Optimization of Cas9 activity through the addition of cytosine extensions to single-guide RNAs.
Optimization of Cas9 activity through the addition of cytosine extensions to single-guide RNAs.
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DOI：
10.1038/s41551-023-01011-7
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发表时间：
2023-05
影响因子：
28.1
通讯作者：
中科院分区：
文献类型：
--
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对Cas9活动的精确调控对于安全和高效的编辑至关重要。在这里，我们证明了Cas9的基因组编辑活性可以通过在传统的单引导RNA(SgRNAs)的5‘端添加胞嘧啶延伸来限制。这种与Cas12a以及通过CRISPR(簇状规则间隔短回文重复序列)进行基因激活和干扰的系统兼容的“保护sgRNA”策略，导致了对功能性Cas9复合体形成的长度依赖的抑制。短暂的胞嘧啶延长降低了人类多能干细胞中P53的激活和细胞毒性，并在保持双等位基因编辑的同时增强了同源定向修复。更长的延伸进一步降低了靶上的活性，但提高了单等位基因编辑的特异性和精确度。通过基于荧光的等位基因特异性系统和计算模拟监测INDELs，我们确定了Cas9活性的最佳窗口，用于许多基因组编辑应用，包括双等位基因和单等位基因编辑，以及通过同源定向修复产生和纠正与疾病相关的单核苷酸替换。保障sgRNA策略可能会提高基因组编辑的安全性和适用性。基于Cas9的标准基因组编辑系统的活动可以通过在传统的单引导RNA的5‘端添加胞嘧啶延伸来限制。
The precise regulation of the activity of Cas9 is crucial for safe and efficient editing. Here we show that the genome-editing activity of Cas9 can be constrained by the addition of cytosine stretches to the 5′-end of conventional single-guide RNAs (sgRNAs). Such a ‘safeguard sgRNA’ strategy, which is compatible with Cas12a and with systems for gene activation and interference via CRISPR (clustered regularly interspaced short palindromic repeats), leads to the length-dependent inhibition of the formation of functional Cas9 complexes. Short cytosine extensions reduced p53 activation and cytotoxicity in human pluripotent stem cells, and enhanced homology-directed repair while maintaining bi-allelic editing. Longer extensions further decreased on-target activity yet improved the specificity and precision of mono-allelic editing. By monitoring indels through a fluorescence-based allele-specific system and computational simulations, we identified optimal windows of Cas9 activity for a number of genome-editing applications, including bi-allelic and mono-allelic editing, and the generation and correction of disease-associated single-nucleotide substitutions via homology-directed repair. The safeguard-sgRNA strategy may improve the safety and applicability of genome editing. The activity of standard Cas9-based genome-editing systems can be constrained by the addition of cytosine stretches to the 5′-end of conventional single-guide RNAs.
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影响因子：
48
作者：
Chavez A;Tuttle M;Pruitt BW;Ewen-Campen B;Chari R;Ter-Ovanesyan D;Haque SJ;Cecchi RJ;Kowal EJK;Buchthal J;Housden BE;Perrimon N;Collins JJ;Church G
通讯作者：
Church G
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
影响因子：
5.9
作者：
Kagita A;Lung MSY;Xu H;Kita Y;Sasakawa N;Iguchi T;Ono M;Wang XH;Gee P;Hotta A
通讯作者：
Hotta A
影响因子：
3.7
作者：
Kim JH;Lee SR;Li LH;Park HJ;Park JH;Lee KY;Kim MK;Shin BA;Choi SY
通讯作者：
Choi SY
DOI：
10.1016/j.bbagrm.2012.10.006
发表时间：
2013-03
影响因子：
4.7
作者：
Arimbasseri, Aneeshkumar G.;Rijal, Keshab;Maraia, Richard J.
通讯作者：
Maraia, Richard J.