Several Streptococcus pyogenes Cas9 (SpCas9) variants have been developed to improve an enzyme’s specificity or to alter or broaden its protospacer-adjacent motif (PAM) compatibility, but selecting the optimal variant for a given target sequence and application remains difficult. To build computational models to predict the sequence-specific activity of 13 SpCas9 variants, we first assessed their cleavage efficiency at 26,891 target sequences. We found that, of the 256 possible four-nucleotide NNNN sequences, 156 can be used as a PAM by at least one of the SpCas9 variants. For the high-fidelity variants, overall activity could be ranked as SpCas9 ≥ Sniper-Cas9 > eSpCas9(1.1) > SpCas9-HF1 > HypaCas9 ≈ xCas9 >> evoCas9, whereas their overall specificities could be ranked as evoCas9 >> HypaCas9 ≥ SpCas9-HF1 ≈ eSpCas9(1.1) > xCas9 > Sniper-Cas9 > SpCas9. Using these data, we developed 16 deep-learning-based computational models that accurately predict the activity of these variants at any target sequence.
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We have made the source code for DeepSpCas9variants and the custom Python scripts used for the indel frequency calculations available on Github at https://github.com/NahyeKim/DeepSpCas9variants and https://github.com/CRISPRJWCHOI/CRISPR_toolkit/tree/master/Indel_searcher_2.
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We would like to thank Younggwang Kim, S. Park and Younghye Kim for assisting with the experiments. This work was supported in part by the National Research Foundation of Korea (grants 2017R1A2B3004198 (to H.H.K.), 2017M3A9B4062403 (to H.H.K.) and 2018R1A5A2025079 (to H.H.K.)), Brain Korea 21 Plus Project (Yonsei University College of Medicine), the Institute for Basic Science (IBS-R026-D1) and the Korean Health Technology R&D Project, Ministry of Health and Welfare, Republic of Korea (grants HI17C0676 (to H.H.K.) and HI16C1012 (to S.-R.C. and H.H.K.)).
Yonsei University has filed a patent based on this work, in which N.K., H.K.K. and H.H.K. are the co-inventors (patent no. 10-2019-0127304).
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Supplementary Text, Supplementary Discussions 1 and 2, Supplementary Figs. 1–17 and Supplementary Notes 1–3.
Library A design and indel frequencies from the library.
Library B design and indel frequencies from the library.
Library C design and indel frequencies from the library.
Frequency of shuffling between sgRNA-encoding and barcode-target sequences.
Average indel frequencies associated with all possible 4-nt PAM sequences.
PAM compatibilities determined using the 30 fixed protospacers from library A and a wide range of protospacers from library B.
Indel frequencies at 30 perfectly matched target sequences used for analyzing specificity. Of 30 guide RNAs, 8 were selected.
Data sets used for the development and evaluation of DeepSpCas9variants.
Primer sequences used for experiments.
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Kim, N., Kim, H.K., Lee, S. et al. Prediction of the sequence-specific cleavage activity of Cas9 variants. Nat Biotechnol 38, 1328–1336 (2020). https://doi.org/10.1038/s41587-020-0537-9
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