小柯机器人

加拿大多伦多大学Evgueni A. Ivakine研究组取得一项新突破。他们提出使用 CRISPR 引导编辑的饱和变异解释。相关论文于2022年2月21日发表在《自然—生物技术》杂志上。

他们将 CRISPR 引导编辑用于高通量变异分类，并将其与允许任何基因座单倍体化的策略相结合，从而简化了变异解释。他们通过将饱和引导编辑 (SPE) 应用于 NPC 细胞内胆固醇转运蛋白 1 基因 (NPC1) 来证明其实用性，该基因突变会导致溶酶体贮积症C 型 Niemann-Pick 病。他们的数据表明 NPC1 对遗传扰动非常敏感，706 种错义突变中有 410 种被归类为有害突变，并且变异的衍生功能评分反映了多种分子缺陷。

他们进一步将他们的方法应用于 BRCA2 基因，证明 SPE 可通过适当的细胞分析转化为其他基因。总之，他们表明 SPE 允许对遗传变异进行有效、准确的功能表征。

据了解，迄今为止，只有依靠同源定向修复的方法才能对其内源性基因座中的遗传变异进行高通量功能表征，这些方法受到低编辑效率的限制。

附：英文原文

Title: Saturation variant interpretation using CRISPR prime editing

Author: Erwood, Steven, Bily, Teija M. I., Lequyer, Jason, Yan, Joyce, Gulati, Nitya, Brewer, Reid A., Zhou, Liangchi, Pelletier, Laurence, Ivakine, Evgueni A., Cohn, Ronald D.

Issue&Volume: 2022-02-21

Abstract: High-throughput functional characterization of genetic variants in their endogenous locus has so far been possible only with methods that rely on homology-directed repair, which are limited by low editing efficiencies. Here, we adapted CRISPR prime editing for high-throughput variant classification and combined it with a strategy that allows for haploidization of any locus, which simplifies variant interpretation. We demonstrate the utility of saturation prime editing (SPE) by applying it to the NPC intracellular cholesterol transporter 1 gene (NPC1), mutations in which cause the lysosomal storage disorder Niemann–Pick disease type C. Our data suggest that NPC1 is very sensitive to genetic perturbation, with 410 of 706 assayed missense mutations being classified as deleterious, and that the derived function score of variants is reflective of diverse molecular defects. We further applied our approach to the BRCA2 gene, demonstrating that SPE is translatable to other genes with an appropriate cellular assay. In sum, we show that SPE allows for efficient, accurate functional characterization of genetic variants.

DOI: 10.1038/s41587-021-01201-1

Source: https://www.nature.com/articles/s41587-021-01201-1

Nature Biotechnology：《自然—生物技术》，创刊于1996年。隶属于施普林格·自然出版集团，最新IF：68.164
官方网址：https://www.nature.com/nbt/
投稿链接：https://mts-nbt.nature.com/cgi-bin/main.plex