A thermostable type I-B CRISPR-Cas system for orthogonal and multiplexed genetic engineering

Abstract

Thermophilic cell factories have remarkably broad potential for industrial applications, but are limited by a lack of genetic manipulation tools and recalcitrance to transformation. Here, we identify a thermophilic type I-B CRISPR-Cas system from Parageobacillus thermoglucosidasius and find it displays highly efficient transcriptional repression or DNA cleavage activity that can be switched by adjusting crRNA length to less than or greater than 26 bp, respectively, without ablating Cas3 nuclease. We then develop an orthogonal tool for genome editing and transcriptional repression using this type I-B system in both thermophile and mesophile hosts. Empowered by this tool, we design a strategy to screen the genome-scale targets involved in transformation efficiency and established dynamically controlled supercompetent P. thermoglucosidasius cells with high efficiency ( ~ 108 CFU/μg DNA) by temporal multiplexed repression. We also demonstrate the construction of thermophilic riboflavin cell factory with hitherto highest titers in high temperature fermentation by genome-scale identification and combinatorial manipulation of multiple targets. This work enables diverse high-efficiency genetic manipulation in P. thermoglucosidasius and facilitates the engineering of thermophilic cell factories.

A highly efficient method for genomic deletion across diverse lengths in thermophilic Parageobacillus thermoglucosidasius

“…To expedite the assessment of these selected NHEJ enzymes, we devised an assay system that comprises a test plasmid coupled with a Y2 strain [ 20 ] that stably harbors the sfgfp gene within its genome. In the plasmid, the individual NHEJ enzymes and the xylose-inducible gRNA were designed to simultaneously expresses.…”

“…For assessing the ligation activity of NHEJ enzymes, the plasmid pZL02-NHEJ was utilized. This plasmid employs pZL02 as its backbone, mirroring the functionality of pZH01, a construct previously developed by our laboratory [ 20 ]. For entailed information on pZL02 sequence, refer to Supplementary Note 1.…”

“…Competent cell preparation of P. thermoglucosidasius NCIMB 11955 was conducted as described previously [ 20 ]. Below is a concise overview of the process.…”

“…This scenario has facilitated genetic editing of non-model microorganisms by harnessing their native CRISPR-Cas3 systems [ [14] , [15] , [16] , [17] , [18] , [19] ]. We have previously developed efficient tools for genetic manipulation in P. thermoglucosidasius based on the endogenous type I CRISPR-Cas system, enabling capabilities in genetic editing, gene regulation, CRISPRi (CRISPR interference) library, and the dynamically controlled supercompetent cells [ 20 ]. Although exhibiting excellent efficiency, we found that this type I CRISPR systems failed to generate long-range genomic deletion in P. thermoglucosidasius .…”

Multiplex CRISPR-Cas Genome Editing: Next-Generation Microbial Strain Engineering

“…If the expression of the Cas protein, which has a high affinity for nucleic acids, is increased unnecessarily, it will inevitably burden the cell . Therefore, in multiplex genome editing, the endogenous CRISPR-Cas system could be used to alleviate the toxicity of heterologous Cas proteins. ,− Other types of CRISPR-Cas systems, such as Cas12a (also known as Cpf1, 1200–1500 amino acids) could be used in organisms like Corynebacterium glutamicum and cyanobacteria, where editing is challenging due to the toxicity of Cas9 (900–1700 aa) . Furthermore, the easily deliverable miniature Cas12f (400–700 aa) and Cas12j (700–800 aa) could also be used for multiplex genome editing.…”