CRISPR/Cas13 effectors have differing extents of off-target effects that limit their utility in eukaryotic cells.
CRISPR/Cas13 effectors have differing extents of off-target effects that limit their utility in eukaryotic cells.
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DOI：
10.1093/nar/gkac159
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发表时间：
2022-06-24
影响因子：
14.9
通讯作者：
Wilusz, Jeremy E.
中科院分区：
文献类型：
--
作者：
Ai, Yuxi;Liang, Dongming;Wilusz, Jeremy E.
CRISPR/Cas 13效应物作为检测和消耗感兴趣的RNA的容易定制的工具，已经获得了越来越多的关注。Cas 13核糖核酸酶活性的激活需要靶RNA和Cas 13相关的指导RNA之间的近乎完美的互补性。尽管如此，真核细胞中Cas 13效应子的特异性一直存在争议，因为Cas 13核酸酶结构域可以暴露在酶表面上，从而提供了对附近RNA的混杂切割（所谓的附带损伤）的可能性。在这里，使用果蝇和人类细胞中的共转染试验，我们发现RxCas 13 d（一种常用的Cas 13效应子）的脱靶效应可以与靶RNA敲低水平一样强。脱靶效应的程度与靶RNA表达水平正相关，即使在降低RxCas 13 d/指导RNA水平后也可以观察到附带损伤。PspCas 13 b效应子显示出改善的特异性，并且与RxCas 13 d不同，可用于耗尽果蝇环状RNA而不影响相关线性RNA的表达。尽管如此，PspCas 13 b仍然可以具有脱靶效应，我们特别发现Cas 13效应子的脱靶效应程度取决于所检查的细胞类型和靶RNA。总之，这些结果强调了在设计和解释基于Cas 13的敲低实验时需要谨慎。
CRISPR/Cas13 effectors have garnered increasing attention as easily customizable tools for detecting and depleting RNAs of interest. Near perfect complementarity between a target RNA and the Cas13-associated guide RNA is required for activation of Cas13 ribonuclease activity. Nonetheless, the specificity of Cas13 effectors in eukaryotic cells has been debated as the Cas13 nuclease domains can be exposed on the enzyme surface, providing the potential for promiscuous cleavage of nearby RNAs (so-called collateral damage). Here, using co-transfection assays in Drosophila and human cells, we found that the off-target effects of RxCas13d, a commonly used Cas13 effector, can be as strong as the level of on-target RNA knockdown. The extent of off-target effects is positively correlated with target RNA expression levels, and collateral damage can be observed even after reducing RxCas13d/guide RNA levels. The PspCas13b effector showed improved specificity and, unlike RxCas13d, can be used to deplete a Drosophila circular RNA without affecting the expression of the associated linear RNA. PspCas13b nonetheless still can have off-target effects and we notably found that the extent of off-target effects for Cas13 effectors differs depending on the cell type and target RNA examined. In total, these results highlight the need for caution when designing and interpreting Cas13-based knockdown experiments.
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影响因子：
46.9
作者：
Jackson, AL;Bartz, SR;Linsley, PS
通讯作者：
Linsley, PS
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
CRISPR-C2C2的两种不同的RNase活性启用了指导RNA处理和RNA检测。
影响因子：
64.8
作者：
East-Seletsky A;O'Connell MR;Knight SC;Burstein D;Cate JH;Tjian R;Doudna JA
通讯作者：
Doudna JA
影响因子：
11.8
作者：
Kushawah, Gopal;Hernandez-Huertas, Luis;Moreno-Mateos, Miguel A.
通讯作者：
Moreno-Mateos, Miguel A.
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle