Reprogramming the endogenous type I CRISPR‐Cas system for simultaneous gene regulation and editing in Haloarcula hispanica. Issue 1 (17th March 2022)

• Record Type: Journal Article
• Authors: Du, Kaixin; Gong, Luyao; Li, Ming; Yu, Haiying; Xiang, Hua
• Is Part Of: MLife. Volume 1:Issue 1(2022)
• Page Start: 40
• Page End: 50
• Publication Date: 2022-03-17
• Subjects: CRISPR‐Cas system -- gene editing -- gene regulation -- type I; Microbiology -- Periodicals; Microbiology; Periodicals; 579; 579
• DOI: 10.1002/mlf2.12010
• Languages: English
• ISSNs: 2770-100X

Abstract

Abstract: The type I system is the most widely distributed CRISPR‐Cas system identified so far. Recently, we have revealed the natural reprogramming of the type I CRISPR effector for gene regulation with a crRNA‐resembling RNA in halophilic archaea. Here, we conducted a comprehensive study of the impact of redesigned crRNAs with different spacer lengths on gene regulation with the native type I‐B CRISPR system in Haloarcula hispanica . When the spacer targeting the chromosomal gene was shortened from 36 to 28 bp, transformation efficiencies of the spacer‐encoding plasmids were improved by over three orders of magnitude, indicating a significant loss of interference. However, by conducting whole‐genome sequencing and measuring the growth curves of the hosts, we still detected DNA cleavage and its influence on cell growth. Intriguingly, when the spacer was shortened to 24 bp, the transcription of the target gene was downregulated to 10.80%, while both interference and primed adaptation disappeared. By modifying the lengths of the spacers, the expression of the target gene could be suppressed to varying degrees. Significantly, by designing crRNAs with different spacer lengths and targeting different genes, we achieved simultaneous gene editing ( cdc6E ) and gene regulation ( crtB ) for the first time with the endogenous type I CRISPR‐Cas system. Impact statement: This is the first systematic study of the effects of spacer length on different CRISPR functionalities, including target interference, primed adaptation, and transcription inhibition. As the type I system is widespread among prokaryotes, this study has provided a novel and significant strategy in a wide‐range of archaea and bacteria with endogenous CRISPR‐Cas systems, for delicate gene regulation or editing, just by modulating the crRNA length for the target gene.