Next Generation Prokaryotic Engineering: The CRISPR-Cas Toolkit

The increasing demand for environmentally friendly production processes of green chemicals and fuels has stimulated research in microbial metabolic engineering. CRISPR-Cas-based tools for genome editing and expression control have enabled fast, easy, and accurate strain development for established production platform organisms, such as Escherichia coli and Saccharomyces cerevisiae. However, the growing interest in alternative production hosts, for which genome editing options are generally limited, requires further developing such engineering tools. In this review, we discuss established and emerging CRISPR-Cas-based tools for genome editing and transcription control of model and non-model prokaryotes, and we analyse the possibilities for further improvement and expansion of these tools for next generation prokaryotic engineering. The increasing demand for environmentally friendly production processes of green chemicals and fuels has stimulated research in microbial metabolic engineering. CRISPR-Cas-based tools for genome editing and expression control have enabled fast, easy, and accurate strain development for established production platform organisms, such as Escherichia coli and Saccharomyces cerevisiae. However, the growing interest in alternative production hosts, for which genome editing options are generally limited, requires further developing such engineering tools. In this review, we discuss established and emerging CRISPR-Cas-based tools for genome editing and transcription control of model and non-model prokaryotes, and we analyse the possibilities for further improvement and expansion of these tools for next generation prokaryotic engineering. SpyCas9 has recently been established as an efficient counterselection system in combination with homologous recombination-based strategies for bacterial genome editing. Besides the traditionally used SpyCas9, other CRISPR-Cas systems (both heterologous and native) are currently being evaluated in bacteria for their editing potential. Catalytically inactive variants of CRISPR-Cas systems are used for transcriptional control in bacteria with great potential for fundamental research and applications. SpyCas9 has recently been established as an efficient counterselection system in combination with homologous recombination-based strategies for bacterial genome editing. Besides the traditionally used SpyCas9, other CRISPR-Cas systems (both heterologous and native) are currently being evaluated in bacteria for their editing potential. Catalytically inactive variants of CRISPR-Cas systems are used for transcriptional control in bacteria with great potential for fundamental research and applications. a bacterial or archaeal DNA array constituted of small (30–45 nt long) sequences, usually of foreign origin, which are separated by (almost) identical repeat sequences of similar size. enzymes encoded by cas genes that generally reside in close proximity to a CRISPR array, taking part in any of the three stages of the CRISPR-Cas-based immunity. a bacterial or archaeal DNA locus constituted of a CRISPR array, its corresponding Cas genes and possible ancillary modules. an RNA molecule that guides the targeting of CRISPR-Cas systems. It originates from the CRISPR locus and is composed of the processed transcript of a spacer and a CRISPR array repeat. damage of the DNA such that the backbones of both strands are cleaved simultaneously. recombineering based on double-stranded linear DNA fragments. DSDR requires the expression of a phage-derived exonuclease (Exo/α or RecE) and an ssDNA-binding protein (Beta or RecT). In the case of the λ-Red system, also Gam (γ) can be added, which inhibits the DNA exonucleases of the hosts. an error-prone DSDB repair mechanism, based on the concerted activities of DNA-binding protein Ku and ligase LigD. a DNA sequence, targeted for cleavage by a CRISPR-Cas system, that is identical to a spacer sequence of the corresponding CRISPR array. the short (2–8 nt) conserved sequence adjacent to a protospacer, mandatory for recognition and targeting by CRISPR-Cas systems (except type III), as a means to discriminate self (CRISPR spacer) from non-self (invader protospacer). genetic engineering tool mediated by phage-derived recombination systems, such as phage λ-derived Red αβγ or Rac prophage-derived RecET, that facilitate the homologous recombination between ssDNA or dsDNA fragments with complementary genome sequences. the first 8–12 bp of a protospacer immediately adjacent to the PAM sequence; required together with the PAM for successful targeting. a synthetic chimera combining the crRNA and tracrRNA into a single CRISPR guide. cleavage of the DNA such that the backbone of one of the strands is nicked while the other one remains intact. recombineering based on single-stranded linear DNA fragments. SSDR only requires expression of the phage-derived ssDNA-binding protein Beta or RecT. short (30–45 nt) DNA sequences derived from target genetic elements; located in the CRISPR array and flanked by repeat sequences. an RNA molecule encoded by type II CRISPR loci that hybridizes to the repeat parts of crRNAs. The crRNA:tracrRNA hybrid is required for Cas9 activation.