Large scale validation of an efficient CRISPR/Cas-based multi gene editing protocol in Escherichia coli

Abstract

BackgroundThe exploitation of the CRISPR/Cas9 machinery coupled to lambda (λ) recombinase-mediated homologous recombination (recombineering) is becoming the method of choice for genome editing in E. coli. First proposed by Jiang and co-workers, the strategy has been subsequently fine-tuned by several authors who demonstrated, by using few selected loci, that the efficiency of mutagenesis (number of mutant colonies over total number of colonies analyzed) can be extremely high (up to 100%). However, from published data it is difficult to appreciate the robustness of the technology, defined as the number of successfully mutated loci over the total number of targeted loci. This information is particularly relevant in high-throughput genome editing, where repetition of experiments to rescue missing mutants would be impractical. This work describes a “brute force” validation activity, which culminated in the definition of a robust, simple and rapid protocol for single or multiple gene deletions.ResultsWe first set up our own version of the CRISPR/Cas9 protocol and then we evaluated the mutagenesis efficiency by changing different parameters including sequence of guide RNAs, length and concentration of donor DNAs, and use of single stranded and double stranded donor DNAs. We then validated the optimized conditions targeting 78 “dispensable” genes. This work led to the definition of a protocol, featuring the use of double stranded synthetic donor DNAs, which guarantees mutagenesis efficiencies consistently higher than 10% and a robustness of 100%. The procedure can be applied also for simultaneous gene deletions.ConclusionsThis work defines for the first time the robustness of a CRISPR/Cas9-based protocol based on a large sample size. Since the technical solutions here proposed can be applied to other similar procedures, the data could be of general interest for the scientific community working on bacterial genome editing and, in particular, for those involved in synthetic biology projects requiring high throughput procedures.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-017-0681-1) contains supplementary material, which is available to authorized users.

“Polar mutagenesis of bacterial transcriptional units using Cas12a”

“…Our results show that this sequence was easily engineered using the Cas12a-encoding plasmids designed by Yan et al (29). These plasmids are easy to use and allow multiple rounds of mutagenesis similar to previously reported Cas9 approaches (6). Cas12a also brings the advantage that it processes its own crRNAs, which we think will allow certain flexibility in the future when dealing with organisms with poorly characterised promoter sequences.…”

“…The efficiency of genome engineering strains has been shown to depend on the efficiencies of gRNAs to direct Cas9 DNA cleavage and donor oligos (6). Using the same crRNA (crRNA1) to generate several mutations at the same target sequence we saw that each mutation was produced with efficiencies ranging from 10% to 100%.…”

“…CRISPR/Cas genome editing in E. coli (5), opened the possibility of marker-less genetic engineering with single base resolution. Therefore, it is possible to now generate multi-scale libraries of mutants in a few d a y s (6). The objective of the following study was to design a strategy to shut-down the transcription of entire operons using a synthetic terminator sequence (7) in E. coli.…”

Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

“…Gomaa et al reported that recombination events had occurred between direct repeats of the artificial CRISPR‐array on the plasmid, losing the genome targeting spacer sequences. Other studies also found similar recombination‐induced spacer loss in the CRISPR array on plasmids . Moreover, under the genome‐targeting stress, the mutation or deletion that disrupts the cas genes would also allow the bacteria to survive .…”

Serine integrase recombinational engineering (SIRE): A versatile toolbox for genome editing

“…The nuclease aided methods make use of DNA cleaving enzymes such as the meganuclease I-SceI (Kuhlman & Cox, 2010) or the endonuclease Cas9 in CRISPR set-ups (Li et al, 2015;Ng, Hung, Kao, Zhou, & Zhang, 2016;Zerbini et al, 2017;Zhao et al, 2016), which both induce a lethal double-stranded break at the place of recognition. Repair is done by recombination with an introduced DNA fragment using the same λ-Red genes.…”