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High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells
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High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells
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High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells
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Journal Article
High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells
2020
Overview
The applications of clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing can be limited by a lack of compatible protospacer adjacent motifs (PAMs), insufficient on-target activity and off-target effects. Here, we report an extensive comparison of the PAM-sequence compatibilities and the on-target and off-target activities of Cas9 from
Streptococcus pyogenes
(SpCas9) and the SpCas9 variants xCas9 and SpCas9-NG (which are known to have broader PAM compatibility than SpCas9) at 26,478 lentivirally integrated target sequences and 78 endogenous target sites in human cells. We found that xCas9 has the lowest tolerance for mismatched target sequences and that SpCas9-NG has the broadest PAM compatibility. We also show, on the basis of newly identified non-NGG PAM sequences, that SpCas9-NG and SpCas9 can edit six previously unedited endogenous sites associated with genetic diseases. Moreover, we provide deep-learning models that predict the activities of xCas9 and SpCas9-NG at the target sequences. The resulting deeper understanding of the activities of xCas9, SpCas9-NG and SpCas9 in human cells should facilitate their use.
A comparison of compatibilities in protospacer adjacent motifs and of on-target and off-target activities of
Streptococcus pyogenes
Cas9 variants at endogenous sites in human cells enables the editing of new genomic sites associated with genetic diseases.
Publisher
Nature Publishing Group UK,Nature Publishing Group
