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Sang-Yeon Seo, Seonwoo Min, Sungtae Lee, Jung Hwa Seo, Jinman Park, Hui Kwon Kim, Myungjae Song, Dawoon Baek, Sung-Rae Cho, and Hyongbum Henry Kim*
Massively Parallel Evaluation and Computational Prediction of the Activities and Specificities of 17 Small Cas9s
Nat. Methods, 20, 999–1009
Date: May 15, 2023

Recently, various small Cas9 orthologs and variants have been reported for use in in vivo delivery applications. Although small Cas9s are particularly suited for this purpose, selecting the most optimal small Cas9 for use at a specific target sequence continues to be challenging. Here, to this end, we have systematically compared the activities of 17 small Cas9s for thousands of target sequences. For each small Cas9, we have characterized the protospacer adjacent motif and determined optimal single guide RNA expression formats and scaffold sequence. High-throughput comparative analyses revealed distinct high- and low-activity groups of small Cas9s. We also developed DeepSmallCas9, a set of computational models predicting the activities of the small Cas9s at matched and mismatched target sequences. Together, this analysis and these computational models provide a useful guide for researchers to select the most suitable small Cas9 for specific applications.

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