A CRISPR/Cas9-based single-stranded DNA recombineering system for genome editing of Rhodococcus opacus PD630

Abstract

Genome engineering of Rhodococcus opacus PD630, an important microorganism used for the bioconversion of lignin, is currently dependent on inefficient homologous recombination. Although a CRISPR interference procedure for gene repression has previously been developed for R. opacus PD630, a CRISPR/Cas9 system for gene knockout has yet to be reported for the strain. In this study, we found that the cytotoxicity of Cas9 and the deficiency in pathways for repairing DNA double-strand breaks (DSBs) were the major causes of the failure of conventional CRISPR/Cas9 technologies in R. opacus , even when augmented with the recombinases Che9c60 and Che9c61. We successfully developed an efficient single-stranded DNA (ssDNA) recombineering system coupled with CRISPR/Cas9 counter-selection, which facilitated rapid and scarless editing of the R. opacus genome. A two-plasmid system, comprising Cas9 driven by a weak Rhodococcus promoter Pniami, designed to prevent cytotoxicity, and a single-guide RNA (sgRNA) under the control of a strong constitutive promoter, was proven to be appropriate with respect to cleavage function. A novel recombinase, Rr RecT derived from a Rhodococcus ruber prophage, was identified for the first time, which facilitated recombination of short ssDNA donors (40–80 nt) targeted to the lagging strand and enabled us to obtain a recombination efficiency up to 10 3 -fold higher than that of endogenous pathways. Finally, by incorporating Rr RecT and Cas9 into a single plasmid and then co-transforming cells with sgRNA plasmids and short ssDNA donors, we efficiently achieved gene disruption and base mutation in R. opacus , with editing efficiencies ranging from 22 % to 100 %. Simultaneous disruption of double genes was also confirmed, although at a lower efficiency. This effective genome editing tool will accelerate the engineering of R. opacus metabolism.

Production of biopolymer precursors beta-alanine and L-lactic acid from CO2 with metabolically versatile Rhodococcus opacus DSM 43205

“…Recently, fast-growing interest in metabolic versatility and use of R. opacus as a platform for biocatalysis, biodegradation and biosynthesis has driven development of more genome engineering tools for R. opacus . New genetic parts have been validated and most importantly CRISPR/Cas9-based gene knockout system is reported ( Liang et al, 2021 ; Grechishnikova et al, 2022 ). These tools are anticipated to facilitate and accelerate the engineering of R. opacus DSM 43025 to yield outstanding strains for the production of biochemicals and biofuels from CO 2 .…”

CRISPR Tools in Bacterial Whole-Cell Biocatalysis

“…For instance, the NHEJ pathway was identified in Rhodococcus opacus PD630 but presented a very low repair efficiency (0% to 25% depending on the sgRNA) and did not permit further applications. 157 This repair system was also identified in Clostridium cellulolyticum. However, a CRISPR-mediated editing attempt by Xu et indicating the lethality of the generated DSB, probably related to a low expression of key enzymes in the system.…”

“…Examples of the use of these particular CRISPR-mediated techniques in biotransformations are scarce. For instance, the NHEJ pathway was identified in Rhodococcus opacus PD630 but presented a very low repair efficiency (0% to 25% depending on the sgRNA) and did not permit further applications . This repair system was also identified in Clostridium cellulolyticum .…”

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

“…To increase homologous recombination of the donor template DNA, phage-derived recombinase can be overexpressed. In addition to the widely used λ-red protein or RecET derived from Rac prophage, efforts are underway to identify new recombinases that can further increase editing efficiency. , Additionally, there have been reports that overexpression of proteins, such as mutated NgAgo ( Natronobacterium gregoryi Argonaute protein without enzyme activity), AtpD (β-subunit of ATP synthase), and hBrex27 (exon 27 domain of human BRCA2) can increase the efficiency of homologous recombination, thereby improving the efficiency of multiplex genome editing.…”