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

Abstract

Parageobacillus thermoglucosidasius is emerging as a highly promising thermophilic organism for metabolic engineering. The utilization of CRISPR-Cas technologies has facilitated programmable genetic manipulation in P. thermoglucosidasius. However, the absence of thermostable NHEJ enzymes limited the capability of the endogenous type I CRISPR-Cas system to generate a variety of extensive genomic deletions. Here, two thermophilic NHEJ enzymes were identified and combined with the endogenous type I CRISPR-Cas system to develop a genetic manipulation tool that can achieve long-range genomic deletion across various lengths. By optimizing this tool-through adjusting the expression level of NHEJ enzymes and leveraging our discovery of a negative correlation between GC content of the guide RNA (gRNA) and deletion efficacy-we streamlined a comprehensive gRNA selection manual for whole-genome editing, achieving a 100 % success rate in randomly selecting gRNAs. Notably, using just one gRNA, we achieved genomic deletions spanning diverse length, exceeding 200 kilobases. This tool will facilitate the genomic manipulation of P. thermoglucosidasius for both fundamental research and applied engineering studies, further unlocking its potential as a thermophilic cell factory.

Keywords: Long-range genomic deletions; Thermophile; Thermostable NHEJ enzymes; Type I CRISPR system.

Fig. 1

Identification of thermostable NHEJ enzymes. (a) Phylogenetic analysis of Ku proteins was conducted using MAGE software for sequence comparison and tree construction. The neighbor-joining algorithm was employed to build the evolutionary tree. Sequences examined in this study are denoted by red dots. (b) Overview of the assay system used to evaluate NHEJ system activity. (c) Assessing cleavage activity of type I-B CRISPR system activated by xylose. (d) Analysis of deletion size distribution in three independently created sfgfp deletion strains conducted via Sanger sequencing. (e) Similar to (d), except with NHEJ_Bme enzymes replaced by NHEJ_Bth. Data are the mean of three biological repeats and are expressed as mean ± SD. Statistical significance is calculated based on two-tailed Student's t-test (***P < 0.001).

Fig. 2

Enhancement of genome editing efficiency with glucose and xylose supplementation. (a) Assessing the editing efficiency by adding xylose into liquid medium either immediately or after a delay. (b) Analysis of the sfgfp gene expression driven by a xylose-induced promoter in a liquid medium supplemented with both glucose and xylose. (c) The workflow of genome editing. (d) Comparison of editing efficiencies in strains cultivated in media supplemented with either xylose alone or a combination of xylose and glucose. Data are the mean of three biological repeats and are expressed as mean ± SD. Statistical significance is calculated based on two-tailed Student's t-test (***P < 0.001).

Fig. 3

Evaluation of genome editing efficiencies using various gRNAs. (a) Locations of gRNAs across multiple genes. (b) Negative correlation between gRNAs editing efficiency and their GC contents. (c) Selection of gRNAs from sfgfp gene based on varying GC contents. (d) Comparison of editing efficiency for gRNAs with diverse GC contents. Data are the mean of three biological repeats and are expressed as mean ± SD.

Fig. 4

Assessing editing efficiency across the whole genome. (a) Development of a gRNA selection manual for each gene within the genome, followed by testing through the random sampling of several gRNAs. (b) Evaluation of the genome editing efficacy for each of the seven chosen gRNAs. The Y2 parental strain is used as a control (wt). (c) Determination of the maximum deletion lengths achieved by each of the seven chosen gRNAs through sanger sequencing. The bold TTA sequence indicated the PAM sequence. (d) A gRNA targeting BCV53_13685 gene generating large genomic deletions with varying lengths.