小柯机器人

北京大学魏文胜课题组开发出基于碱基编辑的新型高通量功能性筛选方法。相关论文于2021年6月21日在线发表于国际学术期刊《自然—生物技术》。

研究人员表示，经典CRISPR基因敲除（KO）筛选依赖于Cas9引起的DNA双链断裂（DSB）来生成靶向基因KO。这些方法可能会产生扭曲的结果，因为DSB相关效应通常被错误地认为是基因扰动本身的后果，尤其是当靶向高拷贝数位点时。

研究人员报告了一种不依赖于DSB的全基因组CRISPR筛选方法，称为iBARed胞嘧啶碱基编辑介导的基因KO（BARBEKO）。通过扰乱基因起始密码子或剪接位点，或通过引入过早终止密码子，该方法利用CRISPR胞嘧啶碱基编辑器进行基因组规模的KO筛选。此外，它与iBAR集成，这是研究人员为提高筛选质量和效率而设计的策略。通过以高感染多样性（最多10个）慢病毒感染构建这样的细胞文库，研究人员获得了有效且准确的筛选结果，同时大大减少了起始细胞。

更重要的是，与Cas9介导的筛选相比，BARBEKO筛选不再受HeLa、K562或DSB敏感的视网膜色素上皮1细胞中DNA断裂诱导的细胞毒性影响。研究人员认为，BARBEKO会提供一种有价值的工具来补充各种条件下的CRISPR-KO筛选。

 附：英文原文

Title: Genome-wide interrogation of gene functions through base editor screens empowered by barcoded sgRNAs

Author: Ping Xu, Zhiheng Liu, Ying Liu, Huazheng Ma, Yiyuan Xu, Ying Bao, Shiyou Zhu, Zhongzheng Cao, Zeguang Wu, Zhuo Zhou, Wensheng Wei

Issue&Volume: 2021-06-21

Abstract: Canonical CRISPR–knockout (KO) screens rely on Cas9-induced DNA double-strand breaks (DSBs) to generate targeted gene KOs. These methodologies may yield distorted results because DSB-associated effects are often falsely assumed to be consequences of gene perturbation itself, especially when high copy-number sites are targeted. In the present study, we report a DSB-independent, genome-wide CRISPR screening method, termed iBARed cytosine base editing-mediated gene KO (BARBEKO). This method leverages CRISPR cytosine base editors for genome-scale KO screens by perturbing gene start codons or splice sites, or by introducing premature termination codons. Furthermore, it is integrated with iBAR, a strategy we devised for improving screening quality and efficiency. By constructing such a cell library through lentiviral infection at a high multiplicity of infection (up to 10), we achieved efficient and accurate screening results with substantially reduced starting cells. More importantly, in comparison with Cas9-mediated fitness screens, BARBEKO screens are no longer affected by DNA cleavage-induced cytotoxicity in HeLa-, K562- or DSB-sensitive retinal pigmented epithelial 1 cells. We anticipate that BARBEKO offers a valuable tool to complement the current CRISPR–KO screens in various settings.

DOI: 10.1038/s41587-021-00944-1

Source: https://www.nature.com/articles/s41587-021-00944-1

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