Coupling the CRISPR/Cas9 System with Lambda Red Recombineering Enables Simplified Chromosomal Gene Replacement in Escherichia coli

Abstract

cTo date, most genetic engineering approaches coupling the type II Streptococcus pyogenes clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system to lambda Red recombineering have involved minor single nucleotide mutations. Here we show that procedures for carrying out more complex chromosomal gene replacements in Escherichia coli can be substantially enhanced through implementation of CRISPR/Cas9 genome editing. We developed a three-plasmid approach that allows not only highly efficient recombination of short single-stranded oligonucleotides but also replacement of multigene chromosomal stretches of DNA with large PCR products. By systematically challenging the proposed system with respect to the magnitude of chromosomal deletion and size of DNA insertion, we demonstrated DNA deletions of up to 19.4 kb, encompassing 19 nonessential chromosomal genes, and insertion of up to 3 kb of heterologous DNA with recombination efficiencies permitting mutant detection by colony PCR screening. Since CRISPR/Cas9-coupled recombineering does not rely on the use of chromosome-encoded antibiotic resistance, or flippase recombination for antibiotic marker recycling, our approach is simpler, less labor-intensive, and allows efficient production of gene replacement mutants that are both markerless and "scar"-less. Since its inception in 1998, phage recombinase-mediated homologous recombination, also known as recombineering, has revolutionized bacterial genetics, synthetic biology, and metabolic engineering (1, 2). Relying on either RecET from the Rac prophage (2) or the bacteriophage lambda Red proteins, Exo, Beta, and Gam (1), recombineering allows simple and efficient construction of gene knockout mutants via homologous recombination of a double-stranded DNA (dsDNA) PCR product with bacterial chromosomes (3). This work has led to the construction of an Escherichia coli K-12 single-gene knockout library (the Keio collection [4]), enabled widespread use of chromosome-encoded expression platforms for metabolic and strain engineering (5-11), and simplified conventional recombinant DNA technology through the advent of ligation-independent DNA cloning (2, 12). Automated, high-throughput, and multiplexed genome editing applications have also been envisioned, including multiplexed automated genome engineering (MAGE), generating up to 4.3 billion genomic mutants per day (13). Finally, phage-mediated recombineering systems have been identified and exploited for use in an array of bacterial genera (14-19).Recombineering can be carried out using either singlestranded DNA (ssDNA) or dsDNA substrates. Since recombineering occurs through an entirely ssDNA intermediate (20), ssDNA recombineering requires only an ssDNA recombinase (RecT or Beta) for recombination (21), whereas recombination of dsDNA substrates requires both an exonuclease (RecE or Exo) and its associated recombinase (RecT or Beta). For this reason, recombination of ssDNA substrates, such as oligonucleotides, is simpler, more efficient, and better understood ...

A CRISPR-Cas9 Assisted Non-Homologous End-Joining Strategy for One-step Engineering of Bacterial Genome

“…The CRISPR-Ca9 system has been broadly adopted in markerless genome engineering, and its utility and flexibility in genetic manipulation of the bacterial genome have improved dramatically34373839404142. Recently, Beier et al .…”

Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis

“…7C) was expected given that increasing insertion size has been shown to correlate with decreasing editing efficiency in CRISPR-Cas9 systems (10,14). Moreover, the reduced recombination frequency of the HA operon KI cassette could have been exacerbated by an excessive metabolic burden associated with HA synthesis, which is an energy-and carbon-intensive process directly competing with central metabolism and cell wall synthesis (41).…”

“…The CRISPR-induced double-stranded breaks (DSBs) enable selection of mutants that evade DNA cleavage via recombination of exogenous editing templates. Accordingly, the CRISPR-Cas9 system has proven to be an indispensable tool for genome editing in bacteria (9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22), yeasts (23,24) and higher eukaryotes (25)(26)(27)(28).…”

“…While CRISPR-Cas9 offers potential solutions to these technical issues in E. coli and S. cerevisiae (9,10,23,24), the protocols include multicopy plasmids which must be removed from the cell. In that regard, an ideal CRISPR-Cas9 system should facilitate multiple mutations, either in series or simultaneously, while imposing minimal physiological impact on the host.…”

Editing of the Bacillus subtilis Genome by the CRISPR-Cas9 System

“…So far, the CRISPR-Cas9 system has been applied in very few bacterial species to edit their genomes (9)(10)(11)(12)(13)(14)(15)). An example is the two-plasmid-based system constructed for engineering Escherichia coli (10).…”