CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles
CRISPR/Cas9 cleavages in budding yeast reveal templated insertions and strand-specific insertion/deletion profiles
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DOI：
10.1073/pnas.1716855115
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发表时间：
2018-02-27
影响因子：
11.1
通讯作者：
Haber, James E.
中科院分区：
文献类型：
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作者：
Lemos, Brenda R.;Kaplan, Adam C.;Haber, James E.
利用CRISPR-Cas9技术提供了前所未有的能力，通过DNA双链断裂(DSB)的诱导和修复来修改基因组座位。我们分析了Cas9在芽殖酵母中诱导的非同源末端连接(NHEJ)修复，发现Cas9与其引导RNA(GRNA)结合的方向深刻地影响了裂解部位的插入/缺失(Indels)模式。由Cas9创建的一种常见的Indel是1-BP(+1)插入，似乎是由于Cas9产生了由DNA聚合酶填充并连接的1-NT 5‘端悬垂。通过使用两个gRNA来研究+1插入的起源，其中PAM序列位于相反的DNA链上，但设计为切割相同的序列。这些模板化的+1插入依赖于X家族DNA聚合酶Pol4。删除Pol4也消除了+2和+3插入，这两个插入偏向于同源核苷酸插入。利用倒置的PAM序列，我们还发现总体NHEJ效率和修复谱有显著差异，表明Cas9：gRNA复合体的结合影响了后续的NHEJ加工。与位点特异性HO内切酶诱导的事件一样，CRISPR-Cas9介导的NHEJ修复依赖于Ku异源二聚体和DNA连接酶4。Cas9事件高度依赖于Mre11-Rad50-Xrs2复合体，与Mre11‘S核酸酶活性无关。对哺乳动物细胞中大量Cas9切割事件的结果的检查发现，人类细胞中+1插入的模板来源相似，但类似模板+2插入的频率也很高。
Harnessing CRISPR-Cas9 technology provides an unprecedented ability to modify genomic loci via DNA double-strand break (DSB) induction and repair. We analyzed nonhomologous end-joining (NHEJ) repair induced by Cas9 in budding yeast and found that the orientation of binding of Cas9 and its guide RNA (gRNA) profoundly influences the pattern of insertion/deletions (indels) at the site of cleavage. A common indel created by Cas9 is a 1-bp (+1) insertion that appears to result from Cas9 creating a 1-nt 5' over-hang that is filled in by a DNA polymerase and ligated. The origin of +1 insertions was investigated by using two gRNAs with PAM sequences located on opposite DNA strands but designed to cleave the same sequence. These templated +1 insertions are dependent on the X-family DNA polymerase, Pol4. Deleting Pol4 also eliminated +2 and +3 insertions, which are biased toward homonucleotide insertions. Using inverted PAM sequences, we also found significant differences in overall NHEJ efficiency and repair profiles, suggesting that the binding of the Cas9: gRNA complex influences subsequent NHEJ processing. As with events induced by the site-specific HO endonuclease, CRISPR-Cas9-mediated NHEJ repair depends on the Ku heterodimer and DNA ligase 4. Cas9 events are highly dependent on the Mre11-Rad50-Xrs2 complex, independent of Mre11's nuclease activity. Inspection of the outcomes of a large number of Cas9 cleavage events in mammalian cells reveals a similar templated origin of +1 insertions in human cells, but also a significant frequency of similarly templated +2 insertions.