SRP486012 PRJNA1069067 GSE254241 The genome-wide dual-CRISPR screening identifies essential non-coding regulatory elements Non-coding regulatory elements (NCRE) represent a major fraction of the human genome, play important roles in different biological pathways, and have the potential as genomic medicine targets. We developed a straightforward dual-CRISPR screening system capable of deleting thousands of NCREs genome-wide to study their functions in distinct biological contexts in K562 cells and 293T cells. We show that many NCREs, including ultraconserved elements (UCE), have silencer activity and play essential roles in cell growth and drug response. NCREs with redundant functions could also be identified from the screening data. This dual-CRISPR system is also compatible with single-cell sequencing. We identified that UCE PAX6_Tarzan might be critical in heart development, as removing it from human embryonic stem cells led to defects in cardiomyocyte differentiation. Our study provides further evidence that many NCREs have important biological functions contributing to human biology and diseases, and may serve as future drug targets. Overall design: We have developed a new dual-CRISPR screening system that could delete thousands of NCREs in a systematic and genome-wide fashion. This dual CRISPR screening system is easy to construct and sequence, without the need for additional barcoding. As target regions of more than 200 bp in size are removed from the genome, NCREs could be studied irrespective of their specific biological functions. We designed dual-CRISPR libraries targeting 4,047 UCEs in the human genome from UCNEbase (Dimitrieva and Bucher, 2013), 1,527 in vivo-validated conserved enhancers from VISTA Enhancer Browser (Visel et al., 2007), and all 13,539 predicted enhancers in K562 cells from ENCODE (Yip et al., 2012). Using this system, we studied the biological functions of the UCEs in the human genome and identified regions that would affect cell survival and drug response in K562 and 293T cells. GSE254241 pubmed 38778183