CRISPR enzymes require a protospacer-adjacent motif (PAM) near the target cleavage site, constraining the sequences accessible for editing. In the present study, we combine protein motifs from several orthologs to engineer two variants of Streptococcus canis Cas9—Sc++ and a higher-fidelity mutant HiFi-Sc++—that have simultaneously broad 5′-NNG-3′ PAM compatibility, robust DNA-cleavage activity and minimal off-target activity. Sc++ and HiFi-Sc++ extend the use of CRISPR editing for diverse applications.
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All data needed to evaluate the conclusions in the paper are present in the paper and supplementary tables. All source data can be found at the National Center for Biotechnology Information’s Sequence Read Archive under the accession code PRJNA623032. Nuclease plasmids are available on Addgene.
Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 337, 816–821 (2012).
Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).
Barrangou, R. & Horvath, P. A decade of discovery: CRISPR functions and applications. Nat. Microbiol. 2, 17092 (2017).
Mojica, F. J., Díez-Villaseñor, C., García-Martínez, J. & Almendros, C. Short motif sequences determine the targets of the prokaryotic CRISPR defense system. Microbiology 155, 733–740 (2009).
Shah, S. A., Erdmann, S., Mojica, F. J. & Garrett, R. A. Protospacer recognition motifs: mixed identities and functional diversity. RNA Biol. 10, 891–899 (2013).
Sternberg, S. H., Redding, S., Jinek, M., Greene, E. C. & Doudna, J. A. DNA interrogation by the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–67 (2014).
Jiang, F. et al. RNA complex preorganized for target DNA recognition. Science 384, 1477–1481 (2015).
Komor, 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 533, 420–424 (2016).
Gaudelli, N. M. et al. Programmable base editing of A•T to G•C in genomic DNA without DNA cleavage. Nature 551, 464–471 (2017).
Richardson, C. D., Ray, G. J., DeWitt, M. A., Curie, G. L. & Corn, J. E. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat. Biotechnol. 34, 339–344 (2016).
Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).
Esvelt, K. M. et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat. Methods 520, 186–191 (2013).
Kim, E. et al. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat. Commun. 8, 14500 (2017).
Harrington, L. B. et al. A thermostable Cas9 with increased lifetime in human plasma. Nat. Commun. 8, 1424 (2017).
Edraki, A. et al. A compact, high accuracy Cas9 with a dinucleotide PAM for in vivo genome editing. Mol. Cell 73, 714–726 (2019).
Kleinstiver, B. P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).
Gao, L. et al. Engineered Cpf1 variants with altered PAM specificities. Nat. Biotechnol. 35, 789–792 (2017).
Ma, D. et al. Engineer chimeric Cas9 to expand PAM recognition based on evolutionary information. Nat. Commun. 10, 560 (2019).
Kleinstiver, B. P. et al. Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing. Nat. Biotechnol. 37, 276–282 (2019).
Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).
Nishimasu, H. et al. Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 361, 1259–1262 (2018).
Chatterjee, P., Jakimo, N. & Jacobson, J. M. Minimal PAM specificity of a highly similar SpCas9 ortholog. Sci. Adv. 4, eaau0766 (2018).
Hua, K., Tao, X., P. Han, P., Wang, R. & Zhu, J. K. Genome engineering in rice using Cas9 variants that recognize NG PAM sequences. Mol. Plant 12, 1003–1014 (2019).
Zhong, Z. et al. Improving plant genome editing with high-fidelity xCas9 and non-canonical PAM-targeting Cas9-NG. Mol. Plant 12, 1027–1036 (2019).
Guo, M. et al. Structural insights into a high fidelity variant of SpCas9. Cell Res. 29, 183–192 (2019).
Henikoff, S. & Henikoff, J. G. Amino acid substitution matrices from protein blocks. Proc. Natl Acad. Sci. USA 89, 10915–10919 (1992).
Chen, X., Zaro, J. & Shen, W. C. Fusion protein linkers: property, design and functionality. Adv. Drug Deliv. Rev. 65, 1357–1369 (2012).
Leenay, R. T. et al. Identifying and visualizing functional pam diversity across CRISPR-Cas systems. Mol. Cell 62, 137–147 (2016).
Crooks, G. E. et al. WebLogo: a sequence logo generator. Genome Res. 14, 1188–1190 (2004).
Wheeler, T. J., Clements, J. & Finn, R. D. Skylign: a tool for creating informative, interactive logos representing sequence alignments and profile hidden Markov models. BMC Bioinform. 15, 7 (2014).
Hsiau, T. et al. Inference of CRISPR edits from Sanger trace data. Preprint at bioRxiv https://doi.org/10.1101/251082 (2019).
Tsai, S. Q. et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat. Biotechnol. 33, 187–197 (2015).
Vakulskas, C. A. et al. A high-fidelity Cas9 mutant delivered as a ribonucleoprotein complex enables efficient gene editing in human hematopoietic stem and progenitor cells. Nat. Med. 24, 1216–1224 (2018).
Chen, J. S. et al. Enhanced proofreading governs CRISPR-Cas9 targeting accuracy. Nature 550, 407–410 (2017).
Clement, K. et al. CRISPResso2 provides accurate and rapid genome editing sequence analysis. Nat. Biotechnol. 37, 224–226 (2019).
Miller, S. M. et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nat. Biotechnol. 38, 471–481 (2020).
Walton, R. T., Christie, K. A., Whittaker, M. N. & Kleinstiver, B. P. Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants. Science, 368, 290–296 (2020).
Brinkman, E. K., Chen, T., Amendola, M. & Steensel, B. V. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014).
Rodriguez, T. C., Pratt, H. E., Liu, P., Amrani, N. & Zhu, L. J. GS-preprocess: containerized GUIDE-seq data analysis tools with diverse sequencer compatibility. Preprint at bioRxiv https://doi.org/10.1101/2020.01.26.914861 (2020).
Zhu, L. J. et al. GUIDEseq: a bioconductor package to analyze GUIDE-Seq datasets for CRISPR-Cas nucleases. BMC Genomics 18, 379 (2017).
We thank E. Boyden for access to cell culture, in addition to N. Gershenfeld and S. Zhang for shared lab equipment. We further thank A. Hennes and M. Topalli for technical assistance. This work was supported by the consortium of sponsors of the MIT Media Lab and the MIT Center for Bits and Atoms, as well as the CHDI Foundation. GUIDE-seq work was supported by a grant (no. GM115911) to E.J.S. from the US National Institutes of Health.
P.C., N.J. and J.M.J. are listed as inventors for US Patent Application no. 20190218532: Streptococcus canis Cas9 as a genome engineering platform with novel PAM specificity.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figs. 1–3.
SgRNA sequences used in this study, grouped by editing context. Primers used for genomic amplification are also specified.
Curated dataset of indel formation efficiencies at each site (annotated by target sequence, genomic locus and PAM), for Fig. 2a.
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Chatterjee, P., Jakimo, N., Lee, J. et al. An engineered ScCas9 with broad PAM range and high specificity and activity. Nat Biotechnol 38, 1154–1158 (2020). https://doi.org/10.1038/s41587-020-0517-0
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