Authors
Creative Biogene
Summary
The CRISPR-Cas9 system was repurposed to edit bacterial genomes after its discovery and biochemical characterization. It can now be considered as a tool of choice to engineer probiotic strains for additive therapies. Genome editing strategies rely on the use of a guide RNA designed to target a chromosomal sequence of interest where Cas9 will cut.
Introduction
CRISPR Editing of Streptococcus
CRISPR-Cas9 could be employed to achieve precise scar-less genome editing in bacteria. E.g., through natural transformation and recombination, Cas9 protein from Streptococcus pyogenes was integrated into the chromosome of Streptococcus pneumoniae, an opportunistic pathogen commonly present in the respiratory tract, sinuses and nasal cavities of healthy carriers. Cas9 can be used to select for the introduction of mutations at desired positions without the need to leave a selection marker or a scar at the edited position.
CRISPR Editing of E. coli
Less recombinogenic bacteria such as E. coli requires the use of the phage lambda red recombination system to promote editing and repair of the Cas9-mediated breaks. CRISPR-Cas9 editing strategies typically rely on the expression of guide RNAs, Cas9 and the lambda red genes from one or several plasmids. Template DNA can be provided as short single-stranded DNA, short or long double stranded DNA (typically PCR products), or cloned on a plasmid. In all cases, Cas9 is guided to introduce a break at a position of interest, which leads to cell death unless the target DNA was modified, or unless it can be repaired by recombination with the template DNA. With the I-SceI nuclease, a non-replicative vector can be integrated through homologous recombination into the locus of interest, followed by Cas9 cleavage of the vector backbone leading to recombination and recovery of the desired scar-less mutation. High-throughput modifications of many positions in parallel have been performed. Pools of oligonucleotides designed to carry both a homologous repair cassette and a sgRNA can be cloned on a vector, yielding a library that can be used to perform multiplexed recombineering. The replacement of large fragments of the E. coli genome with synthetic DNA has also been available. Multiple guide RNAs were used simultaneously in the same E. coli cell to cleave two positions in the E. coli chromosome and two positions on a plasmid carrying a synthetic DNA fragment, triggering the replacement of the chromosomal DNA through homologous recombination. E. coli strains, like Nissle 1917, have been engineered to express antigens, antimicrobial compounds, enzymes to disperse biofilms, quorum sensing molecules that control pathogen virulence, metabolic functions of interest and more.
A novel and powerful approach in the field of genome editing is the use of the catalytic dead variant of Cas9 (dCas9) fused to a cytosine deaminase or an adenosine deaminases in order to convert C•G to T•A or A•T to G•C at specific target positions without the need to introduce a DNA break. Cytosines or adenosines located within a small window 15–25 bp from the protospacer adjacent motif (PAM) are modified, leading to a somewhat random mutational outcome when several C or A are present in the target window. Nonetheless, this approach appears to be very efficient and can easily be used to modify many positions in parallel, which has been used to optimize the E. coli genome.
CRISPR Editing of Probiotic Bacteria
The most commonly used probiotic bacteria are Bifidobacteria and Lactobacilli. Engineered Lactobacilli are being developed as targeted therapies against a wide range of diseases including oral mucositis, inflammatory bowel disease, viral and bacterial infections.
CRISPR Editing of Clostridia
Other bacteria of interest include the Clostridia, a diverse class of bacteria that include strains of industrial interest but also many commensals of the gut microbiome, of which a few—and most notoriously Clostridium difficile—can be opportunistic pathogens. Several reports have demonstrated the use of CRISPR tools to modify species of biotechnological interest including Clostridium acetobutylicum, Clostridium beijerinckii and Clostridium cellulolyticum.
CRISPR-Cas9 tools have also been developed for Staphylococcus aureus, an opportunistic pathogen commonly found on the skin. Staphylococci can either be beneficial commensals or pathogens depending on the genetic makeup of specific strains. In addition to these microbiome-associated bacteria, CRISPR-Cas9 tools have been developed for other bacterial species including Bacillus subtilis, Bacillus licheniformis, Bacillus thuringiensis, Pichia pastoris, etc.
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