CRISPR-Cas adaptation in Escherichia coli

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

CRISPR-Cas immunity, DNA repair and genome stability

© 2018 The Author(s). Co-opting of CRISPR-Cas ‘Interference’ reactions for editing the genomes of eukaryotic and prokaryotic cells has highlighted crucial support roles for DNA repair systems that strive to maintain genome stability. As front-runners in genome editing that targets DNA, the class 2 CRISPR-Cas enzymes Cas9 and Cas12a rely on repair of DNA double-strand breaks (DDSBs) by host DNA repair enzymes, using mechanisms that vary in how well they are understood. Data are emerging about the identities of DNA repair enzymes that support genome editing in human cells. At the same time, it is becoming apparent that CRISPR-Cas systems functioning in their native environment, bacteria or archaea, also need DNA repair enzymes. In this short review, we survey how DNA repair and CRISPR-Cas systems are intertwined. We consider how understanding DNA repair and CRISPR-Cas interference reactions in nature might help improve the efficacy of genome editing procedures that utilise homologous or analogous systems in human and other cells

Cas3 Protein—A Review of a Multi-Tasking Machine

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 element