Re-engineered guide RNA enables DNA loops and contacts modulating repression in E. coli

Abstract RNA serves as information media as well as molecular scaffold in nature and synthetic systems. The single guide RNA (sgRNA) widely applied in CRISPR techniques exemplifies both functions, with a guide region bearing DNA base-pairing information, and a structural motif for Cas9 protein scaffolding. The scaffold region has been modified by fusing RNA aptamers to the tetra-stem loop. The guide region is typically not regarded as a pluggable module as it encodes the essential function of DNA sequence recognition. Here, we investigate a chimera of two sgRNAs, with distinct guide sequences joined by an RNA linker (dgRNA), regarding its DNA binding function and loop induction capability. First, we studied the sequence bi-specificity of the dgRNA and discovered that the RNA linker allows distal parts of double-stranded DNA to be brought into proximity. To test the activity of the dgRNA in organisms, we used the LacZ gene as a reporter and recapitulated the loop-mediated gene inhibition by LacI in E. coli. We found that the dgRNA can be applied to target distal genomic regions with comparable levels of inhibition. The capability of dgRNA to induce DNA contacts solely requires dCas9 and RNA, making it a minimal system to remodel chromosomal conformation in various organisms.