Department of Molecular Genetics and Biochemistry, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel; Felsenstein Medical Research Center, Tel Aviv University, Tel Aviv, Israel.
Mol Ther. 2021 Mar 3;29(3):937-948. doi: 10.1016/j.ymthe.2020.11.009. Epub 2020 Nov 26.
The CRISPR-Cas system holds great promise in the treatment of diseases caused by genetic variations. The Cas protein, an RNA-guided programmable nuclease, generates a double-strand break at precise genomic loci. However, the use of the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas system to distinguish between single-nucleotide variations is challenging. The promiscuity of the guide RNA (gRNA) and its mismatch tolerance make allele-specific targeting an elusive goal. This review presents a meta-analysis of previous studies reporting position-dependent mismatch tolerance within the gRNA. We also examine the conservativity of the seed sequence, a region within the gRNA with stringent sequence dependency, and propose the existence of a subregion within the seed sequence with a higher degree of specificity. In addition, we summarize the reports on high-fidelity Cas nucleases with improved specificity and compare the standard gRNA design methodology to the single-nucleotide polymorphism (SNP)-derived protospacer adjacent motif (PAM) approach, an alternative method for allele-specific targeting. The combination of the two methods may be advantageous in designing CRISPR-based therapeutics and diagnostics for heterozygous patients.
CRISPR-Cas 系统在治疗由基因突变引起的疾病方面具有巨大的潜力。Cas 蛋白是一种 RNA 指导的可编程核酸酶，可在精确的基因组位置产生双链断裂。然而，利用成簇规律间隔短回文重复序列 (CRISPR)-Cas 系统区分单核苷酸变异是具有挑战性的。引导 RNA (gRNA) 的混杂性及其对错配的容忍度使得等位基因特异性靶向成为难以实现的目标。本综述对以前报道 gRNA 中位置依赖性错配容忍度的研究进行了荟萃分析。我们还检查了种子序列的保守性，种子序列是 gRNA 中具有严格序列依赖性的区域，并提出了种子序列内存在具有更高特异性的亚区域的假设。此外，我们总结了关于具有更高特异性的高保真 Cas 核酸酶的报道，并将标准 gRNA 设计方法与单核苷酸多态性 (SNP) 衍生的前导间隔基序 (PAM) 方法进行了比较，后者是等位基因特异性靶向的替代方法。这两种方法的结合可能有利于设计基于 CRISPR 的治疗和诊断方法，用于杂合子患者。
