Extended Data Fig. 1: Design and testing of the dual-CRISPR screening system.
a, The expression of the two guide RNAs was driven by the human U6 and H1 promoters, respectively. The transcription driven by the U6 and H1 promoters is in a convergent direction, with transcription termination sequences at the end of each guide RNA scaffold sequence. The plasmid also expresses the S.pyogenes Cas9 (SpCas9) nuclease and the puromycin resistance gene to help select transfected cells. Created with BioRender.com. b, K562 cells were transiently transfected with the designed plasmids containing two guide RNAs flanking the testing sequences. In the left panel, the PCR amplicon of 600 bp in size (intact region) indicates the original DNA sequence in the genome, and the genomic sequence knockout (KO) by the two guide RNAs is shown as the PCR amplicon of 350 bp in size (KO region). The differences in transcription and editing efficiency driven by U6 and H1 promoters were tested by swapping the respective gRNAs, indicated as U6_gRNA1/H1_gRNA2 and U6_gRNA2/H1_gRNA1. Similar editing efficiency was observed driven by the two different promoters. In the right panel, the PCR amplicon of 1500 bp in size (intact region) indicates the original DNA sequence upstream of the Top2a gene, and the genomic sequence knockout by the two guide RNAs is shown as the PCR amplicon of 1000 bp in size (KO region). c, Sanger sequencing results of the PCR fragments in (b, dual-CRISPR-1) show complete knockout targeted by the designed two guide RNAs. d, Cloning strategy of the dual-CRISPR screening libraries. To make the dual-CRISPR libraries, oligo pools that contain dual crRNAs and additional restriction enzyme recognition sites were cloned between the two promoters. Then the plasmids containing the crRNAs were amplified and digested again to insert the dual-tracrRNA scaffolds to form the complete dual-CRISPR plasmid library. Created with BioRender.com. e, Cells containing dual-CRISPR libraries can be used to study different biological pathways. Genomic DNA from different cell populations is then isolated, and the dual-CRISPR libraries can be amplified by direct PCR reactions, ready for next-generation sequencing (NGS). The changes in abundance of dual CRISPRs are calculated by different algorithms to identify potential NCRE hits. Created with BioRender.com.
