Dataset Information

CRISPR/Cas9-deaminase enables robust base editing in Rhodobacter sphaeroides 2.4.1.

ABSTRACT:

Background

CRISPR/Cas9 systems have been repurposed as canonical genome editing tools in a variety of species, but no application for the model strain Rhodobacter sphaeroides 2.4.1 was unveiled.

Results

Here we showed two kinds of programmable base editing systems, cytosine base editors (CBEs) and adenine base editors (ABEs), generated by fusing endonuclease Cas9 variant to cytosine deaminase PmCDA1 or heterodimer adenine deaminase TadA-TadA*, respectively. Using CBEs, we were able to obtain C-to-T mutation of single and double targets following the first induction step, with the efficiency of up to 97% and 43%; while the second induction step was needed in the case of triple target, with the screening rate of 47%. Using ABEs, we were only able to gain A-to-G mutation of single target after the second induction step, with the screening rate of 30%. Additionally, we performed a knockout analysis to identify the genes responsible for coenzyme Q10 biosynthesis and found that ubiF, ubiA, ubiG, and ubiX to be the most crucial ones.

Conclusions

Together, CBEs and ABEs serve as alternative methods for genetic manipulation in Rhodobacter sphaeroides and will shed light on the fundamental research of other bacteria that are hard to be directly edited by Cas9-sgRNA.

SUBMITTER: Luo Y

PROVIDER: S-EPMC7183636 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

CRISPR/Cas9-deaminase enables robust base editing in Rhodobacter sphaeroides 2.4.1.

Luo Yufeng Y Ge Mei M Wang Bolun B Sun Changhong C Wang Junyi J Dong Yuyang Y Xi Jianzhong Jeff JJ

Microbial cell factories 20200425 1

<h4>Background</h4>CRISPR/Cas9 systems have been repurposed as canonical genome editing tools in a variety of species, but no application for the model strain Rhodobacter sphaeroides 2.4.1 was unveiled.<h4>Results</h4>Here we showed two kinds of programmable base editing systems, cytosine base editors (CBEs) and adenine base editors (ABEs), generated by fusing endonuclease Cas9 variant to cytosine deaminase PmCDA1 or heterodimer adenine deaminase TadA-TadA*, respectively. Using CBEs, we were abl ...[more]

PMID: 32334589

Similar Datasets

Project description:BACKGROUND:Rhodobacter sphaeroides is a metabolically versatile bacterium that serves as a model for analysis of photosynthesis, hydrogen production and terpene biosynthesis. The elimination of by-products formation, such as poly-?-hydroxybutyrate (PHB), has been an important metabolic engineering target for R. sphaeroides. However, the lack of efficient markerless genome editing tools for R. sphaeroides is a bottleneck for fundamental studies and biotechnological exploitation. The Cas9 RNA-guided DNA-endonuclease from the type II CRISPR-Cas system of Streptococcus pyogenes (SpCas9) has been extensively employed for the development of genome engineering tools for prokaryotes and eukaryotes, but not for R. sphaeroides. RESULTS:Here we describe the development of a highly efficient SpCas9-based genomic DNA targeting system for R. sphaeroides, which we combine with plasmid-borne homologous recombination (HR) templates developing a Cas9-based markerless and time-effective genome editing tool. We further employ the tool for knocking-out the uracil phosphoribosyltransferase (upp) gene from the genome of R. sphaeroides, as well as knocking it back in while altering its start codon. These proof-of-principle processes resulted in editing efficiencies of up to 100% for the knock-out yet less than 15% for the knock-in. We subsequently employed the developed genome editing tool for the consecutive deletion of the two predicted acetoacetyl-CoA reductase genes phaB and phbB in the genome of R. sphaeroides. The culturing of the constructed knock-out strains under PHB producing conditions showed that PHB biosynthesis is supported only by PhaB, while the growth of the R. sphaeroides ?phbB strains under the same conditions is only slightly affected. CONCLUSIONS:In this study, we combine the SpCas9 targeting activity with the native homologous recombination (HR) mechanism of R. sphaeroides for the development of a genome editing tool. We further employ the developed tool for the elucidation of the PHB production pathway of R. sphaeroides. We anticipate that the presented work will accelerate molecular research with R. sphaeroides.

Dataset Information

CRISPR/Cas9-deaminase enables robust base editing in Rhodobacter sphaeroides 2.4.1.

Background

Results

Conclusions

Publications

CRISPR/Cas9-deaminase enables robust base editing in Rhodobacter sphaeroides 2.4.1.

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