Genome-wide target specificities of CRISPR RNA-guided programmable deaminases
Genome-wide target specificities of CRISPR RNA-guided programmable deaminases
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DOI：
10.1038/nbt.3852
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发表时间：
2017-05-01
影响因子：
46.9
通讯作者：
Kim, Jin-Soo
中科院分区：
文献类型：
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作者：
Kim, Daesik;Lim, Kayeong;Kim, Jin-Soo
与Cas9连接的脱氨酶，也称为碱基编辑器，能够实现真核生物基因组中单个核苷酸的靶向突变。然而，它们的脱靶活性在很大程度上是未知的。在此，我们对消化基因组测序（Digenome - seq）进行改进，以评估由Cas9切口酶（nCas9）和人基因组中的脱氨酶APOBEC1组成的可编程脱氨酶的特异性。在体外，用碱基编辑器和DNA修饰酶混合物处理基因组DNA，以便在含尿嘧啶的位点产生DNA双链断裂（DSBs）。然后从全基因组测序数据中通过计算识别脱靶位点。通过测试七种不同的单向导RNA（sgRNAs），我们发现rAPOBEC1 - nCas9碱基编辑器具有高度特异性，对于每种sgRNA，在人基因组中仅在18±9个位点诱导胞嘧啶到尿嘧啶的转换。Digenome - seq足够灵敏，能够捕获替代频率为0.1%的脱靶位点。值得注意的是，碱基编辑器的脱靶位点通常与单独的Cas9的脱靶位点不同，这就需要对它们在全基因组范围内的特异性进行独立评估。
Cas9-linked deaminases, also called base editors, enable targeted mutation of single nucleotides in eukaryotic genomes. However, their off-target activity is largely unknown. Here we modify digested-genome sequencing (Digenome-seq) to assess the specificity of a programmable deaminase composed of a Cas9 nickase (nCas9) and the deaminase APOBEC1 in the human genome. Genomic DNA is treated with the base editor and a mixture of DNA-modifying enzymes in vitro to produce DNA double-strand breaks (DSBs) at uracil-containing sites. Off-target sites are then computationally identified from whole genome sequencing data. Testing seven different single guide RNAs (sgRNAs), we find that the rAPOBEC1-nCas9 base editor is highly specific, inducing cytosine-to-uracil conversions at only 18 +/- 9 sites in the human genome for each sgRNA. Digenome-seq is sensitive enough to capture off-target sites with a substitution frequency of 0.1%. Notably, off-target sites of the base editors are often different from those of Cas9 alone, calling for independent assessment of their genome-wide specificities.