Low incidence of SNVs and indels in trio genomes of Cas9-mediated multiplex edited sheep.
Low incidence of SNVs and indels in trio genomes of Cas9-mediated multiplex edited sheep.
复制标题
Cas9 介导的多重编辑绵羊三基因组中 SNV 和插入缺失的发生率较低
DOI：
10.1186/s12864-018-4712-z
复制
发表时间：
2018-05-25
期刊：
影响因子：
4.4
通讯作者：
Chen Y
中科院分区：
文献类型：
--
作者：
Wang X;Liu J;Niu Y;Li Y;Zhou S;Li C;Ma B;Kou Q;Petersen B;Sonstegard T;Huang X;Jiang Y;Chen Y
背景CRISPR/Cas9系统的简单性使其在建立动物模型、功能基因组筛选以及治疗遗传和传染病方面得到了广泛的应用。然而，非靶标CRISPR/Cas9核酸酶活性产生的意外突变可能会导致负面后果。尤其是最近的一项研究发现，基因编辑可以将数百种意想不到的突变引入基因组，并引起了广泛关注。结果为了解决人们对非靶标的担忧，迫切需要对CRISPR/Cas9介导的非靶标突变进行表征。在这里，我们利用我们之前产生的基因编辑的绵羊，进行了基于家族三元组的全基因组测序，能够区分编辑的后代中的变异，这些变异是遗传的、自然产生的或通过基因改造诱导的。在较高的基因组覆盖深度(~ 25.8x)下对三个家系进行了重新测序。在建立了一条管道来综合分析基因组中从头开始的单核苷酸变异、插入和结构变异的序列数据后，我们只发现了一个由MSTN基因座上两个靶点之间的sgRNA靶点之间的特异性双链断裂引起的2.4kb倒置的意外事件，发生率很低。结论我们首次报道了基于CRISPR的绵羊基因组修饰的保真度，该修饰同时针对三个编码序列位置的基因突变。基于TRIO的测序方法揭示了几乎可以忽略的脱靶修饰，为CRISPR/Cas9体内基因组编辑的安全应用提供了及时的证据。
BackgroundThe simplicity of the CRISPR/Cas9 system has enabled its widespread applications in generating animal models, functional genomic screening and in treating genetic and infectious diseases. However, unintended mutations produced by off-target CRISPR/Cas9 nuclease activity may lead to negative consequences. Especially, a very recent study found that gene editing can introduce hundreds of unintended mutations into the genome, and have attracted wide attention.ResultsTo address the off-target concerns, urgent characterization of the CRISPR/Cas9-mediated off-target mutagenesis is highly anticipated. Here we took advantage of our previously generated gene-edited sheep and performed family trio-based whole genome sequencing which is capable of discriminating variants in the edited progenies that are inherited, naturally generated, or induced by genetic modification. Three family trios were re-sequenced at a high average depth of genomic coverage (~ 25.8×). After developing a pipeline to comprehensively analyze the sequence data for de novo single nucleotide variants, indels and structural variations from the genome; we only found a single unintended event in the form of a 2.4 kb inversion induced by site-specific double-strand breaks between two sgRNA targeting sites at theMSTNlocus with a low incidence.ConclusionsWe provide the first report on the fidelity of CRISPR-based modification for sheep genomes targeted simultaneously for gene breaks at three coding sequence locations. The trio-based sequencing approach revealed almost negligible off-target modifications, providing timely evidences of the safe application of genome editing in vivo with CRISPR/Cas9.
登录
查看更多内容
影响因子：
12.3
作者：
Gao Y;Wu H;Wang Y;Liu X;Chen L;Li Q;Cui C;Liu X;Zhang J;Zhang Y
通讯作者：
Zhang Y
使用 CRISPR/Cas9 系统有效敲除山羊基因
影响因子：
3.7
作者：
Ni W;Qiao J;Hu S;Zhao X;Regouski M;Yang M;Polejaeva IA;Chen C
通讯作者：
Chen C
DOI：
10.1126/science.1252806
发表时间：
2014-06-06
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Jiang Y;Xie M;Chen W;Talbot R;Maddox JF;Faraut T;Wu C;Muzny DM;Li Y;Zhang W;Stanton JA;Brauning R;Barris WC;Hourlier T;Aken BL;Searle SMJ;Adelson DL;Bian C;Cam GR;Chen Y;Cheng S;DeSilva U;Dixen K;Dong Y;Fan G;Franklin IR;Fu S;Guan R;Highland MA;Holder ME;Huang G;Ingham AB;Jhangiani SN;Kalra D;Kovar CL;Lee SL;Liu W;Liu X;Lu C;Lv T;Mathew T;McWilliam S;Menzies M;Pan S;Robelin D;Servin B;Townley D;Wang W;Wei B;White SN;Yang X;Ye C;Yue Y;Zeng P;Zhou Q;Hansen JB;Kristensen K;Gibbs RA;Flicek P;Warkup CC;Jones HE;Oddy VH;Nicholas FW;McEwan JC;Kijas J;Wang J;Worley KC;Archibald AL;Cockett N;Xu X;Wang W;Dalrymple BP
通讯作者：
Dalrymple BP
影响因子：
12.3
作者：
Lawrenson T;Shorinola O;Stacey N;Li C;Østergaard L;Patron N;Uauy C;Harwood W
通讯作者：
Harwood W
影响因子：
64.8
作者：
通讯作者：
--