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通过配对单引导RNA文库进行全基因组Cas9介导的必需非编码调控元件筛选。

Genome-wide Cas9-mediated screening of essential non-coding regulatory elements via libraries of paired single-guide RNAs.

作者信息

Li Yufeng, Tan Minkang, Akkari-Henić Almira, Zhang Limin, Kip Maarten, Sun Shengnan, Sepers Jorian J, Xu Ningning, Ariyurek Yavuz, Kloet Susan L, Davis Richard P, Mikkers Harald, Gruber Joshua J, Snyder Michael P, Li Xiao, Pang Baoxu

机构信息

Department of Cell and Chemical Biology, Leiden University Medical Center, Leiden, the Netherlands.

Leiden Genome Technology Center, Department of Human Genetics, Leiden University Medical Center, Leiden, the Netherlands.

出版信息

Nat Biomed Eng. 2024 Jul;8(7):890-908. doi: 10.1038/s41551-024-01204-8. Epub 2024 May 22.

DOI:10.1038/s41551-024-01204-8
PMID:38778183
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC11310080/
Abstract

The functions of non-coding regulatory elements (NCREs), which constitute a major fraction of the human genome, have not been systematically studied. Here we report a method involving libraries of paired single-guide RNAs targeting both ends of an NCRE as a screening system for the Cas9-mediated deletion of thousands of NCREs genome-wide to study their functions in distinct biological contexts. By using K562 and 293T cell lines and human embryonic stem cells, we show that NCREs can have redundant functions, and that many ultra-conserved elements have silencer activity and play essential roles in cell growth and in cellular responses to drugs (notably, the ultra-conserved element PAX6_Tarzan may be critical for heart development, as removing it from human embryonic stem cells led to defects in cardiomyocyte differentiation). The high-throughput screen, which is compatible with single-cell sequencing, may allow for the identification of druggable NCREs.

摘要

构成人类基因组主要部分的非编码调控元件（NCREs）的功能尚未得到系统研究。在此，我们报告了一种方法，该方法涉及针对NCRE两端的配对单导向RNA文库，作为全基因组范围内Cas9介导的数千个NCRE缺失的筛选系统，以研究它们在不同生物学背景下的功能。通过使用K562和293T细胞系以及人类胚胎干细胞，我们表明NCREs可以具有冗余功能，并且许多超保守元件具有沉默子活性，并在细胞生长和细胞对药物的反应中发挥重要作用（值得注意的是，超保守元件PAX6_Tarzan可能对心脏发育至关重要，因为从人类胚胎干细胞中去除它会导致心肌细胞分化缺陷）。这种与单细胞测序兼容的高通量筛选可能有助于识别可成药的NCREs。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/08c3/11310080/350c0ea7b544/41551_2024_1204_Fig9_ESM.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/08c3/11310080/a36ced51d722/41551_2024_1204_Fig8_ESM.jpg
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https://cdn.ncbi.nlm.nih.gov/pmc/blobs/08c3/11310080/6c5407f8bd75/41551_2024_1204_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/08c3/11310080/98a7905929d4/41551_2024_1204_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/08c3/11310080/40b5461a4191/41551_2024_1204_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/08c3/11310080/76376e70a898/41551_2024_1204_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/08c3/11310080/6f92828aeea3/41551_2024_1204_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/08c3/11310080/7db9be5f5173/41551_2024_1204_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/08c3/11310080/39635a76db42/41551_2024_1204_Fig7_ESM.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/08c3/11310080/a36ced51d722/41551_2024_1204_Fig8_ESM.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/08c3/11310080/350c0ea7b544/41551_2024_1204_Fig9_ESM.jpg

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