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• W3173166568 abstract "•BESS method was proposed to focus and characterize the functional PAMs in BE•New non-canonical PAMs were identified to have BE activity•PAM patterns identified by BESS warn of the potential off-target editing Base editing (BE) is a promising genome engineering tool for modifying DNA or RNA and has been widely used in various microorganisms as well as eukaryotic cells. Despite the proximal protospacer adjacent motif (PAM) is critical to the targeting range and off-target effect of BE, there is still lack of a specific approach to analyze the PAM pattern in BE systems. Here, we developed a base editing-coupled survival screening method. Using dCas9 from Streptococcus pyogenes (SpdCas9) and its variants xdCas9 3.7 and dCas9 NG as example, their PAM patterns in BE systems were extensively characterized using the NNNN PAM library with high sensitivity. In addition to the typical PAM recognition features, we observed more unique PAMs exhibiting BE activity. These PAM patterns will boost the finding of potential off-target editing event arising from non-canonical PAMs and provide the guidelines for PAM usage in the BE system. Base editing (BE) is a promising genome engineering tool for modifying DNA or RNA and has been widely used in various microorganisms as well as eukaryotic cells. Despite the proximal protospacer adjacent motif (PAM) is critical to the targeting range and off-target effect of BE, there is still lack of a specific approach to analyze the PAM pattern in BE systems. Here, we developed a base editing-coupled survival screening method. Using dCas9 from Streptococcus pyogenes (SpdCas9) and its variants xdCas9 3.7 and dCas9 NG as example, their PAM patterns in BE systems were extensively characterized using the NNNN PAM library with high sensitivity. In addition to the typical PAM recognition features, we observed more unique PAMs exhibiting BE activity. These PAM patterns will boost the finding of potential off-target editing event arising from non-canonical PAMs and provide the guidelines for PAM usage in the BE system. Base editing (BE) is an important genome editing technique that harnesses catalytically impaired dead Cas9 (dCas9) or nicking Cas9 (nCas9)-guided nucleotide modification enzymes to modify of specific bases (Komor et al., 2016Komor A.C. Kim Y.B. Packer M.S. Zuris J.A. Liu D.R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature. 2016; 533: 420-424Crossref PubMed Scopus (1726) Google Scholar; Nishida et al., 2016Nishida K. Arazoe T. Yachie N. Banno S. Kakimoto M. Tabata M. Mochizuki M. Miyabe A. Araki M. Hara K.Y. et al.Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.Science. 2016; 353: aaf8729Crossref PubMed Scopus (565) Google Scholar). In contrast to Cas9 endonuclease-mediated homologous recombination (Zhou et al., 2019bZhou D. Jiang Z. Pang Q. Zhu Y. Wang Q. Qi Q. CRISPR/Cas9-Assisted seamless genome editing in Lactobacillus plantarum and its application in N-acetylglucosamine production.Appl. Environ. Microbiol. 2019; 85: e01367-19Crossref PubMed Scopus (19) Google Scholar), the BE system based on dCas9 does not cause DNA double-strand breaks (DSBs), which are highly genotoxic for the host, and is therefore regarded as a more promising genome editing tool for applications in basic research and gene therapy (Mention et al., 2019Mention K. Santos L. Harrison P.T. Gene and base editing as a therapeutic option for cystic FibrosisLearning from other diseases.Genes. 2019; 10: 387-404Crossref Scopus (17) Google Scholar; Rees and Liu, 2018Rees H.A. Liu D.R. Base editing: precision chemistry on the genome and transcriptome of living cells.Nat. Rev. Genet. 2018; 19: 770-788Crossref PubMed Scopus (471) Google Scholar). However, in addition to interrogating DNA sequences that are complementary to sgRNA, the complexes must recognize a short protospacer adjacent motif (PAM) located upstream of the DNA target sequence for efficient BE (Jinek et al., 2012Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (7538) Google Scholar; Mojica et al., 2005Mojica F.J. Diez-Villasenor C. Garcia-Martinez J. Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements.J. Mol. Evol. 2005; 60: 174-182Crossref PubMed Scopus (1089) Google Scholar). PAM compatibility not only determines the sequence accessibility in the BE system, the recognition of non-canonical PAMs also would increase the risk of potential off-target editing. The most commonly applied Cas9 nuclease from Streptococcus pyogenes (SpCas9) recognized NGG as the optimal PAM (canonical PAM) (Mali et al., 2013Mali P. Yang L. Esvelt K.M. Aach J. Guell M. DiCarlo J.E. Norville J.E. Church G.M. RNA-guided human genome engineering via Cas9.Science. 2013; 339: 823-826Crossref PubMed Scopus (5647) Google Scholar). Several non-canonical PAMs including NAG, NGA, and NCG have also been reported to support cleavage of DNA by SpCas9, albeit less efficiently than the canonical NGG PAM (Hu et al., 2019Hu Z. Wang D. Zhang C. Wang S. Gao S. Hou L. Wang H. Wang Y. Diverse noncanonical PAMs recognized by SpCas9 in human cells.bioRxiv. 2019; https://doi.org/10.1101/671503Crossref Scopus (0) Google Scholar; Jiang et al., 2013Jiang W. Bikard D. Cox D. Zhang F. Marraffini L.A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Nat. Biotechnol. 2013; 31: 233-239Crossref PubMed Scopus (1455) Google Scholar). The extensive PAM recognition will not only expand the genomic target scope but also may increase the risk of off-target editing. Tsai et al. have reported that non-canonical PAMs such as NAG, NGA, NAA, NGT, NGC, and NCG would trigger genome-wide off-target DSBs (Tsai et al., 2015Tsai S.Q. Zheng Z. Nguyen N.T. Liebers M. Topkar V.V. Thapar V. Wyvekens N. Khayter C. Iafrate A.J. Le L.P. et al.GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases.Nat. Biotechnol. 2015; 33: 187-197Crossref PubMed Scopus (1069) Google Scholar). However, researchers generally used the canonical NGG PAM in the SpdCas9-based BE system. It is currently unknown whether the BE system can use other non-canonical PAMs and result in off-target editing. On the other hand, off-target editing is the major bottleneck for the reliable application of BE, especially for gene therapy (Fu et al., 2013Fu Y. Foden J.A. Khayter C. Maeder M.L. Reyon D. Joung J.K. Sander J.D. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells.Nat. Biotechnol. 2013; 31: 822-826Crossref PubMed Scopus (1941) Google Scholar; Lin et al., 2014Lin Y.N. Cradick T.J. Brown M.T. Deshmukh H. Ranjan P. Sarode N. Wile B.M. Vertino P.M. Stewart F.J. Bao G. CRISPR/Cas9 systems have off-target activity with insertions or deletions between target DNA and guide RNA sequences.Nucleic Acids Res. 2014; 42: 7473-7485Crossref PubMed Scopus (361) Google Scholar; Liu et al., 2018Liu H.Y. Wang L. Luo Y.Z. Blossom of CRISPR technologies and applications in disease treatment.Synth. Syst. Biotechnol. 2018; 3: 217-228Crossref PubMed Scopus (11) Google Scholar). Therefore, it is very essential to comprehensively characterize the usefulness of various PAMs and evaluate the off-target risk in the BE system. Several methods have been developed to determine the PAM sequences for Cas9 nuclease or its variants (Leenay and Beisel, 2017Leenay R.T. Beisel C.L. Deciphering, communicating, and engineering the CRISPR PAM.J. Mol. Biol. 2017; 429: 177-191Crossref PubMed Scopus (75) Google Scholar). These methods can be divided into four categories: (1) in silico identification of PAMs with the natural CRISPR array sequences through Basic Local Alignment Search Tool (BLAST) search and flanking sequence alignment (Savitskaya et al., 2013Savitskaya E. Semenova E. Dedkov V. Metlitskaya A. Severinov K. High-throughput analysis of type I-E CRISPR/Cas spacer acquisition in E. coli.RNA Biol. 2013; 10: 716-725Crossref PubMed Scopus (79) Google Scholar). (2) In vitro DNA cleavage assay using the purified Cas proteins, in vitro transcribed guide RNAs, and a target DNA library with randomized PAM sequences (Karvelis et al., 2015Karvelis T. Gasiunas G. Young J. Bigelyte G. Silanskas A. Cigan M. Siksnys V. Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements.Genome Biol. 2015; 16: 253Crossref PubMed Scopus (109) Google Scholar). PAMs that depleted after the cleavage reaction are considered to be functional PAMs. (3) In vivo DNA cleavage assay, also known as plasmid clearance assay (Jiang et al., 2013Jiang W. Bikard D. Cox D. Zhang F. Marraffini L.A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Nat. Biotechnol. 2013; 31: 233-239Crossref PubMed Scopus (1455) Google Scholar). The Cas effector proteins, guide RNAs, and the target plasmid library with randomized PAM sequences were expressed in a convenient host and subjected to the cleavage reaction in vivo. The plasmids with functional PAMs would be cleared and show a significant decline in the high-throughput sequencing results (Esvelt et al., 2013Esvelt K.M. Mali P. Braff J.L. Moosburner M. Yaung S.J. Church G.M. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.Nat. Methods. 2013; 10: 1116-1121Crossref PubMed Scopus (542) Google Scholar). (4) PAM SCreen Achieved by NOT-gated Repression (PAM-SCANR) and PAM-SEARCH are a group of recently developed methods that in vivo analyze the DNA binding ability of dCas with various PAM sequences (Collias et al., 2020Collias D. Leenay R.T. Slotkowski R.A. Zuo Z. Collins S.P. McGirr B.A. Liu J. Beisel C.L. A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9.Sci. Adv. 2020; 6: eabb4054Crossref PubMed Scopus (6) Google Scholar; Leenay et al., 2016Leenay R.T. Maksimchuk K.R. Slotkowski R.A. Agrawal R.N. Gomaa A.A. Briner A.E. Barrangou R. Beisel C.L. Identifying and visualizing functional PAM diversity across CRISPR-cas systems.Mol. Cell. 2016; 62: 137-147Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). By far, the most common used method is the in vivo plasmid clearance assay, which recapitulated the natural feature of CRISPR-Cas system to clean of foreign genetic elements (Elmore et al., 2015Elmore J. Deighan T. Westpheling J. Terns R.M. Terns M.P. DNA targeting by the type I-G and type I-A CRISPR-Cas systems of Pyrococcus furiosus.Nucleic Acids Res. 2015; 43: 10353-10363PubMed Google Scholar; Esvelt et al., 2013Esvelt K.M. Mali P. Braff J.L. Moosburner M. Yaung S.J. Church G.M. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing.Nat. Methods. 2013; 10: 1116-1121Crossref PubMed Scopus (542) Google Scholar; Jiang et al., 2013Jiang W. Bikard D. Cox D. Zhang F. Marraffini L.A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Nat. Biotechnol. 2013; 31: 233-239Crossref PubMed Scopus (1455) Google Scholar). However, BE only requires the DNA binding of dCas9, not the DNA cleavage activity. Therefore, the cleavage-based PAM determination method is not well suited for the BE system. PAM-SCANR enables rapid elucidation of the functional PAMs supporting the DNA binding of dCas9 that based on the NOT-gate repression of fluorescent protein through fluorescence-activated cell sorting (Leenay et al., 2016Leenay R.T. Maksimchuk K.R. Slotkowski R.A. Agrawal R.N. Gomaa A.A. Briner A.E. Barrangou R. Beisel C.L. Identifying and visualizing functional PAM diversity across CRISPR-cas systems.Mol. Cell. 2016; 62: 137-147Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). Some new functional PAMs for B. halodurans type I-C CRISPR system have been identified using this method. In addition to DNA binding by dCas9, BE relies on the base modification enzyme, such as the cytosine deaminase or adenine deaminases (Gaudelli et al., 2017Gaudelli N.M. Komor A.C. Rees H.A. Packer M.S. Badran A.H. Bryson D.I. Liu D.R. Programmable base editing of A∗T to G∗C in genomic DNA without DNA cleavage.Nature. 2017; 551: 464-471Crossref PubMed Scopus (1212) Google Scholar; Komor et al., 2016Komor A.C. Kim Y.B. Packer M.S. Zuris J.A. Liu D.R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature. 2016; 533: 420-424Crossref PubMed Scopus (1726) Google Scholar), to modify the specific bases. None of these methods can directly reveal the gene editing results of BE, as well the potential risk of off-target editing, for different PAM sequences. Here, we developed a base editing-coupled survival screening method (BESS) and analyzed the PAM compatibility of SpdCas9 and its PAM-broadened variants in the BE system. Most importantly, because of enrichment and screening of the base-edited mutants with 5-fluorouracil (5-Fu), BESS is able to identify certain PAMs with less BE activity in high sensitivity, which is extremely helpful for studying the off-target effect of BE. To evaluate the high-sensitive PAM pattern, we designed a cytimidine BESS (simplified as “BESS”) (Figure 1A). In this method, the counter selective gene upp was modified into upp6tgg by replacing the sixth amino acid valine (encoded by GTC) with a tryptophan (encoded by TGG) (Figure S1). 5-Fu plate experiment demonstrated that the upp6tgg variant has the same 5-Fu sensitivity as the original upp gene (Figure S2). If the inserted TGG codon in upp6tgg mutated into TAA, TGA, or TAG that all encode termination codons by the cytimidine base editor (CBE) (Banno et al., 2018Banno S. Nishida K. Arazoe T. Mitsunobu H. Kondo A. Deaminase-mediated multiplex genome editing in Escherichia coli.Nat. Microbiol. 2018; 3: 423-429Crossref PubMed Scopus (72) Google Scholar), the translation of upp6tgg would be prematurely terminated and the strain will grow on 5-Fu screening plates (Figure 1B); otherwise, 5-Fu is lethal to the strain containing upp6tgg (Andersen et al., 1992Andersen P.S. Smith J.M. Mygind B. Characterization of the upp-gene encoding Uracil phosphoribosyltransferase of escherichia-coli K12.Eur. J. Biochem. 1992; 204: 51-56Crossref PubMed Scopus (58) Google Scholar). To prove this concept, the CBE system consisting of the cytosine deaminase PmCDA1 fused with SpdCas9 (SpdCas9-CDA) was introduced into Escherichia coli strain GB05Red Δupp. In the absence of sgRNA, strain GB05Red Δupp with NGG-upp6tgg could only grow on LB plates without 5-Fu (Figure S2), and after importing sgRNA and inducing CBE, approximately 10% of the cells were able to grow on the 5-Fu plates (Figures 1C and S3). Sanger sequencing showed that 11 of 16 random selected colonies grown on 5-Fu plates contained C⋅G-to-T⋅A base conversation at the third position of TGG codon, and the remaining strains had CC in the antisense chain of TGG mutated to TT (Figure 1D). These demonstrated that BESS can edit NGG-upp6tgg and confer strain 5-Fu resistance. The major advantage of BESS is the efficient enrichment of base-edited mutants through 5-Fu screening. Functional PAMs, such as NGG, are able to edit upp6tgg and terminate its translation. However, invalid PAMs, such as TTT, cannot mutate upp6tgg (Figure 2A). The growth of Δupp strain was slightly impaired in 10 μg/mL of 5-Fu, while the wild-type strain was already unable to grow at the same 5-Fu concentration (Figures 2B and S4). This indicates that only strains carrying functional PAMs and causing nonsense mutation in upp6tgg can be enriched by 5-Fu screening. To evaluate the enrichment capability of BESS for the functional PAM sequences, strain GB05Red Δupp harboring CBE and upp6tgg with the canonical NGG PAM was mixed with the same strain but containing an invalid TTT PAM sequence at a ratio of 1:15. Besides, to visually distinguish the NGG PAM strain, the TTT PAM strain was co-expressed with a red fluorescent protein (Mkate) (Figure 2C). This model can reveal the screening effectiveness of BESS if only NGG in the NNN random PAM library, 1/16 of the total, is able to inactivate upp6tgg. When the mixture was induced, the percentage of NGG PAM strain identified by colony counting increased from 7.3% to 28.1% after 24 hr of cultivation in liquid medium with 10 μg/ul of 5-Fu. After harvesting the colonies for another round of enrichment, the proportion of NGG PAM strain increased to 67.6%, 9-fold enrichment compared to the starting mixture (Figure 2D). To further investigate the sensitivity of BESS, the ratio of functional PAM strain to invalid PAM strain was set to 1:99. With CBE and 5-Fu screening, the proportion of functional PAM strain increased from 1.0% to 6.8%, and after a second round of screening, the proportion even increased to 55.2%, 55 folds enriched than the initial proportion (Figure 2D). This proved that BESS can be used to efficiently enrich strains with functional PAM sequences in the BE system. The above results showed that BESS can be used to enrich and evaluate the activity of functional PAMs. As the most prevalent Cas effector protein used in the BE system (Rees and Liu, 2018Rees H.A. Liu D.R. Base editing: precision chemistry on the genome and transcriptome of living cells.Nat. Rev. Genet. 2018; 19: 770-788Crossref PubMed Scopus (471) Google Scholar; Zhao et al., 2020Zhao D.D. Li J. Li S.W. Xin X. Hu M.Z. Price M.A. Rosser S.J. Bi C.H. Zhang X.L. Glycosylase base editors enable C-to-A and C-to-G base changes.Nat. Biotechnol. 2020; 39: 35-40Crossref PubMed Scopus (53) Google Scholar), the PAM compatibility of SpdCas9 was first subjected to BESS screening. To do that, NNNN PAM libraries containing 256 various PAM sequences were constructed ahead the target site of upp6tgg, forming 6TGG PAM library. Four nucleotides were introduced between the Shine Dalgarno region and the coding sequence, which possibly impacts the translation of upp6tgg. We co-expressed a β-lactamase in tandem with upp6tgg during the construction of the PAM library and balanced the impact of the inserted sequence on translation by imposing ampicillin pressure. The NGS sequencing results showed that the inserted NNNN sequences did not cause significant negative consequences on gene expression, and the PAM sequences were normally distributed in the initial PAM library after ampicillin selection (Figure S5). Then, 6TGG PAM library was introduced into the GB05Red Δupp strain for BESS. After two rounds of screening, the 152 bp DNA fragment containing the PAM and sgRNA targeting sequence was PCR amplified for high-throughput sequencing (Figure 1A). For the first round of enrichment, an average of 19.8% sequencing reads appeared C⋅G-to-T⋅A base conversion at the TGG codon. After second round of enrichment, the proportion increased to 37.2% (Figure 3A). Among these mutant reads, TGG mutate to TGA accounted for the highest proportion, followed by TAA, and TAG was the least (Figure 3B). Besides, there were no significant changes in the mutation patterns as the rounds of enrichment increased. These results are consistent with the previously reported one that cytosine at the 18th of sgRNA is more efficient relative to the 17th in the CBE system (Nishida et al., 2016Nishida K. Arazoe T. Yachie N. Banno S. Kakimoto M. Tabata M. Mochizuki M. Miyabe A. Araki M. Hara K.Y. et al.Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.Science. 2016; 353: aaf8729Crossref PubMed Scopus (565) Google Scholar). In the two biological replicates, there were 122 and 118 PAMs detected with more than 1% of sequencing reads carrying the C⋅G-to-T⋅A base conversion on the TGG codon (Figure 3C). That means almost half of PAMs in the NNNN PAM library can undergo BE. This amount is far beyond the original consensus that SpCas9 only recognizes a few PAM sequences such as NGG, NAG, and NGA. Although the number of functional PAMs did not increase with the number of BESS rounds, the overall percentage of the base-edited reads for these functional PAMs significantly increased after the second round of BESS enrichment (Figure 3C). These results demonstrated that BESS can effectively enrich functional PAMs with BE activity among the PAM library, and as the rounds of BESS screening increased, the BE efficiency of these PAMs would be further improved. After two rounds of BESS, 37 and 42 PAMs were enriched in the two duplicate PAM libraries, respectively (Figure 3D). These enriched PAM sequences included all of the 16 NGGN sequences, 14 NAGN sequences, 4 NGAN, and 6 other sequences (Figures 3E and S6, Data S1). Among them, all NGG sequences had the enrichment factors greater than 5 and BE efficiencies higher than 77%, indicating that NGG was the most effective PAM sequence for SpdCas9 in the BE system (Figures 3E and S6). In addition, we found that four of the six other PAM sequences can be categorized into NNGG, and the BE efficiency of ACGG, TCGG, and TTGG even exceeded 70% (Figure 3E). To further confirm this result, upp6tgg with the ACGG, TTGG, or GTGG PAM sequence was separately constructed for BE experiment. After 24 hr of induction, nonsense mutations of upp6tgg were observed in these strains, demonstrating that certain NNGG sequences enabled to support SpdCas9 for BE (Figure S7). It has been reported that SpCas9 was able to utilize NNGG PAM to cleave a certain target DNA with limited activity in bacteria and eukaryotic cells and had the potential off-target editing risk (Jiang et al., 2013Jiang W. Bikard D. Cox D. Zhang F. Marraffini L.A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Nat. Biotechnol. 2013; 31: 233-239Crossref PubMed Scopus (1455) Google Scholar; Kim et al., 2020Kim H.K. Lee S. Kim Y. Park J. Min S. Choi J.W. Huang T.P. Yoon S. Liu D.R. Kim H.H. High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells.Nat. Biomed. Eng. 2020; 4: 111-124Crossref PubMed Scopus (35) Google Scholar). BESS screening revealed that all NNGG sequences could serve as functional PAMs for BE (Figure 4A). In addition to the well-known functional NAGG and NGGG PAMs, all NTGG and NCGG have more than 20% of sequencing reads that showed C⋅G-to-T⋅A base conversion. In particular, the BE efficiency of ATGG even reached 91%, which is higher than that of the canonical NGG PAM. This result implied that SpdCas9 appeared to be able to tolerate one base backward shift of the core NGG PAM. Since BE relies on the DNA binding capacity of dCas9, we investigated the performance of NNGG as the PAM sequences in CRISPRi assay that also only require DNA binding of dCas9. CRISPRi experiment also demonstrated that most of the NNGG sequences, except GCGG and CAGG, were able to repress the expression of gfp in varying degrees (Figure S8). Importantly, we found that one base forward of the NGGN PAM, namely GGNN, was capable of converting cytosine to thymine at the TGG codon as well (Figure 4B). Although GGCN and GGTN were less efficient than GGAN and GGGN, but with the exception of GGTT and GGTG, all of the remaining GGNN PAMs showed C⋅G-to T⋅A-base conversation in more than 20% of the sequencing reads. These demonstrated that SpdCas9 can actually tolerate one base forward or backward shift of the NGG PAM in the BE system. The above BESS results showed that in addition to NGG, SpdCas9 can utilize a number of other PAMs, such as NAG, NGA, NNGG, and GGNN, for efficient BE. However, these 65 PAMs only account for about half of the BESS-identified PAMs that with BE activity (Figure 3C). There are still a large number of previously unnoticed potential PAM sequences that have been identified by BESS screening (Data S1). In order to comprehensively investigate the PAM pattern of SpdCas9, we plotted the BE efficiency heatmap for the 256 different PAM sequences (Figure 5A). Sequence logos indicated that the best PAM for SpdCas9 identified by BESS was NGG (Figure S9), which was consistent with the previous reported one (Jiang et al., 2013Jiang W. Bikard D. Cox D. Zhang F. Marraffini L.A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Nat. Biotechnol. 2013; 31: 233-239Crossref PubMed Scopus (1455) Google Scholar; Mojica et al., 2009Mojica F.J.M. Diez-Villasenor C. Garcia-Martinez J. Almendros C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system.Microbiology. 2009; 155: 733-740Crossref PubMed Scopus (880) Google Scholar). We found that the PAM sequences of NGCB (B = G/T/C), NTGN, NCGY (Y = T/C), GTCT, TTTC, and AAAG also allowed SpdCas9 to edit of upp6tgg at different degrees (Figure 5A). The editing efficiency of certain NTGN PAMs was even comparable to that of the canonical NGG PAM, with a maximum of about 60% (Figure 5B). It is worth to note that these PAM sequences have never been reported to support SpdCas9 for BE. The PAM recognition properties of SpCas9 have been extensively studied with various methods (Karvelis et al., 2015Karvelis T. Gasiunas G. Young J. Bigelyte G. Silanskas A. Cigan M. Siksnys V. Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements.Genome Biol. 2015; 16: 253Crossref PubMed Scopus (109) Google Scholar; Leenay et al., 2016Leenay R.T. Maksimchuk K.R. Slotkowski R.A. Agrawal R.N. Gomaa A.A. Briner A.E. Barrangou R. Beisel C.L. Identifying and visualizing functional PAM diversity across CRISPR-cas systems.Mol. Cell. 2016; 62: 137-147Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar). We thus intended to investigate whether a similar pattern was evidenced in the previously published data sets. The most widely used plasmid clearance experiment showed significant depletion of NGG in the library, and certain NAG and NNGG sequences also had some degree of depletion (Jiang et al., 2013Jiang W. Bikard D. Cox D. Zhang F. Marraffini L.A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Nat. Biotechnol. 2013; 31: 233-239Crossref PubMed Scopus (1455) Google Scholar) (Figure S10). However, other potential PAMs identified by BESS could not be captured in this DNA cleavage-based negative selection method. Recently, the BE activity of SpdCas9 with various PAM sequences has been extensively characterized in 46 sgRNA target sites (Miller et al., 2020Miller S.M. Wang T. Randolph P.B. Arbab M. Shen M.W. Huang T.P. Matuszek Z. Newby G.A. Rees H.A. Liu D.R. Continuous evolution of SpCas9 variants compatible with non-G PAMs.Nat. Biotechnol. 2020; 38: 471-481Crossref PubMed Scopus (92) Google Scholar). Due to the lack of selection and enrichment for the BE events, only 4.4% of sequencing reads on average yielded C⋅G-to-T⋅A base conversation at the target sites, much lower than the 37.2% of overall BE frequency in the second round of BESS (Figure 3A). Comprehensive evaluation of the PAM pattern of these 46 sgRNA target sites revealed that NGCG and GTGN had weak BE activity, which is consistent with our results identified by BESS (Figures 5A and S10). However, most previously unnoticed PAMs were not captured in this PAM library but identified by BESS, possibly due to the weak sensitivity to the low-activity PAMs. To validate the PAM pattern obtained by BESS, two potential PAMs, GTGC and GTGT, with the highest base conversion efficiency among NTGN sequence (Figure 5B), were individually placed in front of upp6tgg and used to demonstrate the BE activity. After 12 hr of incubation, colonies emerged on the 5-Fu plates. Sanger sequencing demonstrated that both of GTGC and GTGT had actually supported SpdCas9 to generate C⋅G-to-T⋅A base conversion on the TGG codon of upp6tgg (Figure 5D). This is the first report that certain NTGN sequences can serve as PAMs for SpdCas9 to base edit the target DNA. Notably, CRISPRi experiments showed that neither GTGT nor GTGC could support SpdCas9 for efficiently repressing the transcription of target gene (Figure 5C). The results suggested that certain potential PAMs might only activate SpdCas9-CDA to transiently, rather than persistently, bind the target DNA and convert C⋅G to T⋅A in some particular conformations. This is why other PAM determination methods fail to identify these potential PAMs that only support BE. To further demonstrate the effectiveness of the identified PAMs, sgRNA sequences with different PAMs were designed to introduce the termination codon into the genomic lacZ gene through the C⋅G-to-T⋅A base conversion by BE. We constructed a total of nine sgRNA sites for the three major groups of PAMs identified by BESS (Figure S11), including the NGCB PAMs (TGCT, CGCC, TGCC), NTGN PAMs (ATGT, GTGC, CTGC), and NCGY PAMs (GCGC, CCGC, CCGT). All the sgRNA sites were able to efficiently inactivate lacZ in the E. coli MG1655 strain as shown by blue-white screening (Figure S12) and Sanger sequencing proved that the expected cytosines in the lacZ-inactivated strains were converted to thymines (Figure S13). Recently, more and more Cas9 variants with broader PAM recognition capabilities have been developed and used in BE (Hu et al., 2018Hu J.H. Miller S.M. Geurts M.H. Tang W. Chen L. Sun N. Zeina C.M. Gao X. Rees H.A. Lin Z. et al.Evolved Cas9 variants with broad PAM compatibility and high DNA specificity.Nature. 2018; 556: 57-63Crossref PubMed Scopus (668) Google Scholar; Nishimasu et al., 2018Nishimasu H. Shi X. Ishiguro S. Gao L. Hirano S. Okazaki S. Noda T. Abudayyeh O.O. Gootenberg J.S. Mori H. et al.Engineered CRISPR-Cas9 nuclease with expanded targeting space.Science. 2018; 361: 1259-1262Crossref PubMed Scopus (370) Google Scholar; Walton et al., 2020Walton R.T. Christie K.A. Whittaker M.N. Kleinstiver B.P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants.Science. 2020; 368: 290-296Crossref PubMed Scopus (209) Google Scholar). To further confirm the effectiveness of BESS, PAM patterns of t" @default.
• W3173166568 created "2021-07-05" @default.
• W3173166568 creator A5024696419 @default.
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• W3173166568 title "Base editing-coupled survival screening enabled high-sensitive analysis of PAM compatibility and finding of the new possible off-target" @default.
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