RNA-guided editing of bacterial genomes using CRISPR-Cas systems

Abstract

The targeting of nucleases to specific DNA sequences facilitates genome editing. Recent work demonstrated that the CRISPR-associated (Cas) nuclease Cas9 can be targeted to sequences in vitro simply by modifying a short7 CRISPR RNA (crRNA) guide. Here we use this CRISPR-Cas system to introduce marker-free mutations in Streptococcus pneumoniae and Escherichia coli. The approach involves re-programming Cas9 by using a crRNA complementary to a target chromosomal locus and introducing a template DNA harboring a desired mutation and an altered crRNA recognition site for recombination with the target locus. We exhaustively analyze Cas9 target requirements to define the range of targetable sequences and show strategies for editing sites that do not meet these requirements. Alone or together with recombineering, CRISPR assisted editing induces recombination at the targeted locus and kills non-edited cells leading to a recovery of close to a 100% of edited cells. Multiple crRNA can be used to modify several loci simultaneously. Our results show that CRISPR-mediated genome editing only requires programming of the crRNA and template sequences and thus constitutes a useful tool for genetic engineering.

A standardized workflow for surveying recombinases expands bacterial genome‐editing capabilities

“…Several single nucleotide mutations to the rpsL gene, which encodes the ribosomal protein S12, have been characterized as conferring Sm resistance in different bacteria (Funatsu and Wittmann, 1972; Timms et al ., 1992; Gregory et al ., 2001). This array of modifications make the antibiotic marker a robust tool for genomic mutagenesis in E. coli (Jiang et al ., 2013) and P. syringae (Swingle et al ., 2010). Given the negligible occurrence rates of spontaneous mutants observed in P. putida (Jatsenko et al ., 2010), we chose streptomycin resistance as conferred by oligo‐mediated mutagenesis to the P. putida rpsL gene as a measure of putative recombinase activity.…”

“…SR generates an A‐G mismatch (poorly detected by MMR) that introduces a single nucleotide change (A→C) in codon K43 (AAA‐Lys), resulting in T43 (ACA‐Thr). The K43T missense mutation is analogous to the classical K42T mutation of the rpsL gene of E. coli (Timms et al ., 1992; Jiang et al ., 2013), which confers resistance to streptomycin.…”

Mitigating amphibian chytridiomycoses in nature

“…We do not dismiss the possibility that assisted selection might provide conservation benefits, only caution that the current knowledge base indicates significant research is still required before natural and assisted selection can be applied widely to chytrid mitigation. If genetic determinants of host resistance are identified in multiple amphibian species and new technologies for genetic manipulation prove amenable to immunogenetic modification of susceptible amphibian species, the situation might change, but it will also open up new ethical issues for conservationists [60][61][62]. Clearly, it is imperative to continue investigating the genetic basis of amphibian resistance and novel means by which it can be augmented.…”

Directed evolution combined with synthetic biology strategies expedite semi-rational engineering of genes and genomes

“…More recently, the clustered, regularly interspaced, short palindromic repeats (CRISPR)-Cas9 (endonuclease)-mediated genome-editing technology, which depends on specific homologous recombination and nuclease-specific cleavage, has been developed and applied in genome engineering. 21,22 To simplify the CRISPR process and broaden its applications, the small guide RNA, a hybrid of the trans-activating crRNA and the precursor CRISPR RNA, was constructed and employed in genome editing such as gene inactivation, precise mutations, and insertions. 22,23 In addition, the CRISPR-Cas9 system was also engineered to down-regulate gene expression at the transcriptional level by inactivating the Cas9 nuclease, 23 demonstrating its versatile applications in genome engineering.…”

“…21,22 To simplify the CRISPR process and broaden its applications, the small guide RNA, a hybrid of the trans-activating crRNA and the precursor CRISPR RNA, was constructed and employed in genome editing such as gene inactivation, precise mutations, and insertions. 22,23 In addition, the CRISPR-Cas9 system was also engineered to down-regulate gene expression at the transcriptional level by inactivating the Cas9 nuclease, 23 demonstrating its versatile applications in genome engineering. Consequently, constructing an optimized biosynthesis pathway at the genome level by applying these bioengineering tools is a promising and attractive option for the near future (Fig.…”