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

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.

Prime Editing: An All-Rounder for Genome Editing

“…Attempts have been made in the field of microbiology. Tong et al successfully performed large fragment deletions (up to 97 bp) and insertions (up to 33 bp) in Escherichia coli, revealing the industrial application potential of PE in microbial cell factories [ 95 ]. The successful employment of PE for precision editing among different species is encouraging and reveals that PE can be applied to generate purposeful editing in a wide range of organisms; however, such interspecies compatibility requires urgent verification through experiments.…”

Reprogramming Microbial CO2-Metabolizing Chassis With CRISPR-Cas Systems

“…As a one-step forward, prime-editing was invented via a combination of nCas9 and a reverse-transcriptase, allowing the insertion of small DNA fragments without generating DNA double-strand breaks or requiring a donor DNA ( Anzalone et al, 2019 ) ( Figure 2B ). Tong et al (2021) further adapted this method for bacteria, making possible the introduction of deletions, insertions, and nucleotide substitutions with prime editing in E. coli . More specifically, an up to 97 bp of DNA fragment was deleted, and an up to 33 bp of DNA fragment was inserted into the genome of E. coli with high fidelity and efficiency ( Tong et al, 2021 ).…”

“… Tong et al (2021) further adapted this method for bacteria, making possible the introduction of deletions, insertions, and nucleotide substitutions with prime editing in E. coli . More specifically, an up to 97 bp of DNA fragment was deleted, and an up to 33 bp of DNA fragment was inserted into the genome of E. coli with high fidelity and efficiency ( Tong et al, 2021 ). To enable large DNA insertion, a more recent study invented a twin-prime system with a prime editing system and serine recombinase.…”

In Vivo Rapid Investigation of CRISPR-Based Base Editing Components in Escherichia coli (IRI-CCE): A Platform for Evaluating Base Editing Tools and Their Components

“…In eukaryotes, double-strand breaks (DSBs) generated by CRISPR/Cas tool are repaired by error-prone nonhomologous end-joining (NHEJ) pathway leading to the gene disruption. Most bacteria lack the efficient NHEJ pathway, thus restricting the DSB-dependent CRISPR/Cas applications [3]. The DSB-free DNA base editors (BEs) are emerging tools to install precise mutations at target regions in bacteria [4].…”