Reprogramming the endogenous type I CRISPR‐Cas system for simultaneous gene regulation and editing in Haloarcula hispanica

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.

Engineered minimal type I CRISPR-Cas system for transcriptional activation and base editing in human cells

“…Second, the Cascade complex is very flexible. Modifying the spacer length can alter the Cas protein stoichiometry within the Cascade complex and influence the extent of the R-loop region, potentially impacting its functionality 16, 27, 53, 54, 64 . For transcriptional activation, significant activation was detected with VPR fused to Cas7 (Fig.…”

“…The crRNA-binding complex of type I is called Cascade (CRISPR-associated complex for antiviral defense), which binds to the crRNA for target recognition, facilitates duplex formation between the crRNA and its complementary target DNA for R-loop formation, and recruits Cas3 nuclease to degrade DNA processively [4][5][6][7] . Type I systems have been developed into various 2 microbial genome manipulation tools, such as tools for genome editing with homologous repair templates [8][9][10] , transcriptional regulation 11,12 , large fragment deletion 13,14 , large fragment integration without requiring homologous recombination 15 , and simultaneous genome editing and gene regulation with Cascade-Cas3 16 . Since 2019, several type I subtypes have been successfully used in eukaryotic genome manipulation.…”

Type I CRISPR-Cas-mediated microbial gene editing and regulation

“…In C. butyricum , expression of Cas9 resulted in extremely poor transformation, owing to its toxicity, while the endogenous type I-B system dramatically increased the plasmid delivery rate and successfully deleted genes with 100% efficiency [62] . Moreover, gene editing using endogenous type I-B systems was also demonstrated in multiple other bacterial or archaeal species such as C. tyrobutyricum [63] , C. difficile [64] , the industrial biofuel-producing microorganism Thermoanaerobacterium aotearoense [65] and the polyploid haloarchaeon Haloarcula hispanica [66] , [67] .…”

“…It was shown that spacer length could affect CRISPR-Cas functionality such as DNA interference and adaptation [110] , [111] . Therefore, Du et al systematically examined the effect of spacer lengths on CRISPR-Cas-mediated DNA cleavage and adaptation in H. hispanica , which revealed that CRISPR-Cas functionalities including DNA interference and adaptation are significantly reduced with the decreasing length of spacers [66] . Interestingly, it was found that target gene expression could be significantly inhibited by the endogenous type I-B system even with an active Cas3 nuclease when the spacer length was reduced to 24 bp ( Figure 4B ).…”

“…Interestingly, it was found that target gene expression could be significantly inhibited by the endogenous type I-B system even with an active Cas3 nuclease when the spacer length was reduced to 24 bp ( Figure 4B ). By expressing crRNAs with different spacer lengths to target different genes, the endogenous type I-B system was designed to simultaneously achieve desired gene repression and gene editing [66] .…”