A Cas12a-Mediated Crispri Screen for Small Noncoding RNAs in the Human Commensal Bacteroides Thetaiotaomicron

Gram-negative, obligate anaerobic Bacteroides thetaiotaomicron a dominant member of the intestinal bacterial consortium in healthy human beings. In the past, transposon mutant libraries of B. thetaiotaomicron were screened to identify genetic determinants required for colonization of germ-free (Goodman et al, CH&M, 2009) and gnotobiotic (Wu et al, Science, 2015) mice. This approach, whose underlying principle is that the insertion of a transposon disrupts the function of the targeted gene, has however been largely restricted the screening of protein-coding genes and operons.

Small noncoding RNAs (sRNAs) are widely used within bacteria to adapt gene expression in response to myriads of external and intrinsic cues. We recently identified hundreds of sRNA candidates in the B. thetaiotaomicron genome, whose functions are currently not known. However, due to their short length (typically 50-500 nt), sRNAs are less likely to be hit by random mutagenesis and, consequently, are poorly reflected in existing Tn-seq (transposon sequencing) datasets.

For this reason, in this work we seek to employ CRISPR interference (CRISPRi) to identify and characterize B. thetaiotaomicron sRNAs essential for growth under defined conditions. We in-silico screened several protospacer adjacent motifs (PAMs) to identify the one with the highest occurrence among the sRNA pool. The analysis resulted in the AT-rich 5’-TTV PAM recognized by Cas12a (Cpf1), in line with the low GC content of the B. thetaiotaomicron genome. We also employed a cell-free transcription and translation system (TXTL) to screen potential nucleases to express with our library. Finally, by using a luciferase reporter, we assessed how the position and strandness of the targeted region inside the reporter gene affect the extent of transcriptional repression by Cas12a.

The CRISPRi approach described in this study will allow us to pinpoint functionally important sRNAs in B. thetaiotaomicron grown under defined conditions, including the ones that mimic the bacterium’s in-vivo niche.