Response to "Unexpected mutations after CRISPR-Cas9 editing in vivo".
Response to "Unexpected mutations after CRISPR-Cas9 editing in vivo".
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DOI：
10.1038/nmeth.4541
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发表时间：
2018-04
期刊：
影响因子：
48
通讯作者：
Pinello L
中科院分区：
文献类型：
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作者：
Lareau CA;Clement K;Hsu JY;Pattanayak V;Joung JK;Aryee MJ;Pinello L
致编辑：Schaefer等人1最近指出，CRISPR-Cas9核酸酶可以在基因组位点诱导脱靶改变，这些基因组位点与预期的靶位点不相似。这种新提出的CRISPR-Cas9脱靶活性与先前发表的工作（主要在细胞中进行，但也在小鼠中进行）相反，如果作者是正确的，可能对研究和治疗应用产生深远的影响。然而，我们在这里证明了Schaefer等人1中数据的最简单解释是，两只CRISPR-Cas9处理的小鼠在遗传上比对照小鼠更密切相关。这强烈表明，所谓的“意外突变”只是代表了核酸酶处理前存在的共享单核苷酸多态性（SNP）和插入缺失。Schaefer等人1的结论，即基因组编辑的F03和F05小鼠共有的序列变体（并且在对照未处理的FVB小鼠中未发现）是由CRISPR-Cas9引起的，这一结论关键地取决于所有这些小鼠最初在遗传上是相同的假设。如果这种克隆性假设是正确的，那么可以预期所有三只小鼠在dbSNP中发现的常见变体方面应该几乎相同（图1a中所示的假设结果）。然而，在用GATK最佳实践对这些小鼠进行基因分型后，我们在dbSNP位点上总共鉴定出31，079个高质量变异，这些变异在两只小鼠中一致，但在检查所有可能的成对组合时与第三只小鼠不同（图1b和补充说明1）。此外，每只小鼠中33-46%的这些高置信度基因分型变体是杂合的（补充表1），作者认为在高度近交系小鼠中不应该是这种情况7。因此，这三只小鼠既不是克隆的，也不是完全同基因的。即使在一个更现实和宽松的等距离模型下，允许罕见和私人突变（图1c），我们的重新分析仍然揭示了F03和F05小鼠在遗传上比对照小鼠更接近
To the Editor: Schaefer et al. 1 recently stated that CRISPR–Cas9 nuclease can induce off-target alterations at genomic loci that do not resemble the intended on-target site. This new proposed CRISPR–Cas9 off-target activity runs contrary to previously published work (performed mostly in cells, but also in mice) 2–6 and, if the authors are correct, could have profound implications for research and therapeutic applications. However, we here demonstrate that the simplest interpretation of data in Schaefer et al. 1 is that the two CRISPR–Cas9-treated mice are genetically more closely related to each other than to the control mouse. This strongly suggests that the so-called ‘unexpected mutations’ simply represent shared single-nucleotide polymorphisms (SNPs) and indels that existed before nuclease treatment. The conclusion of Schaefer et al. 1 that the sequence variants shared by the genome-edited F03 and F05 mice (and not found in the control untreated FVB mouse) are caused by CRISPR–Cas9 critically depends upon the assumption that all of these mice were initially genetically identical. If this clonality assumption were true, one would expect that all three mice should be nearly identical for common variants found in dbSNP (a hypothetical result represented in Fig. 1a). However, after genotyping these mice with GATK best practices, we identified a total of 31,079 high-quality variants at dbSNP loci that were concordant in two mice but distinct from the third when examining all possible pairwise combinations (Fig. 1b and Supplementary Note 1). Furthermore, 33–46% of these highconfidence-genotyped variants in each mouse are heterozygous (Supplementary Table 1), which the authors have argued should not be the case in highly inbred mice7. Thus, the three mice are neither clonal nor completely isogenic. Even under a more realistic and relaxed equal-distance model that allows rare and private mutations (Fig. 1c), our reanalysis still reveals that the F03 and F05 mice are genetically more closely related to each other than to the control
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影响因子：
46.9
作者：
Tsai, Shengdar Q.;Zheng, Zongli;Nguyen, Nhu T.;Liebers, Matthew;Topkar, Ved V.;Thapar, Vishal;Wyvekens, Nicolas;Khayter, Cyd;Iafrate, A. John;Le, Long P.;Aryee, Martin J.;Joung, J. Keith
通讯作者：
Joung, J. Keith
影响因子：
48
作者：
Kim, Daesik;Bae, Sangsu;Kim, Jin-Soo
通讯作者：
Kim, Jin-Soo
影响因子：
16
作者：
van Overbeek, Megan;Capurso, Daniel;May, Andrew P.
通讯作者：
May, Andrew P.
影响因子：
23.9
作者：
Veres, Adrian;Gosis, Bridget S.;Ding, Qiurong;Collins, Ryan;Ragavendran, Ashok;Brand, Harrison;Erdin, Serkan;Cowan, Chad A.;Talkowski, Michael E.;Musunuru, Kiran
通讯作者：
Musunuru, Kiran
影响因子：
46.9
作者：
通讯作者：
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