Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification.
Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification.
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DOI：
10.1038/nbt.2909
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发表时间：
2014-06
影响因子：
46.9
通讯作者：
Liu, David R.
中科院分区：
文献类型：
--
作者：
Guilinger, John P.;Thompson, David B.;Liu, David R.
通过Cas9进行的基因组编辑，其在由短的单向导RNA（sgRNA）编程的序列处切割双链DNA，可能导致脱靶DNA修饰，这在某些应用中可能是有害的。为了提高DNA切割特异性，我们产生了无催化活性的Cas9和FokI核酸酶（fCas9）的融合物。fCas9的DNA切割需要两个fCas9单体的缔合，这两个fCas9单体同时结合相距约15或25个碱基对的靶位点。在人类细胞中，fCas9修饰的靶DNA位点的特异性比野生型Cas9高>140倍，并且效率类似于成对的Cas9“切口”，最近工程化的变体每个单体仅切割一条DNA链。在具有高度相似的脱靶位点的基因座处，fCas9的特异性比配对切口酶的特异性高至少4倍。符合fCas9底物要求的靶位点在人类基因组中平均每34 bp出现一次，表明这种方法对于高度特异性的全基因组编辑具有广泛的通用性。
Genome editing by Cas9, which cleaves double-stranded DNA at a sequence programmed by a short single-guide RNA (sgRNA), can result in off-target DNA modification that may be detrimental in some applications. To improve DNA cleavage specificity, we generated fusions of catalytically inactive Cas9 and FokI nuclease (fCas9). DNA cleavage by fCas9 requires association of two fCas9 monomers that simultaneously bind target sites ~15 or 25 base pairs apart. In human cells, fCas9 modified target DNA sites with >140-fold higher specificity than wild-type Cas9 and with an efficiency similar to that of paired Cas9 ‘nickases’, recently engineered variants that cleave only one DNA strand per monomer. The specificity of fCas9 was at least 4-fold higher_than that of paired nickases at loci with highly similar off-target sites. Target sites that conform to the substrate requirements of fCas9 occur on average every 34 bp in the human genome, suggesting the broad versatility of this approach for highly specific genome-wide editing.
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