Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes.
Double strand break repair by capture of retrotransposon sequences and reverse-transcribed spliced mRNA sequences in mouse zygotes.
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DOI：
10.1038/srep12281
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发表时间：
2015-07-28
影响因子：
4.6
通讯作者：
Ishino F
中科院分区：
文献类型：
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作者：
Ono R;Ishii M;Fujihara Y;Kitazawa M;Usami T;Kaneko-Ishino T;Kanno J;Ikawa M;Ishino F
CRISPR/Cas 系统在单个引导 RNA (sgRNA) 指定的基因组位点有效引入双链断裂 (DSB)。 DSB 随后通过非同源末端连接 (NHEJ) 或同源重组 (HR) 进行修复。在这里，我们证明通过 CRISPR/Cas 系统引入小鼠受精卵的 DSB 通过捕获来自逆转录转座子、基因组 DNA、mRNA 和 sgRNA 的 DNA 序列进行修复。在分析的 93 只小鼠中，57 只携带突变等位基因，其中 22 只在 DSB 引入位点有长从头插入；两个是没有内含子的 Pcnt 和 Inadl 的剪接 mRNA，表明涉及逆转录 (RT)。十五个等位基因包括逆转录转座子、mRNA 和其他没有 RT 证据的序列。另外两种是 sgRNA，其中一种含有 T7 启动子衍生序列，表明 PCR 产物是其起源。总之，在小鼠受精卵中鉴定出 RT 产物介导的 DSB 修复 (RMDR) 和非 RMDR 修复。我们还证实 RMDR 和非 RMDR 都发生在 CRISPR/Cas 转染的 NIH-3T3 细胞中。最后，由于 C57BL/6 小鼠中的两个从头 MuERV-L 插入在自然条件下被证明具有 RMDR 的特征，我们假设 RMDR 有助于进化过程中新 DNA 序列的出现。
The CRISPR/Cas system efficiently introduces double strand breaks (DSBs) at a genomic locus specified by a single guide RNA (sgRNA). The DSBs are subsequently repaired through non-homologous end joining (NHEJ) or homologous recombination (HR). Here, we demonstrate that DSBs introduced into mouse zygotes by the CRISPR/Cas system are repaired by the capture of DNA sequences deriving from retrotransposons, genomic DNA, mRNA and sgRNA. Among 93 mice analysed, 57 carried mutant alleles and 22 of them had long de novo insertion(s) at DSB-introduced sites; two were spliced mRNAs of Pcnt and Inadl without introns, indicating the involvement of reverse transcription (RT). Fifteen alleles included retrotransposons, mRNAs, and other sequences without evidence of RT. Two others were sgRNAs with one containing T7 promoter-derived sequence suggestive of a PCR product as its origin. In conclusion, RT-product-mediated DSB repair (RMDR) and non-RMDR repair were identified in the mouse zygote. We also confirmed that both RMDR and non-RMDR take place in CRISPR/Cas transfected NIH-3T3 cells. Finally, as two de novo MuERV-L insertions in C57BL/6 mice were shown to have characteristic features of RMDR in natural conditions, we hypothesize that RMDR contributes to the emergence of novel DNA sequences in the course of evolution.