High-throughput analysis of the activities of xCas9, SpCas9-NG and SpCas9 at matched and mismatched target sequences in human cells
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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DOI：
10.1038/s41551-019-0505-1
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发表时间：
2020-01-14
影响因子：
28.1
通讯作者：
Kim, Hyongbum Henry
中科院分区：
文献类型：
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作者：
Kim, Hui Kwon;Lee, Sungtae;Kim, Hyongbum Henry
在人类细胞中的内源性位点处，化脓链球菌Cas9变体的原型间隔子邻近基序的相容性以及在靶和脱靶活性的比较使得能够编辑与遗传疾病相关的新基因组位点基于成簇的规则间隔短回文重复序列（CRISPR）的基因组编辑的应用可能受到缺乏相容的原型间隔子邻近基序（PAM）的限制，靶向活性不足和脱靶效应。在这里，我们报告了来自化脓链球菌（SpCas 9）的Cas9和SpCas 9变体xCas 9和SpCas 9-NG（已知其具有比SpCas 9更宽的PAM相容性）在人细胞中的26，478个慢病毒整合靶序列和78个内源靶位点处的PAM序列相容性以及靶向和脱靶活性的广泛比较。我们发现xCas 9对错配靶序列的耐受性最低，而SpCas 9-NG具有最广泛的PAM相容性。我们还表明，基于新鉴定的非NGG PAM序列，SpCas 9-NG和SpCas 9可以编辑与遗传疾病相关的六个先前未编辑的内源性位点。此外，我们还提供了深度学习模型，可以预测xCas 9和SpCas 9-NG在靶序列上的活性。由此产生的对xCas 9、SpCas 9-NG和SpCas 9在人类细胞中的活性的更深入理解应该有助于它们的使用。
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.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.