美国普林斯顿大学Britt Adamson团队近期取得重要工作进展，他们研究建立了用于对遗传变异进行多重功能筛选的，基准高效先导编辑平台。相关研究成果2024年11月19日在线发表于《自然—方法学》杂志上。

据介绍，先导编辑以最小脱靶概率对基因组进行精确编辑，但低且多变的编辑效率使该方法，在高通量功能基因组学中的应用变得复杂。

研究人员组装了一个能够高效替代编辑的先导编辑平台，适用于小遗传变异的功能性研究。研究人员使用一个包含约24万个工程化引物编辑引导RNA（epegRNA）的库，对该平台进行了基准测试，该库针对约1.7万个密码子进行1-3 bp的替换，用于基因功能丧失筛查。

通过比较筛选样本中这些epegRNA的丰度，研究人员鉴定出了7996个针对1149个必需基因的无义突变，和破坏3′外显子边界剪接位点基序的同义突变的负选择表型。对密码子匹配对照的严格评估表明，这些表型对预期的编辑具有高度特异性。

总之，这一研究建立了一种先导编辑方法，通过简单的读数对遗传变异进行多重功能表征。

附：英文原文

Title: A benchmarked, high-efficiency prime editing platform for multiplexed dropout screening

Author: Cirincione, Ann, Simpson, Danny, Yan, Weihao, McNulty, Ryan, Ravisankar, Purnima, Solley, Sabrina C., Yan, Jun, Lim, Fabian, Farley, Emma K., Singh, Mona, Adamson, Britt

Issue&Volume: 2024-11-19

Abstract: Prime editing installs precise edits into the genome with minimal unwanted byproducts, but low and variable editing efficiencies have complicated application of the approach to high-throughput functional genomics. Here we assembled a prime editing platform capable of high-efficiency substitution editing suitable for functional interrogation of small genetic variants. We benchmarked this platform for pooled, loss-of-function screening using a library of ~240,000 engineered prime editing guide RNAs (epegRNAs) targeting ~17,000 codons with 1–3bp substitutions. Comparing the abundance of these epegRNAs across screen samples identified negative selection phenotypes for 7,996 nonsense mutations targeted to 1,149 essential genes and for synonymous mutations that disrupted splice site motifs at 3′ exon boundaries. Rigorous evaluation of codon-matched controls demonstrated that these phenotypes were highly specific to the intended edit. Altogether, we established a prime editing approach for multiplexed, functional characterization of genetic variants with simple readouts.

DOI: 10.1038/s41592-024-02502-4

Source: https://www.nature.com/articles/s41592-024-02502-4