D Silva, Jovita2025-01-052025-01-05https://hdl.handle.net/1885/733731498Prokaryotes have developed an array of defence mechanisms against invading mobile genetic elements (MGE's) such as bacteriophages and plasmids. Among them, CRISPR-Cas (Clustered regularly interspaced short palindromic repeats- CRISPR associated proteins) is the only known adaptive defence system. CRISPR-Cas systems generate an RNA from a memory cassette of past infections, which guides the Cas protein to bind to and interfere with complementary invading DNA during a subsequent infection. This RNA guided nuclease activity is programmable and thus over the last decade has been explored for gene editing and molecular diagnostics. CRISPR-Cas systems are numerous and diverse, thus classified into 2 classes and 6 types. The type V systems, also called CRISPR-Cas12 embodies an effector protein with a conserved RuvC catalytic site that causes double-stranded breaks in the target DNA (some Cas12 harbour inactive RuvC). The Cas12 family by itself is very diverse and over the last few years, there has been a growing interest in understanding the evolution of the type V CRISPR Cas systems to unravel how these systems have evolved? Several CRISPR-Cas systems have been evolutionarily linked to transposable elements. Cas12 systems have evolved from a transposon associated element IS200/IS605 or IS607, called 'TnpB'. TnpB is also an RNA guided nuclease, but instead of defence, it functions to identify its targeted site for transposition. This raises the question: how did a transposon such as TnpB evolve to acquire the ability to protect its host against foreign genetic elements? In order to understand the evolution of TnpB into a fully functional Cas12 system (such as Cas12a), this thesis focusses on the identification and characterisation of their evolutionary intermediates. I undertook a computational analysis identifying Cas12 and TnpB effectors from a large dataset of assembled metagenomes which was then subjected to phylogenetic analysis. Evolutionary intermediates sub-types of Cas12 were identified as those that were closely linked to typical TnpB's and did not occur with adaptation proteins such as Cas1 and Cas2. Three such sub-types, type V-U1, type V-U2 and type V-U4 were then investigated by selectively characterising representative of each sub-type. All systems were structurally aligned with pre-existing structures to identify similarities and unique features that tied them to TnpB. Their loci were heterologously expressed to study their expression profiles and identify the small RNA require for interference. The candidate effectors were then subjected to in vitro assays, bacteriophage infection assays and plasmid interference assays to assess their programmability, interference, to understand their mechanism of action and validate their ability to function as ancestral defence systems. Through functional characterisation of three different evolutionary intermediate sub-types, the key findings emphasised on the independent evolution of each subtype from a different TnpB on multiple independent occurrences. I characterised the sub-type V-U1 to have evolved from be a nuclease inactive variant, that interfered with dsDNA bacteriophages and plasmidic elements through binding to its target sequence thereby causing CRISPR interference. Conversely, type V-U2, did not exhibit any bacteriophage defence against bacteriophages, but harboured a RuvC active catalytic centre that facilitated plasmidic interference. Type V-U4 was most similar to the only characterised TnpB, ISDra2. It harboured a RuvC intact catalytic domain that together with small functional RNAs targeted double-stranded DNA bacteriophage. Overall, identifying and understanding the elemental characteristics of three independent evolutionary inter-mediatory Cas12 subtypes, revealed properties that are ancestral and most essential in the functioning of Cas12 systems. This works more predominantly unravels the blueprint of Cas12's evolution from TnpB transposable elements.en-AU202510.25911/JBW5-GG96