Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency.
Optimizing sgRNA structure to improve CRISPR-Cas9 knockout efficiency.
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DOI：
10.1186/s13059-015-0846-3
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发表时间：
2015-12-15
期刊：
影响因子：
12.3
通讯作者：
Wu H
中科院分区：
文献类型：
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作者：
Dang Y;Jia G;Choi J;Ma H;Anaya E;Ye C;Shankar P;Wu H
单向导RNA（Single-guide RNA，sgRNA）是成簇规则间隔短回文重复序列（clustered regularly interspaced short palindromic repeats，CRISPR）-Cas9基因组编辑系统的两个关键组分之一。与天然细菌CRISPR RNA（crRNA）-反式激活crRNA（tracrRNA）双链体相比，目前常用的sgRNA结构具有缩短的双链体，并且含有胸腺嘧啶的连续序列，其是RNA聚合酶III的暂停信号，因此可能降低转录效率。在这里，我们系统地研究了这两个元件对敲除效率的影响，并表明通过延长双链体长度并将胸腺嘧啶连续序列的第四个胸腺嘧啶突变为胞嘧啶或鸟嘌呤来修饰sgRNA结构显著地，有时显着地提高了细胞中的敲除效率。此外，优化的sgRNA结构还显著提高了更具挑战性的基因组编辑程序的效率，例如基因缺失，这对于诱导非编码基因的功能丧失非常重要。通过对sgRNA结构的系统研究，我们发现将双链体延伸约5个碱基对，并将第4位的胸腺嘧啶连续序列突变为胞嘧啶或鸟嘌呤，可以显着提高基于CRISPR-Cas9的基因组编辑实验中的基因敲除效率。本文的在线版本（doi：10.1186/s13059-015-0846-3）包含补充材料，可供授权用户使用。
Single-guide RNA (sgRNA) is one of the two key components of the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing system. The current commonly used sgRNA structure has a shortened duplex compared with the native bacterial CRISPR RNA (crRNA)–transactivating crRNA (tracrRNA) duplex and contains a continuous sequence of thymines, which is the pause signal for RNA polymerase III and thus could potentially reduce transcription efficiency. Here, we systematically investigate the effect of these two elements on knockout efficiency and showed that modifying the sgRNA structure by extending the duplex length and mutating the fourth thymine of the continuous sequence of thymines to cytosine or guanine significantly, and sometimes dramatically, improves knockout efficiency in cells. In addition, the optimized sgRNA structure also significantly increases the efficiency of more challenging genome-editing procedures, such as gene deletion, which is important for inducing a loss of function in non-coding genes. By a systematic investigation of sgRNA structure we find that extending the duplex by approximately 5 bp combined with mutating the continuous sequence of thymines at position 4 to cytosine or guanine significantly increases gene knockout efficiency in CRISPR-Cas9-based genome editing experiments. The online version of this article (doi:10.1186/s13059-015-0846-3) contains supplementary material, which is available to authorized users.