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 fo...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,RrRecT 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 103-fold higher than that of endogenous pathways.Finally,by incorporating RrRecT 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.展开更多
基金supported by the National Key R&D Program of China(2018YFA0902200)Natural Science Foundation of China(No.21776157,No.22078173)。
文摘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,RrRecT 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 103-fold higher than that of endogenous pathways.Finally,by incorporating RrRecT 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.
