers et al., 2011) to generate multiple sequence alignment and tree files (Newick format). Phylogenetic trees were visualized using FigTree 1.4.3 (http://tree.bio.ed.ac.uk/software/).Plasmid construction DNA sequences encoding anti-CRISPR genes were cloned into pCDF-1b (Novagen) for in vivo phage Mu targeting experiments, luminescence assays, and co-expression in E. coli. Mutations were introduced into the AcrIIA5 open reading frame contained in the pCDF-1b-derived plasmid by site-directed mutagenesis. For each mutation, two 40-bp complementary primers containing desired mutations were designed. The PCR reaction was conducted using Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific), followed by Dpnl digestion. The resulting DNA product was used to transform E.coli DH5α cells. Plasmids were isolated from streptomycin resistant colonies and all mutations were verified by sequencing.The plasmids expressing GeoCas9 and HpaCas9 used for phage targeting assays were previously described (Harrington et al., 2017; Lee et al., 2018). The non-targeting plasmids expressing Cas9 orthologs were constructed from pGeoCas9-sgRNA (Harrington et al., 2017), replacing the Cas9 coding sequence and the sgRNA scaffolds (listed in Table S1). The Cas9 homologs from Kiloniella laminariae and Brackiella oedipodis, which have not been previously described, were cloned and expressed in the same manner as the other Cas9 proteins. sgRNA and PAM sequences were determined using the same approach as used previously by our group (Lee et al., 2018). The detailed characterization of these Cas9 homologs will be described in a future publication.The sgRNA scaffolds were ordered as gblocks (IDT) and were cloned into the plasmid using a Gibson Assembly reaction (NEB). A unique BsaI restriction site was included in the non-targeting plasmids to clone DNA fragments encoding a crRNA that targets phage Mu or the J23119 promoter (http://parts.igem.org/Part:BBa_J23119). These DNA fragments were generated by phosphorylation and annealing of ssDNA oligos (Eurofins Genomics).The AcrIIA5 mammalian expression vectors were generated by cloning a codon-optimized anti-CRISPR sequence into pCDest2 (pEJS1004-pCDest-ACRIIA5-FLAG-NLS) or a lentiviral vector (pEJS1005-pLenti-AcrIIA5-FLAG/NLS-HygR) using Hifi-Assembly (NEB). The plasmid used for the mouse cell line genome editing experiments was previously described for AcrIIC1 (Pawluk et al., 2016; Addgene #85679). The same plasmid was used for expression of AcrIIA4 and AcrIIA5. Addgene plasmids were used for the SpyCas9-sgRNA (#62988) and SauCas9-sgRNA (#61591) experiments.Expression plasmid 6x-His-tagged SpyCas9-sgRNA was constructed from 6x-His-tagged Nme1Cas9-sgRNA in pMCSG7 (Pawluk et al., 2016), replacing the Cas9 coding sequence and the sgRNA scaffolds. Both plasmids were used for the co-expression and co-purification experiments.In vivo phage Mu plaquing assays E. coli BB101 cells were co-transformed with plasmids expressing Cas9-sgRNA combinations targeting phage Mu and a pCDF-1b plasmid expressing the different anti-CRISPR proteins. Cells containing both plasmids were sub-cultured in LB supplemented with chloramphenicol and streptomycin and grown for two hours, at which point anti-CRISPR expression was induced with 0.01mM IPTG for 3h. Cells were then mixed with soft LB-agar and top-plated on LB supplemented with both antibiotics and 200 ng/mL aTc, 0.2% arabinose, and 10 mM MgSO4. Serial dilutions of phage Mu were spotted on top and the plates were incubated overnight at 37°C. Experiments were performed in triplicate. To confirm anti-CRISPR expression in E. coli, 500 μL of cells after IPTG induction were collected by centrifugation, resuspended in 100 μL SDS loading buffer and analyzed by SDS-PAGE on a 15% Tris-Tricine gel, followed by Coomassie Blue staining.Cell culture, transfection, and stable cell line construction Cells were cultured in Dulbecco’s modified Eagle’s minimum essential medium (DMEM; Life Technologies) supplemented with 10% (vol/vol) FBS (Sigma) and 1% P/S (Life Technologies). Plasmids transfection was performed using Polyfect reagent as described (Pawluk et al., 2016). Transient transfection of 100 ng Cas9, 100 ng sgRNA, and either 200 ng or 300 ng of anti-CRISPR were used for HEK293T and a Traffic Light Reporter (TLR) cell line (TLR-MCV1, unpublished), respectively. For the “NoAcr” conditions 200 ng or 300 ng of a stuffer plasmid was included in the transfection to keep the total amount of DNA constant. Lentiviral transduction was performed as described (Ma et al., 2016). Briefly, viruses were produced and collected by transfecting HEK293T (ATCC) with the lentiviral vector plasmid, pEJS1005-pLenti-c.o.AcrIIA5-FLAG-NLS-HygR that expresses AcrIIA5 (driven by the EF1-a promoter) and packaging helper plasmids (VSV-G and ΔR8.2). HEK293T and HEK293T-TLR-MCV1 target cells were transduced with viruses and then selected with hygromycin, resulting in HEK293T-AcrIIA5 and HEK293T-TLR-MCV1-AcrIIA5 cell lines.Neuro-2a cells cultured at 37°C, 5% CO2 in DMEM + 10% FBS+ 1% Penicillin/Streptomycin (GIBCO) were transiently transfected with 250 ng of either SpyCas9/sgRNA targeting the NPC1 locus or SauCas9/sgRNA targeting the NPC1 locus, and 250 ng of anti-CRISPR expressing plasmid.Electroporation of mammalian cells The HEK293T cells or HEK293T-AcrIIA5 cells were electroporated using the Neon transfection system (ThermoFisher) with an in vitro-formed ribonucleoprotein complex of SpyCas9, crRNAs (C0 or C20), and tracrRNAs (T0 or T2) that were synthesized as described previously (Mir et al., 2018a). Briefly, 80 pmol SpyCas9, 100 pmol crRNA, and 100 pmol tracrRNA were incubated in Buffer R at room temperature for 30 minutes and electroporated into 100,000 cells.Flow cytometry The mCherry-positive TLR-MCV1 cells were analyzed on a MACSQuant® VYB from Miltenyi Biotec using a yellow laser with a 561 nm excitation and emission 615/20 nm filter. FlowJo® v10.4.1. was used for gating single cells based on FSC-A and FSC-H after removal of debris. The percentage of cells expressing mCherry was used to estimate the Cas9-mediated editing efficiency.Indel analysis by T7E1 and TIDE Genomic DNA from cells was harvested using DNeasy Blood and Tissue kit (QIAGEN) according to the manufacturer protocol. PCR was used to amplify the locus surrounding the targeted site DreamTaq Green PCR Master Mix 2X (Thermo Scientific). The PCR reactions were subsequently used for T7E1 assay as previously described (Pawluk et al., 2016). The amplicons were also sequenced by Sanger sequencing using the forward amplification primer. TIDE decomposition software (https://tide.deskgen.com/) was used to assess editing percentage for all transfections (Brinkman et al., 2014).Luminescence assay DNA encoding a crRNA targeting the constitutive promoter region of J23119 was cloned into the BsaI site of the pCRISPathBrick plasmid (Cress et al., 2015). This plasmid was co-transformed into E. coli BL21 cells with pCM-str, a plasmid in which the J23119 artificial promoter drives constitutive expression of the luxCDABE operon from Photorhabdus luminescens (Winson et al., 1998). These cells were then co-transformed with a pCDF-1b plasmid expressing the anti-CRISPR proteins and a protospacer targeting the J23119 promoter. Cells containing the three plasmids were grown in LB supplemented with kanamycin, chloramphenicol and streptomycin until they reached an optical density at 600 nm (OD600) of 0.6. The cultures were then diluted to an OD600 of 0.1 in LB containing 200 ng/mL aTc, 0.2% arabinose and 0.01 mM IPTG, and 100 μl was dispensed into a 96-well plate. The plate was incubated with shaking at 37°C using a Synergy H1 reader controlled by Gen5 2.09 software (BioTek Instruments Inc.), and the OD600 and luminescence was monitored for 24 hours.Co-expression and co-purification of Nme1Cas9/sgRNA and anti-CRISPR E. coli BB101 cells were co-transformed with 6x-His-tagged Nme1Cas9/sgRNA in pMCSG7 (Pawluk et al., 2016) or 6x-His-tagged SpyCas9/sgRNA in pMCSG7 and a pCDF-1b vector encoding untagged anti-CRISPR protein. Cells were grown in LB at 37°C to an OD of 0.8. Protein expression was induced by the addition of 1mM IPTG, and the cells were incubated for an additional 3 hours at 37°C. Cells were collected by centrifugation, resuspended in binding buffer 50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 5% glycerol, 20 mM imidazole, and lysed by sonication. Clarified lysates were incubated with Ni-NTA agarose (QIAGEN) for 30 minutes at 4°C, washed with binding buffer supplemented with 30 mM imidazole, and bound protein was eluted with binding buffer supplemented with 300 mM imidazole. The purified ribonucleoprotein complexes were analyzed by SDS-PAGE using a 15% Tris-Tricine gel, and the proteins were visualized using Coomassie stain. The co-purifying sgRNA was examined using a denaturing 12.5% polyacrylamide/Urea gel and visualized by SYBR™ Gold (ThermoFisher Scientific) staining.For size exclusion chromatography experiments, a Superdex 200 10/300 column was used for Nme1Cas9-sgRNA and Nme1Cas9sgRNA purified from cells expressing the AcrIIA5. Fractions were analyzed on a 15% PAGE gel and stained with Coomassie Blue. The co-purifying sgRNA was examined using a denaturing 12.5% polyacrylamide/Urea gel and visualized by SYBR™ Gold (ThermoFisher Scientific) staining.In vitro DNA cleavage assays DNA cleavage reactions were conducted in Cleavage Buffer 75 mM NaCl, 20 mM Tris (pH 7.5), 5 mM MgCl2, 1 mM TCEP at 37°C. Cas9/sgRNA complexes purified from cells expressing anti-CRISPR proteins were added to the reactions at a concentration of 500 nM. Linear DNA substrates generated by restriction digestion were used at a concentration of 20 nM. Samples removed at various time points were quenched by the addition of EDTA to a final concentration of 10 nM. Cleavage products were analyzed on a 1.25% agarose gel stained with RedSafe (FroggaBio).RNA cloning and sequencing sgRNA bound to affinity purified Nme1Cas9 in the presence or absence of AcrIIA5 or in the presence of AcrIIC1 were electrophoresed on a denaturing 12.5% polyacrylamide/Urea gel and visualized by SYBR™ Gold (ThermoFisher Scientific) staining. Bands corresponding to full-length sgRNA were excised for each sample and bands with higher mobility than full-length sgRNA were excised from the sample of Nme1Cas9 purified from cells grown in the presence of AcrIIA5. The gel slices were soaked in 250 μL of DNA Gel Elution Buffer (New England Biolabs) supplemented with 1:100 SUPERase· In RNase Inhibitor (ThermoFisher Scientific) and rotated overnight at 4°C. The eluate was filtered through a Nanosep® MF 0.45 μm column (Pall Laboratory, ODM45C35). The RNA was ethanol precipitated and reconstituted in ultrapure water. Libraries were prepared with the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs) following the protocol provided by the manufacturer. 100 ng of immunoprecipitated RNA was used as starting material. The resulting DNA library was visualized using 8% PAGE and bands corresponding to the sgRNA fragments were excised. The DNA was eluted from the excised bands by rotating overnight in DNA Gel Elution buffer at room temperature. The eluate was filtered through a Nanosep® MF 0.45 μm column and the DNA was ethanol precipitated and resuspended in ultrapure water. DNA fragments were then ligated to the TOPO Blunt vector (ThermoFisher Scientific), DNA was purified from single colonies and inserts were sequenced using the M13F or M13R primers.Assays with chemically modified sgRNA molecules To perform assays with chemically modified sgRNA molecules, we generated a stable human HEK293T cell line that expresses AcrIIA5 through lentiviral vector transduction and analyzed the editing efficiency of a SpyCas9/crRNA/tracrRNA ribonucleoprotein (RNP) complex at a well-validated site within the VEGFA gene (Mir et al., 2018a). The “unmodified” dual crRNA/tracrRNA guide RNA called C0:T0 has three phosphorothioates at each end of both the crRNA (C0) and the tracrRNA (T0) to help protect against cellular exonucleases. The guide RNA referred to as “modified,” called C20:T2, is heavily modified: the C20 crRNA has a mix of 2′-O-methyl and 2′-fluoro residues, and only six unmodified ribose moieties, each of which is adjacent to a phosphorothioate modification and is RNase-resistant. The 67-nucleotide T2 tracrRNA is ~82% modified, with a mix of 55 2′-O-methyl and 2′-fluoro residues, as well as twelve riboses. Those twelve riboses are not phosphorothioated, but they are all buried in the interior of the protein and therefore largely protected from RNases when loaded into the SpyCas9 RNP. These chemical modifications in C20 and T2 do not impair the genome editing efficiencies by RNP in mammalian cells (Mir et al., 2018a).QUANTIFICATION AND STATISTICAL ANALYSISAll experiments were conducted with at least three biological replicates. Number of biological replicates are reported in the individual Figure Legends. Error bars represent the standard deviations (Figures 2A, ​,2B,2B, and ​and2D2D).DATA AND CODE AVAILABILITYThis study did not generate unique datasets or code.

Article TitleAnti-CRISPR AcrIIA5 Potently Inhibits All Cas9 Homologs Used for Genome Editing

Abstract

This study did not generate unique datasets or code.


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