Herring-Nicholas Ashley, Dimig Hillary, Roesing Miranda, Josephs Eric A
Department of Nanoscience, The University of North Carolina at Greensboro, Greensboro, North Carolina, 27401, USA.
Authors contributed equally.
bioRxiv. 2023 Jan 12:2023.01.11.523593. doi: 10.1101/2023.01.11.523593.
For a CRISPR guide RNA (gRNA) with a specific target but activity at known "off-target" sequences, 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 Cas9 gene editing specificity by orders of magnitude with certain 5'- extension sequences, some as-yet-unknown mechanism that makes design of the extension sequence difficult to perform manually-to robustly identify extended gRNAs (x-gRNAs) that have been counter-selected against activity at those off-target sites and that exhibit significantly enhanced Cas9 specificity for their intended targets.
对于具有特定靶点但在已知“脱靶”序列处有活性的CRISPR引导RNA（gRNA），我们提出了一种方法，可筛选数十万个gRNA变体，这些变体在其DNA靶向片段附近有短的、随机的5'核苷酸延伸——这种修饰可以通过某些5'-延伸序列将Cas9基因编辑特异性提高几个数量级，存在一些尚未明确的机制使得延伸序列的设计难以手动完成——从而稳健地鉴定出已针对那些脱靶位点的活性进行反向选择、并对其预期靶点表现出显著增强的Cas9特异性的延伸gRNA（x-gRNA）。
