Selection of Extended CRISPR RNAs with Enhanced Targeting and Specificity.
Selection of Extended CRISPR RNAs with Enhanced Targeting and Specificity.
复制标题
选择具有增强靶向性和特异性的延伸 CRISPR RNA。
DOI：
10.1101/2023.01.11.523593
复制
发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Josephs,EricA
中科院分区：
文献类型：
--
作者：
Herring-Nicholas,Ashley;Dimig,Hillary;Roesing,Miranda;Josephs,EricA
As CRISPR effectors like Cas9 increasingly enter clinical trials for therapeutic gene editing, a future for personalized medicine will require efficient methods to protect individuals from the potential of off-target mutations that may also occur at specific sequences in their genomes that are similar to the therapeutic target. A Cas9 enzyme’s ability to recognize their targets (and off-targets) are determined by the sequence of their RNA-cofactors (their guide RNAs or gRNAs). Here, we present a method to screen hundreds of thousands of gRNA variants with short, randomized 5’ nucleotide extensions near its DNA-targeting segment—a modification that can increase gene editing specificity by orders of magnitude—to identify extended gRNAs (x-gRNAs) that effectively block any activity at those off-target sites while still maintaining strong activity at their intended targets. X-gRNAs that have been selected for specific target / off-target pairs can significantly out-perform other methods that reduce Cas9 off-target activity overall, like using Cas9 variants engineered for higher specificity in general, and we demonstrate their effectiveness in clinically-relevant gRNAs. Our streamlined approach to efficiently identify highly specific and active x-gRNAs provides a way to move beyond a one-size-fits-all model of high-fidelity CRISPR for safer and more effective personalized gene therapies.
