A versatile genetic engineering toolkit for E. coli based on CRISPR-prime editing

Yaojun Tong,Tilmann Weber,Tue S Jørgensen,Sang Yup Lee,Christopher M Whitford

doi:10.1038/s41467-021-25541-3

Abstract

CRISPR base editing is a powerful method to engineer bacterial genomes. However, it restricts editing to single-nucleotide substitutions. Here, to address this challenge, we adapt a CRISPR-Prime Editing-based, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes. It can introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli with high fidelity. Notably, under optimal conditions, the efficiency of 1-bp deletions reach up to 40%. Moreover, deletions of up to 97 bp and insertions up to 33 bp were successful with the toolkit in E. coli, however, efficiencies dropped sharply with increased fragment sizes. With a second guide RNA, our toolkit can achieve multiplexed editing albeit with low efficiency. Here we report not only a useful addition to the genome engineering arsenal for E. coli, but also a potential basis for the development of similar toolkits for other bacteria.

Highlights

CRISPR base editing is a powerful method to engineer bacterial genomes
The PEgRNA is composed of a 20-nt spacer and a 3′ extension containing the primer binding sequence (PBS) and reverse transcription template (RTT) (Fig. 1b)
The third plasmid pCRISPR-prime editing (PE) expresses an E. coli codon optimized fusion protein composed of an engineered reverse transcriptase M-MLV2, a flexible linker, and a Cas9 H840A nickase (Cas9n) (Cas[9] nickase, the H840A mutant of SpyCas9) under a tetracycline-inducible promoter (Fig. 1c)

Summary

Introduction

To address this challenge, we adapt a CRISPR-Prime Editing-based, DSB-free, versatile, and single-nucleotide resolution genetic manipulation toolkit for prokaryotes. It can introduce substitutions, deletions, insertions, and the combination thereof, both in plasmids and the chromosome of E. coli with high fidelity. The classic genetic engineering approaches in prokaryotes often use phage-derived RecET and lambda red recombinase-based recombineering[1,2] They employ the homology-directed integration/replacement of a donor double stranded DNA (dsDNA) or oligonucleotide for making insertions, deletions, and substitutions of the target DNA.

Methods

Results

Conclusion