Microbial treasure trove: Unravelling the potential of class 2 CRISPR–Cas systems

Animals are known to possess a large repertoire of immune systems with a high degree of sophistication. On the other hand, the immune systems found in bacteria and archaea appeared to be much more rudimentary. However, the ground-breaking discovery of a novel immune system, CRISPR-Cas, proved otherwise. CRISPR-Cas, is unique in being both adaptive and heritable, and it relies on small RNA molecules that specifically guide the defence system to matching invader DNA sequences. This natural defence system has been successfully repurposed into a valuable molecular technology which is a robust, efficient, easy-to-use method to precisely alter DNA sequences of living organisms. The current CRISPR-based technologies, mostly employ the Cas9 protein, for diverse biotechnological applications. Nevertheless, the natural diversity of CRISPR-Cas systems is remarkably extensive, including systems that target DNA, systems that target RNA, and systems that target both DNA and RNA. The diverse class 2 CRISPR nucleases have unique molecular features that contribute to an expansive toolbox for genome and transcriptome engineering. These nucleases differ greatly in their structure and mechanisms. These differences could be exploited as complementary applications creating numerous CRISPR-based technologies possibly with favourable specificity, efficiency and/or delivery. This thesis explores the diversity of Class 2 CRISPR–Cas systems and provides mechanistic insights into different class 2 nucleases. In addition, it describes potential applications to expand the current repertoire of CRISPR-based technologies.

Due to the everlasting arms race between prokaryotes and their viruses, the rapid evolution of CRISPR–Cas systems has resulted in extreme structural and functional diversity. As a result, a plethora of distinct CRISPR–Cas systems are represented in genomes of most archaea and almost half of the bacteria. The key players of this system are the crRNA binding effector complexes, and the associated nuclease domains. CRISPR–Cas systems are currently grouped into two classes each of which is subdivided into three types. Class 1 systems (consisting of types I, III, and IV) use a multi-subunit protein complex to achieve interference, and class 2 systems (consisting of types II, V, and VI) utilize a single multi-domain protein, that have been repurposed for genome editing applications in a wide range of organisms. The mechanism of crRNA maturation in CRISPR–Cas12a systems was unravelled during this thesis. Unlike the type II nuclease Cas9, which utilizes a tracrRNA as well as endogenous RNaseIII for maturation of its dual crRNA/tracrRNA guides, pre-crRNA processing in the Cas12a system proceeds in the absence of tracrRNA or other Cas proteins. It was demonstrated that Cas12a nucleases possess a previously unknown RNase domain that is responsible for cleaving the pre-crRNA to generate the mature crRNAs. The typical cleavage pattern revealed that Cas12a recognizes specific secondary structures and/or motifs on its direct repeats. Furthermore, the ability to autonomously process crRNA has significant implications from a genome editing standpoint, as it provides a simple route to editing multiple genomic loci at a time (multiplex editing). Using a single customized CRISPR array up to four genes in mammalian cells ex vivo and up to three genes in mouse brain cells in vivo were shown to be edited simultaneously.

The characterisation of a novel, diminutive type V-U1 Cas protein from Mycolicibacterium mucogenicum (MmuC2c4) was described in this thesis. Type V-U proteins are highly similar to the typical transposon-encoded TnpB-like proteins and each of them (type VU-1 to type VU-5) appear to have originated independently from distinct TnpB families. Akin to most type V proteins, MmuC2c4 was shown to recognize a 5’-TTN-3’ PAM on a double-stranded target DNA.

The characterisation of a type II-C Cas9 orthologue of the thermophilic bacterium Geobacillus thermodenitrificans T12, ThermoCas9 is described. This is one of the first reports that provides fundamental insights into a thermophilic CRISPR–Cas9 family member. It was demonstrated that ThermoCas9 is active in vitro between 20 and 70 ℃, that the structure of its sgRNA influences its activity at elevated temperatures, it has a more stringent PAM-preference at lower temperatures, it does not tolerate extensive spacer-protospacer mismatches, and it preferentially cleaves plasmid DNA compared to linear DNA. Furthermore, ThermoCas9 was employed for pyrF gene deletion and transcriptional silencing of ldhL gene at 55 ℃ in Bacillus smithii ET 138 and for pyrF gene deletion at 37 ℃ in Pseudomonas putida. This is the first time Cas9-based bacterial genome editing and silencing tools were used at temperatures above 42 ℃.

Four Cas12a orthologues were assessed for their salt tolerance as well as pH- and temperature stability using biochemical assays as described. Subsequently, Francisella tularensis subsp. novicida (FnCas12a) and Eubacterium eligens (EeCas12a) were applied for genome editing in a moderate thermophilic bacterium, Bacillus smithii. It is demonstrated that FnCas12a and EeCas12a are sub-optimally active in vivo at temperatures above 45 ℃. The wide growth temperature range of B. smithii ET 138 was employed for the controllable induction of Cas12a expression at temperatures below the 45 ℃ threshold. It was demonstrated that a mutant can be generated within a short span of 2-3 days. This process can be easily adapted for gene editing applications in a wide variety of both mesophilic and moderate thermophilic organisms. potential to harness the activity of anti-CRISPR (Acr) proteins for controllable bacterial genome engineering was also investigated. The Acr protein from Neisseria meningitidis (AcrIIC1Nme) was employed as an “on/off-switch” to control the activity of thermostable Cas9 orthologues from Geobacillus thermodenitrificans T12 (ThermoCas9) and Geobacillus  stearothermophilus (GeoCas9). Initially, it was proven that both ThermoCas9 and GeoCas9 can introduce lethal dsDNA breaks in E. coli at 37 ℃ in a tuneable manner. Next, it was demonstrated that AcrIIC1Nme traps both tested Cas9 orthologues in a DNA-bound, catalytically inactive state. The Cas9/AcrIIC1Nme complexes can promote a transcriptional silencing effect with efficiency comparable to the catalytically “dead” ThermodCas9 and GeodCas9 variants. Finally, a single-vector, tightly controllable and highly efficient Cas9/AcrIIC1Nme-based tool for coupled silencing and targeting in E. coli was developed. This tool may serve as a basis for further developing a controllable genome editing and transcriptional regulation in model as well as non-model microorganisms.

Furthermore, a novel biological role and mechanism for the CRISPR–Cas9 system of the pathogen Campylobacter jejuni (CjeCas9) was uncovered. It was demonstrated that upon C. jejuni infection of human cells, CjeCas9 is secreted into the cytoplasm of the infected cells and it can  autonomously enter the nucleus. Inside the nucleus, it catalyses metal-dependent and sequence-independent nicking of double stranded DNA, eventually leading to cell death. Genome editing using CjeCas9 was compared with the commonly used Cas9 from  Streptococcus pyogenes (SpyCas9), and the latter was shown to be superior in creating indels. It was concluded that the unique catalytic features make CjeCas9 nuclease less suitable for genome editing applications.

In conclusion, the research described in this PhD thesis has uncovered novel molecular requirements and mechanisms of several unique Class 2 CRISPR–Cas nucleases. Besides gaining insights into their biochemical mechanism, the potential of Class 2 nucleases has been harnessed for biotechnological applications. Additionally, a unique role and mechanism of CRISPR–Cas in virulence has been elucidated. The characterisation of nucleases such as FnCas12a, EeCas12a, MmuC2c4 and ThermoCas9 opens up exciting possibilities of utilizing them as genome and transcriptome engineering tools.