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Scalable characterization of the PAM requirements of CRISPR–Cas enzymes using HT-PAMDA

Russell T. Walton, Jonathan Y. Hsu, J. Keith Joung, Benjamin P. Kleinstiver

Published: 2021-02-04 DOI: 10.1038/s41596-020-00465-2

genome editing

CRISPR-Cas enzymes

HT-PAMDA

PAM preferences

scalable characterization

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Supplementary information

Supplementary Information

Supplementary Notes 1 and 2.

Reporting Summary

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References

Supplementary information

Makarova, K. S. et al. Evolutionary classification of CRISPR-Cas systems: a burst of class 2 and derived variants. Nat. Rev. Microbiol. 18, 67–83 (2020).
Anzalone, A. V., Koblan, L. W. & Liu, D. R. Genome editing with CRISPR–Cas nucleases, base editors, transposases and prime editors. Nat. Biotechnol. 38, 824–844 (2020).
Jinek, M. et al. A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).
Marraffini, L. A. & Sontheimer, E. J. Self vs. non-self discrimination during CRISPR RNA-directed immunity. Nature 463, 568–571 (2010).
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).
Gao, L. et al. Engineered Cpf1 variants with altered PAM specificities. Nat. Biotechnol. 35, 789–792 (2017).
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).
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).
Biswas, A., Gagnon, J. N., Brouns, S. J. J., Fineran, P. C. & Brown, C. M. CRISPRTarget. RNA Biol. 10, 817–827 (2013).
Mendoza, B. J. & Trinh, C. T. In silico processing of the complete CRISPR-Cas spacer space for identification of PAM sequences. Biotechnol. J. 13, 1700595 (2018).
Karvelis, T., Gasiunas, G. & Siksnys, V. Methods for decoding Cas9 protospacer adjacent motif (PAM) sequences: a brief overview. Methods 121–122, 3–8 (2017).
Mojica, F. J. M., Díez-Villaseñor, C., García-Martínez, J. & Almendros, C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 155, 733–740 (2009).
Karvelis, T. et al. Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements. Genome Biol. 16, 253 (2015).
Hirano, H. et al. Structure and engineering of Francisella novicida Cas9. Cell 164, 950–961 (2016).
Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).
Hsu, P. D. et al. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat. Biotechnol. 31, 827–832 (2013).
Kleinstiver, B. P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).
Zetsche, B. et al. Cpf1 Is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell 163, 759–771 (2015).
Kleinstiver, B. P. et al. Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAM recognition. Nat. Biotechnol. 33, 1293–1298 (2015).
Hu, J. H. et al. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature 556, 57–63 (2018).
Leenay, R. T. et al. Identifying and visualizing functional PAM diversity across CRISPR-Cas systems. Mol. Cell 62, 137–147 (2016).
Marshall, R. et al. Rapid and scalable characterization of CRISPR technologies using an E. coli cell-free transcription-translation system. Mol. Cell 69, 146–157.e3 (2018).
Miller, S. M. et al. Continuous evolution of SpCas9 variants compatible with non-G PAMs. Nat. Biotechnol. 38, 471–481 (2020).
Kim, H. K. et al. High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells. Nat. Biomed. Eng. 4, 111–124 (2020).
Ondov, B. D., Bergman, N. H. & Phillippy, A. M. Interactive metagenomic visualization in a web browser. BMC Bioinformatics 12, 385 (2011).
Collias, D. et al. A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9. Sci. Adv. 6, eabb4054 (2020).
Kim, D., Kim, D., Lee, G., Cho, S.-I. & Kim, J.-S. Genome-wide target specificity of CRISPR RNA-guided adenine base editors. Nat. Biotechnol. 37, 430–435 (2019).
Rohland, N. & Reich, D. Cost-effective, high-throughput DNA sequencing libraries for multiplexed target capture. Genome Res. 22, 939–946 (2012).
Tareen, A. & Kinney, J. B. Logomaker: beautiful sequence logos in Python. Bioinformatics 36, 2272–2274 (2020).

关键词

genome editing

CRISPR-Cas enzymes

HT-PAMDA

PAM preferences

scalable characterization

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