Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
Program in Genetics and Genome Biology, The Hospital for Sick Children Research Institute, Toronto, ON, Canada.
Nat Biotechnol. 2022 Jun;40(6):885-895. doi: 10.1038/s41587-021-01201-1. Epub 2022 Feb 21.
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.
迄今为止，只有依赖同源定向修复的方法才能实现对遗传变异在其天然位置的高通量功能特征分析，而这些方法的编辑效率较低。在这里，我们对 CRISPR 先导编辑进行了适应性改造，用于高通量变异分类，并结合了一种能够使任何基因座单倍化的策略，从而简化了变异解释。我们通过将其应用于 NPC 细胞内胆固醇转运蛋白 1 基因 (NPC1) 来证明饱和先导编辑 (SPE) 的实用性，该基因的突变会导致溶酶体贮积症尼曼-皮克病 C 型。我们的数据表明，NPC1 对遗传干扰非常敏感，在 706 个检测到的错义突变中有 410 个被归类为有害突变，并且变异的推导功能评分反映了多种分子缺陷。我们还将我们的方法应用于 BRCA2 基因，证明 SPE 可以转化为具有适当细胞测定的其他基因。总之，我们表明 SPE 允许对遗传变异进行高效、准确的功能特征分析。
