Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9.
Efficient inversions and duplications of mammalian regulatory DNA elements and gene clusters by CRISPR/Cas9.
复制标题
通过 CRISPR/Cas9 对哺乳动物调控 DNA 元件和基因簇进行有效倒置和复制
DOI：
10.1093/jmcb/mjv016
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发表时间：
2015-08
影响因子：
5.5
通讯作者：
Wu Q
中科院分区：
文献类型：
--
作者：
Li J;Shou J;Guo Y;Tang Y;Wu Y;Jia Z;Zhai Y;Chen Z;Xu Q;Wu Q
人类基因组包含数百万个DNA调控元件和大量基因簇，其中大部分尚未经过实验测试。用合成的单向导RNA（sgRNA）编程的成簇的规则间隔短回文重复序列（CRISPR）/CRISPR相关核酸酶9（Cas9）作为几乎任何生物体中的基因组编辑方法出现。在这里，我们报告说，通过CRISPR与两个sgRNA可以很容易地在人类和小鼠基因组中实现靶向DNA片段的倒位和复制。具体来说，我们发现，在培养的人类细胞和小鼠中，可以产生大小从几十bp到几百kb的DNA片段的有效精确倒位。此外，DNA片段复制和缺失也可以通过CRISPR通过两个同源染色体（染色单体）上的Cas9诱导的双链断裂（DSB）之间的反式等位基因重组来产生。此外，可以检测到由Cas9与四种sgRNA诱导的原钙粘蛋白（Pcdh）基因簇的组合倒位和重复的连接。在小鼠中，我们通过CRISPR获得了具有精确倒位、重复和可变大小DNA片段缺失的等位基因的创始人。有趣的是，我们发现，非常有效的倒位介导的微同源介导的末端连接（MMEJ）通过短的反向重复序列。我们第一次证明了DNA片段倒位可以通过小鼠的生殖系传播。最后，我们将这种CRISPR方法应用于Pcdh α簇的一个调控元件，并发现了Pcdh γ簇成员调控的新作用。这种简单而有效的方法应该是有用的操纵哺乳动物基因组研究数以百万计的调控DNA元件以及大量的基因簇。
The human genome contains millions of DNA regulatory elements and a large number of gene clusters, most of which have not been tested experimentally. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) programed with a synthetic single-guide RNA (sgRNA) emerges as a method for genome editing in virtually any organisms. Here we report that targeted DNA fragment inversions and duplications could easily be achieved in human and mouse genomes by CRISPR with two sgRNAs. Specifically, we found that, in cultured human cells and mice, efficient precise inversions of DNA fragments ranging in size from a few tens of bp to hundreds of kb could be generated. In addition, DNA fragment duplications and deletions could also be generated by CRISPR through trans-allelic recombination between the Cas9-induced double-strand breaks (DSBs) on two homologous chromosomes (chromatids). Moreover, junctions of combinatorial inversions and duplications of the protocadherin (Pcdh) gene clusters induced by Cas9 with four sgRNAs could be detected. In mice, we obtained founders with alleles of precise inversions, duplications, and deletions of DNA fragments of variable sizes by CRISPR. Interestingly, we found that very efficient inversions were mediated by microhomology-mediated end joining (MMEJ) through short inverted repeats. We showed for the first time that DNA fragment inversions could be transmitted through germlines in mice. Finally, we applied this CRISPR method to a regulatory element of the Pcdhα cluster and found a new role in the regulation of members of the Pcdhγ cluster. This simple and efficient method should be useful in manipulating mammalian genomes to study millions of regulatory DNA elements as well as vast numbers of gene clusters.