CRISPR-Cas9 off-targeting assessment with nucleic acid duplex energy parameters.
CRISPR-Cas9 off-targeting assessment with nucleic acid duplex energy parameters.
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DOI：
10.1186/s13059-018-1534-x
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发表时间：
2018-10-26
期刊：
影响因子：
12.3
通讯作者：
Gorodkin J
中科院分区：
文献类型：
--
作者：
Alkan F;Wenzel A;Anthon C;Havgaard JH;Gorodkin J
CRISPR-Cas9 系统最近的实验工作表明，脱靶结合和切割是该系统所关注的问题，并且这高度依赖于所选的指导 RNA (gRNA) 设计。脱靶的计算预测已被提出作为繁琐的实验工作的一种有吸引力且更可行的替代方案。然而，对大量假定脱靶的准确评分对于计算脱靶评估的成功起着关键作用。我们提出了 Cas9-gRNA-DNA 复合物的近似结合能模型，该模型系统地结合了 RNA-RNA、DNA-DNA 和 RNA-DNA 双链体获得的能量参数。基于该模型，引入了两种用于 CRISPR-Cas9 应用中 gRNA 选择的新型脱靶评估方法：用于为预测脱靶分配置信度分数的 CRISPRoff 和用于测量 gRNA 特异性的 CRISPRspec。我们将这些方法与当前最先进的方法进行了基准测试，结果表明两者都与实验结果更加一致。此外，我们展示了重要的证据支持系统的靶向切割效率和特异性之间的反比关系，其中引入的结合能是关键组成部分。结合能的影响为脱靶机制的进一步研究提供了方向。 CRISPRoff 和 CRISPRspec 的性能可以在任何 CRISPR-Cas9 基因组编辑应用之前对 gRNA 选择进行更准确的脱靶评估。对于给定的 gRNA 序列或给定目标区域中的所有潜在 gRNA，可以通过我们的网络服务器 https://rth.dk/resources/crispr/ 进行基于 CRISPRoff 的脱靶预测和基于 CRISPRspec 的特异性评估。本文的在线版本 (10.1186/s13059-018-1534-x) 包含补充材料，可供授权用户使用。
Recent experimental efforts of CRISPR-Cas9 systems have shown that off-target binding and cleavage are a concern for the system and that this is highly dependent on the selected guide RNA (gRNA) design. Computational predictions of off-targets have been proposed as an attractive and more feasible alternative to tedious experimental efforts. However, accurate scoring of the high number of putative off-targets plays a key role for the success of computational off-targeting assessment. We present an approximate binding energy model for the Cas9–gRNA–DNA complex, which systematically combines the energy parameters obtained for RNA–RNA, DNA–DNA, and RNA–DNA duplexes. Based on this model, two novel off-target assessment methods for gRNA selection in CRISPR-Cas9 applications are introduced: CRISPRoff to assign confidence scores to predicted off-targets and CRISPRspec to measure the specificity of the gRNA. We benchmark the methods against current state-of-the-art methods and show that both are in better agreement with experimental results. Furthermore, we show significant evidence supporting the inverse relationship between the on-target cleavage efficiency and specificity of the system, in which introduced binding energies are key components. The impact of the binding energies provides a direction for further studies of off-targeting mechanisms. The performance of CRISPRoff and CRISPRspec enables more accurate off-target evaluation for gRNA selections, prior to any CRISPR-Cas9 genome-editing application. For given gRNA sequences or all potential gRNAs in a given target region, CRISPRoff-based off-target predictions and CRISPRspec-based specificity evaluations can be carried out through our webserver at https://rth.dk/resources/crispr/. The online version of this article (10.1186/s13059-018-1534-x) contains supplementary material, which is available to authorized users.
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影响因子：
48
作者：
Anderson KR;Haeussler M;Watanabe C;Janakiraman V;Lund J;Modrusan Z;Stinson J;Bei Q;Buechler A;Yu C;Thamminana SR;Tam L;Sowick MA;Alcantar T;O'Neil N;Li J;Ta L;Lima L;Roose-Girma M;Rairdan X;Durinck S;Warming S
通讯作者：
Warming S
CRIS/CAS9活性的生物物理模型，用于基因组编辑和基因调节的合理设计。
影响因子：
4.3
作者：
Farasat I;Salis HM
通讯作者：
Salis HM
影响因子：
7
作者：
Cho SW;Kim S;Kim Y;Kweon J;Kim HS;Bae S;Kim JS
通讯作者：
Kim JS
DOI：
10.1126/science.aaf5573
发表时间：
2016-08-05
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Abudayyeh OO;Gootenberg JS;Konermann S;Joung J;Slaymaker IM;Cox DB;Shmakov S;Makarova KS;Semenova E;Minakhin L;Severinov K;Regev A;Lander ES;Koonin EV;Zhang F
通讯作者：
Zhang F
影响因子：
46.9
作者：
Doench JG;Fusi N;Sullender M;Hegde M;Vaimberg EW;Donovan KF;Smith I;Tothova Z;Wilen C;Orchard R;Virgin HW;Listgarten J;Root DE
通讯作者：
Root DE