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3 CRISPR Cas: From Bacterial Adaptive Immunity to the Swiss Army Knife of Drug Discovery

       Saša Šviković

       Genome Engineering, Discovery Sciences, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden

3.1 Introduction

      Over the course of billions of years of evolution, prokaryotes have developed multiple ways to stay ahead of their fiercest foes, mobile genetic elements. These defense systems include a variety of nucleases, DNA‐modifying enzymes, chemical inhibitors, cell death‐inducing signaling pathways, and some not yet fully understood protection systems (Bernheim and Sorek 2020; Hampton et al. 2020). These systems are incredibly diverse and widespread among prokaryotes, and studies into how these systems function ultimately led to the rise of new tools that have revolutionized biomedical sciences. Similarly to how the discovery of an innate bacterial immune system mediated by restriction endonucleases has ushered a revolution in biotechnology (Roberts 2005), the relatively recent discovery of CRISPR‐Cas systems, an adaptive immune component of prokaryotic defense systems, and their application in genome engineering has completely changed the way how research in life sciences is done. In this Chapter, we will address the basics of CRISPR biology, the diversity and current classification schemes, and how the mechanistic understanding of these diverse systems can give rise to novel tools for biomedical science.

3.2 CRISPR Biology in a Nutshell

      CRISPR, or Clustered Regularly Interspaced Short Palindromic Repeats, represents an adaptive immune system of microbes, able to adapt in response to invasions by mobile genetic elements such as bacteriophages, plasmids, and transposons. The locus encoding for the components of the CRISPR system was discovered as an array of palindromic repeats interrupted by a 20–40 nt sequence downstream of the iap gene in Escherichia coli (Ishino et al. 1987; Nakata et al. 1989). Thanks to the increasing availability of sequences from the microbial species, the structure of CRISPR loci, with many properties relating to their function, was uncovered (Mojica et al. 2000; Jansen et al. 2002).

The identifying trait of the CRISPR locus in an array of alternating identical repeats, interleaved by unique spacersequence (Figure 3.1a), with extreme interspecies diversity in sequence and size, usually in the range of 20–72 bp (Grissa et al. 2007). Bioinformatic analysis of spacer sequences within CRISPR arrays has shown a striking homology to various sequences found in bacteriophages and plasmids (Bolotin et al. 2005; Mojica et al. 2005; Pourcel et al. 2005), which was an early clue that CRISPR systems act as a defense mechanism against mobile genetic elements (Makarova et al. 2006). The collection of the spacers within a microbial species is extremely diverse and dynamic (Weinberger et al. 2012), as the microbes can acquire new spacers upon neutralizing the infection (Barrangou et al. 2007; Marraffini and Sontheimer 2008). The spacer acquisition represents the adaptive component of this immune system, as this confers resistance to reinfection by the same mobile genetic element.

      In most species, repeat monomers vary between 23 and 47 bp in length (Godde and Bickerton 2006), and in most species consist of partially palindromic sequences, able to form stable secondary structures (Kunin et al. 2007). Related species can have similar repeat sequences, but the overall bacterial and archaeal sequence diversity of both spacers and repeats is great.

Figure 3.1 Phases of CRISPR‐mediated immune response. (a) The simplified architecture of an idealized CRISPR locus, containing a CRISPR array consisting of a promoter‐containing leader sequence and spacer and repeat sequences (depicted on the left) and several different cas genes (depicted in different colors on the right), which generate functional components of the CRISPR‐Cas system. During the infection by a mobile genetic element (depicted as a bacteriophage here), invading DNA can be processed by the RecBCD/AddAB systems so that it can be integrated as a spacer by the Cas1:Cas2 adaptation complex as a part of the naïve adaptation phase (b). In the expression phase (c), the CRISPR array is transcribed into pre‐crRNA, which is further processed to crRNA and paired with Cas proteins to form a functional effector complex. Upon a reinfection with phage carrying the same or similar protospacer sequence, the effector complex recognizes the sequence (d) and induces the degradation of the target. Suitable fragments generated during the interference phase can be used as spacers in the primed adaptation phase (e), updating the CRISPR array with the most recent version of suitable spacers.

Immediately adjacent to the CRISPR array is an AT‐rich element, most frequently between 100 and 400 bp long (Jansen et al. 2002; Alkhnbashi et al. 2016). It was subsequently shown that this sequence, termed leader sequence, contains the promoter used to direct transcription of the CRISPR array (Figure 3.1a) and determines the transcriptional start site (Pougach et al. 2010; Pul et al. 2010). Transcription of the CRISPR locus and its subsequent processing ultimately gives rise to CRISPR RNA (crRNA), which provides specificity to the CRISPR systems and guides the degradation of the invading nucleic acids (Brouns et al. 2008). Much like the repeats, leaders are up to 80% identical within a genome, but quite dissimilar among species.

CRISPR arrays are often surrounded by a collection of conserved protein coding genes, termed CRISPR‐associated or cas genes; these genes encode for the protein components of the CRISPR immunological systems. The groups of protein coding genes vary dramatically in composition, order, and sequence between species, underpinning the diversity of CRISPR systems, and their modes of action. Indeed, the complement of cas