Synthesis of an arrayed sgRNA library targeting the human genome
Synthesis of an arrayed sgRNA library targeting the human genome
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DOI：
10.1038/srep14987
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发表时间：
2015-10-08
影响因子：
4.6
通讯作者：
Hornung, Veit
中科院分区：
文献类型：
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作者：
Schmidt, Tobias;Schmid-Burgk, Jonathan L.;Hornung, Veit
聚集规律间隔短回文重复序列（CRISPR）与CRISPR相关蛋白（Cas）结合，可用于在自定义位点上引入双站断裂到哺乳动物基因组中。Cas复合物的内切酶活性可以利用单个引导RNA （sgRNA）靶向特定的基因组区域。为了高效、无偏倚地生成大型sgRNA文库，我们开发了一种连接无关克隆（LIC）组装方法。使用该系统，我们执行了迭代霰弹枪克隆方法，以生成几乎每个蛋白质编码人类基因的一个关键外显子为目标的阵列sgRNA文库。采用正交混合和反卷积方法获得了19,506个独特的序列验证的sgrna（覆盖率为91.4%）。在HEK 293T细胞中测试，该文库构建物的基因组编辑活性中位数为54.6%，使用该文库的sgrna产生敲除细胞的19个基因中有19个成功。
Clustered regularly interspaced short palindromic repeats (CRISPR) in conjunction with CRISPR-associated proteins (Cas) can be employed to introduce double stand breaks into mammalian genomes at user-defined loci. The endonuclease activity of the Cas complex can be targeted to a specific genomic region using a single guide RNA (sgRNA). We developed a ligation-independent cloning (LIC) assembly method for efficient and bias-free generation of large sgRNA libraries. Using this system, we performed an iterative shotgun cloning approach to generate an arrayed sgRNA library that targets one critical exon of almost every protein-coding human gene. An orthogonal mixing and deconvolution approach was used to obtain 19,506 unique sequence-validated sgRNAs (91.4% coverage). As tested in HEK 293T cells, constructs of this library have a median genome editing activity of 54.6% and employing sgRNAs of this library to generate knockout cells was successful for 19 out of 19 genes tested.