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

Yufeng Luo,Bolun Wang,Mei Ge,Junyi Wang,Yuyang Dong,Jianzhong Jeff Xi,Changhong Sun

doi:10.1186/s12934-020-01345-w

Abstract

BackgroundCRISPR/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.ResultsHere 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.ConclusionsTogether, 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.

Highlights

Clustered regularly interspaced short palindromic repeats (CRISPRs)/CRISPR associated protein 9 (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
We demonstrated that cytosine base editors (CBEs) and adenine base editors (ABEs) were robust base editing systems for Rhodobacter sphaeroides 2.4.1 that allowed the efficient modification of multiplex genes in a stringent and chemically inducible manner
For mutants generated using CBEs, if one C was totally mutated to T, we considered it as a good (T) mutant, which was very to be isolated for obtaining pure colonies with a screening rate of nearly 100%; if the areas under the C peaks were equal to or smaller than that of the T peaks, we considered it as a moderate (T≥C) mutant, which was not difficult to be isolated, with the screening rate of more than 50%; if the areas under the C peaks were all greater than that of the T peaks, we considered it as a bad (T

Summary

Introduction

CRISPR/Cas 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. Rhodobacter sphaeroides 2.4.1, originally discovered in 1989, is a gram negative and purple non-sulfur photosynthetic bacterium belonging to the α-3 subgroup of Proteobacteria [13]. As a talented producer of antioxidant coenzyme Q10 (CoQ10), it acts as the chassis microbe for industrial fermentation, demonstrating tremendous medical and commercial value. Mutagenesis strategies usually relied on physical (UV, Ar, C o60, etc.) or chemical (LiCl, NTG, NaN3, etc.) substrates to breed it for a higher titer of CoQ10, which induced random mutations that could not be traced [14].

Methods

Results

Conclusion