美国耶鲁大学Steven K. Reilly和美国斯坦福大学Josh Tycko团队近期取得重要工作进展，他们研究提出了非编码调控元件CRISPRi筛选的多中心集成分析方法。相关研究成果2024年3月19日在线发表于《自然—方法学》杂志上。

据介绍，ENCODE联盟对非编码顺式调控元件（CRE）进行注释的努力促进了人们对基因调控概况的理解。汇集的非编码CRISPR筛选为研究顺式调控机制提供了一种系统的方法。ENCODE4功能表征中心在人类细胞系中进行了108次筛选，包括24.85兆基因组超过54万个扰动。

利用K562细胞中332个经功能确认的CRE基因链接，研究人员建立了利用CRISPR干扰（CRISPRi）筛选内源性非编码元件的准则，包括准确检测表现出可变（通常较低）转录效应的CRE。通过对五种筛选分析工具进行比对，研究人员发现，CASA能生成最保守的CRE调用，而且对低特异性sgRNA的人工影响也很稳定。研究人员发现，转录区域中CRISPRi的微妙DNA链偏差，对筛选设计和分析具有重要意义。

总之，研究人员提供了一个可访问的数据资源，预先设计的sgRNA，用于CRISPRi靶向3275697个ENCODE SCREEN候选CRE，并提供了加速非编码基因组功能表征的筛选指南。

附：英文原文

Title: Multicenter integrated analysis of noncoding CRISPRi screens

Author: Yao, David, Tycko, Josh, Oh, Jin Woo, Bounds, Lexi R., Gosai, Sager J., Lataniotis, Lazaros, Mackay-Smith, Ava, Doughty, Benjamin R., Gabdank, Idan, Schmidt, Henri, Guerrero-Altamirano, Tania, Siklenka, Keith, Guo, Katherine, White, Alexander D., Youngworth, Ingrid, Andreeva, Kalina, Ren, Xingjie, Barrera, Alejandro, Luo, Yunhai, Yardmc, Galip Grkan, Tewhey, Ryan, Kundaje, Anshul, Greenleaf, William J., Sabeti, Pardis C., Leslie, Christina, Pritykin, Yuri, Moore, Jill E., Beer, Michael A., Gersbach, Charles A., Reddy, Timothy E., Shen, Yin, Engreitz, Jesse M., Bassik, Michael C., Reilly, Steven K.

Issue&Volume: 2024-03-19

Abstract: The ENCODE Consortium’s efforts to annotate noncoding cis-regulatory elements (CREs) have advanced our understanding of gene regulatory landscapes. Pooled, noncoding CRISPR screens offer a systematic approach to investigate cis-regulatory mechanisms. The ENCODE4 Functional Characterization Centers conducted 108 screens in human cell lines, comprising >540,000 perturbations across 24.85megabases of the genome. Using 332 functionally confirmed CRE–gene links in K562 cells, we established guidelines for screening endogenous noncoding elements with CRISPR interference (CRISPRi), including accurate detection of CREs that exhibit variable, often low, transcriptional effects. Benchmarking five screen analysis tools, we find that CASA produces the most conservative CRE calls and is robust to artifacts of low-specificity single guide RNAs. We uncover a subtle DNA strand bias for CRISPRi in transcribed regions with implications for screen design and analysis. Together, we provide an accessible data resource, predesigned single guide RNAs for targeting 3,275,697 ENCODE SCREEN candidate CREs with CRISPRi and screening guidelines to accelerate functional characterization of the noncoding genome.

DOI: 10.1038/s41592-024-02216-7

Source: https://www.nature.com/articles/s41592-024-02216-7