Genome Editing with CRISPR-Cas9: Can It Get Any Better?
Genome Editing with CRISPR-Cas9: Can It Get Any Better?
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DOI：
10.1016/j.jgg.2016.04.008
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发表时间：
2016-05-20
期刊：
影响因子：
--
通讯作者：
Concordet JP
中科院分区：
文献类型：
--
作者：
Haeussler M;Concordet JP
CRISPR-Cas革命几乎发生在生命科学的所有领域。利用化脓性链球菌的CRISPR-Cas9系统进行DNA切割已被证明是非常简单和有效的，仅依赖于合成的单向导RNA（sgRNA）的设计及其与Cas9的共表达。本文综述了SgRNA的设计进展。化脓性链球菌和金黄色葡萄球菌Cas9（SpCas 9和SaCas 9）。用于全基因组鉴定脱靶的新测定为体内切割特异性问题提供了重要见解。与此同时，数千名导游的目标活动已经确定。这些数据导致了许多在线工具，这些工具有助于在靶序列中选择指导RNA。对于大多数基础研究应用，似乎可以通过基于计算预测仔细选择指导RNA来最大化切割活性并最小化脱靶。此外，最近对Cas蛋白的研究进一步提高了用于基因组编辑的CRISPR-Cas工具包的灵活性和精确性。受到与靶DNA结合的sgRNA-SpCas 9复合物的晶体结构的启发，最近设计了SpCas 9的几种变体，要么具有新型原型间隔区邻近基序（PAM），要么具有大幅减少的脱靶。新型Cas9和Cas9样蛋白Cpf 1也已从其他细菌中得到表征，并将受益于SpCas 9的见解。CRISPR-Cas9的基因组编辑也可能随着更好地理解和控制Cas9诱导的DNA切割后激活的细胞DNA修复途径而取得进展。
The CRISPR-Cas revolution is taking place in virtually all fields of life sciences. Harnessing DNA cleavage with the CRISPR-Cas9 system of Streptococcus pyogenes has proven to be extraordinarily simple and efficient, relying only on the design of a synthetic single guide RNA (sgRNA) and its co-expression with Cas9. Here, we review the progress in the design of sgRNA from the original dual RNA guide for S. pyogenes and Staphylococcus aureus Cas9 (SpCas9 and SaCas9). New assays for genome-wide identification of off-targets have provided important insights into the issue of cleavage specificity in vivo. At the same time, the on-target activity of thousands of guides has been determined. These data have led to numerous online tools that facilitate the selection of guide RNAs in target sequences. It appears that for most basic research applications, cleavage activity can be maximized and off-targets minimized by carefully choosing guide RNAs based on computational predictions. Moreover, recent studies of Cas proteins have further improved the flexibility and precision of the CRISPR-Cas toolkit for genome editing. Inspired by the crystal structure of the complex of sgRNA-SpCas9 bound to target DNA, several variants of SpCas9 have recently been engineered, either with novel protospacer adjacent motifs (PAMs) or with drastically reduced off-targets. Novel Cas9 and Cas9-like proteins called Cpf1 have also been characterized from other bacteria and will benefit from the insights obtained from SpCas9. Genome editing with CRISPR-Cas9 may also progress with better understanding and control of cellular DNA repair pathways activated after Cas9-induced DNA cleavage.
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影响因子：
46.9
作者：
Doench, John G.;Hartenian, Ella;Graham, Daniel B.;Tothova, Zuzana;Hegde, Mudra;Smith, Ian;Sullender, Meagan;Ebert, Benjamin L.;Xavier, Ramnik J.;Root, David E.
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