Cas3 has essential functions in CRISPR immunity but its other activities and roles, in vitro and in cells, are less widely known. We offer a concise review of the latest understanding and questions arising from studies of Cas3 mechanism during CRISPR immunity, and highlight recent attempts at using Cas3 for genetic editing. We then spotlight involvement of Cas3 in other aspects of cell biology, for which understanding is lacking—these focus on CRISPR systems as regulators of cellular processes in addition to defense against mobile genetic elements.

CRISPR-Cas systems provide adaptive immunity to mobile genetic elements in prokaryotes. In many bacteria, including E. coli, a specialized ribonucleoprotein complex called Cascade enacts immunity by“ an interference reaction" between CRISPR encoded RNA (crRNA) and invader DNA sequences called “protospacers”. Cascade recognizes invader DNA via short “protospacer adjacent motif” (PAM) sequences and crRNA-DNA complementarity. This triggers degradation of invader DNA by Cas3 protein and in some circumstances stimulates capture of new invader DNA protospacers for incorporation into CRISPR as “spacers” by Cas1 and Cas2 proteins, thus enhancing immunity. Co-expression of Cascade, Cas3 and crRNA is effective at giving E. coli cells resistance to phage lysis, if a transcriptional repressor of Cascade and CRISPR, H-NS, is inactivated (Δhns). We present further genetic analyses of the regulation of CRISPR-Cas mediated phage resistance in Δhns E. coli cells. We observed that E. coli Type I-E CRISPR-Cas mediated resistance to phage λ was strongly temperature dependent, when repeating previously published experimental procedures. Further genetic analyses highlighted the importance of culture conditions for controlling the extent of CRISPR immunity in E. coli. These data identified that expression levels of cas3 is an important limiting factor for successful resistance to phage. Significantly, we describe the new identification that cas3 is also under transcriptional control by H-NS but that this is exerted only in stationary phase cells. Regulation of cas3 is responsive to phase of growth, and to growth temperature in E. coli, impacting on the efficacy of CRISPR-Cas immunity in these experimental systems.

Prokaryotic adaptive immunity is established against mobile genetic elements (MGEs) by ‘naïve adaptation’ when DNA fragments from a newly encountered MGE are integrated into CRISPR–Cas systems. In Escherichia coli, DNA integration catalyzed by Cas1–Cas2 integrase is well understood in mechanistic and structural detail but much less is known about events prior to integration that generate DNA for capture by Cas1–Cas2. Naïve adaptation in E. coli is thought to depend on the DNA helicase-nuclease RecBCD for generating DNA fragments for capture by Cas1–Cas2. The genetics presented here show that naïve adaptation does not require RecBCD nuclease activity but that helicase activity may be important. RecA loading by RecBCD inhibits adaptation explaining previously observed adaptation phenotypes that implicated RecBCD nuclease activity. Genetic analysis of other E. coli nucleases and naïve adaptation revealed that 5′ ssDNA tailed DNA molecules promote new spacer acquisition. We show that purified E. coli Cas1–Cas2 complex binds to and nicks 5′ ssDNA tailed duplexes and propose that E. coli Cas1–Cas2 nuclease activity on such DNA structures supports naïve adaptation.

Prokaryotes use the adaptive immunity mediated via the Clustered Regularly Interspaced Short Palindromic Repeat and CRISPR associated (CRISPR-Cas) system for protection against invading elements such as phages and plasmids. The immunity is achieved by capturing small DNA fragments or spacers from foreign nucleic acids (protospacers) and integrating them into the host CRISPR locus. This step of CRISPR-Cas immunity called ‘naïve CRISPR adaptation’ requires the conserved Cas1-Cas2 complex and is often supported by variable host proteins that assist in spacer processing and integration. Bacteria that have acquired new spacers become immune to the same invading elements when reinfected. CRISPR-Cas immunity can also be updated by integrating new spacers from the same invading elements, a process called ‘primed adaptation‘. Only properly selected and integrated spacers are functional in the next steps of CRISPR immunity when their processed transcripts are used for RNA-guided target recognition and interference (target degradation). Capturing, trimming, and integrating new spacers in the correct orientation are universal steps of adaptation to all CRISPR-Cas systems, but some details are CRISPR-Cas type-specific and species-specific. In this review, we provide an overview of the mechanisms of CRISPR-Cas class 1 type I-E adaptation in Escherichia coli as a general model of adaptation processes (DNA capture and integration) that have been studied in detail. We focus on the role of many host non-Cas proteins involved in adaptation, particularly on the role of homologous recombination.

Cas3 nuclease-helicase is part of CRISPR immunity systems in many bacteria and archaea. In type I CRISPR, Cas3 nuclease degrades invader DNA that has been base-paired to crRNA as an R-loop within a “Cascade” complex. An R-loop is a DNA-RNA hybrid that includes a displaced single-strand DNA loop. Purified Cas3 from E. coli and the archaeon M. thermautrophicus can process R-loops without DNA/RNA sequence specificity and without Cascade. This has potential to affect other aspects of microbial biology that involve R-loops. Regulatory RNAs and host cell proteins modulate replication of ColE1 plasmids (e.g., pUC) from R-loop primers. We observed that Cas3 could override endogenous control of a ColE1 replicon, stimulating uncontrolled (“runaway”) replication and resulting in much higher plasmid yields. This effect was absent when using helicase-defective Cas3 (Cas3K320L) or a non-ColE1 plasmid, and was dependent on RNaseHI. Cas3 also promoted formation of plasmid multimers or concatemers, a phenotype consistent with deregulated ColE1 replication and typical of cells lacking RNaseHI. These effects of Cas3 on ColE1 plasmids are inconsistent with it unwinding R-loops in vivo, at least in this assay. We discuss a model of how Cas3 might be able to regulate RNA molecules in vivo, unless it is targeted to CRISPR defense by Cascade, or kept in check by RecG and RNaseHI.