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.

非编码调控元件（NCRE）是人类基因组的主要部分，其功能尚未得到系统研究。在这里，我们报告了一种方法，涉及针对 NCRE 两端的配对单引导 RNA 文库，作为 Cas9 介导的全基因组数千个 NCRE 删除的筛选系统，以研究它们在不同生物环境中的功能。通过使用 K562 和 293T 细胞系以及人胚胎干细胞，我们表明 NCRE 可以具有冗余功能，并且许多超保守元件具有沉默活性，并在细胞生长和细胞对药物的反应中发挥重要作用（特别是，超保守元件） -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）。高通量筛选与单细胞测序兼容，可用于鉴定可成药的 NCRE。