Current and future prospects for CRISPR-based tools in bacteria

Abstract

CRISPR-Cas systems have rapidly transitioned from intriguing prokaryotic defense systems to powerful and versatile biomolecular tools. This article reviews how these systems have been translated into technologies to manipulate bacterial genetics, physiology, and communities. Recent applications in bacteria have centered on multiplexed genome editing, programmable gene regulation, and sequence-specific antimicrobials, while future applications can build on advances in eukaryotes, the rich natural diversity of CRISPR-Cas systems, and the untapped potential of CRISPR-based DNA acquisition. Overall, these systems have formed the basis of an ever-expanding genetic toolbox and hold tremendous potential for our future understanding and engineering of the bacterial world.

Keywords: Cas9; antimicrobials; genetic circuits; genetic control; genome engineering; undomesticated microbes.

Figure 1

Overview of adaptive immunity by CRISPR-Cas systems. Immunity is conferred through three steps: acquisition, expression, and interference. Acquisition: a small piece of the invader DNA is integrated as a new spacer within the CRISPR array. Expression: the CRISPR array is transcribed and undergoes processing by the Cas proteins and accessory factors to form the CRISPR RNA (crRNA). Interference: the spacer portion of the crRNA serves as a recognition element for the Cas proteins to target invading DNA (Type I, II, III, V) or RNA (Type III). Type I, II, and V systems require a protospacer-adjacent motif (PAM, yellow box) for target recognition. The current understanding of Type IV systems is limited to bioinformatics analyses.

Figure 2

Design of the sgRNA. (A) The natural crRNA and tracrRNA are connected by a loop to form a single-guide RNA (sgRNA). (B) Processing of the natural Type II CRISPR array requires two additional factors: RNase III and tracrRNA. (C) Use of sgRNAs bypasses the requirement for RNase III and tracrRNA.

Figure 3

Genome editing with CRISPR-Cas9 in bacteria. (A) DNA cleavage by Cas9 is generally lethal, leading to clearance of cells that did not undergo recombination. (B) Employing a nicking Cas9 appears to either temper the lethality of genome targeting or drive genome editing. (C) Utilizing the bacterial non-homologous end-joining pathway composed of the Ku and LigD can rescue the lethality of Cas9-based genome targeting and drive indel formation.

Figure 4

CRISPR-based gene regulation. (A) A catalytically dead Cas9 (dCas9) can be targeted to the promoter or coding region of a gene, blocking transcription. (B) A fusion between dCas9 and the ω subunit of RNA polymerase (dCas9-ω) can recruit RNA polymerase to activate transcription. (C) Eliminating the cas3 gene from Type I systems can allow targeted DNA binding and transcriptional repression with Cascade. (D) Type III systems can be readily co- opted to bind and cleave target mRNAs. (E) The Francisella novicida Cas9 utilizes a scaRNA to silence an endogenous gene through putative base-pairing interactions. (F) Introducing a DNA oligonucleotide PAMmer allows Cas9 to bind and cleave RNAs complementary to the guide portion of an sgRNA.

Figure 5

CRISPR-based antimicrobials. (A) Targeting the bacterial genome leads to potent and sequence-specific cell killing. (B) Bacteriophages can be employed to deliver plasmids encoding CRISPR-Cas9, leading to targeted killing or plasmid clearance.

Figure 6

Opportunities for CRISPR technologies in bacteria. (A) Building on advances in eukaryotes. The FokI nuclease can be fused to Cas9 as an alternative means of introducing double-stranded breaks as part of genome editing. Fusing binding domains to Cas9 or the 3′ end of the sgRNA can recruit other proteins to regulate gene expression (e.g. the ω subunit of RNA polymerase) or to dynamically image genomic loci (e.g. GFP). (B) Libraries of crRNAs or sgRNAs can be compiled and subjected to specific environmental conditions to rapidly screen for genes that either promote or inhibit growth. (C) Acquisition could be used to count transient events such as an environmental stimulus. (D) Acquisition could also be harnessed to integrate large pieces of synthetic DNA into the genome.