Greta Bigelyte

Prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems provide adaptive immunity against foreign nucleic acids. Guided by RNAs, CRISPR-Cas nucleases theoretically can cleave any double-stranded (ds) DNA target of interest and this flexibility was utilized to adopt these nucleases as novel tools for genome editing. However, the size of commonly used Cas9 and Cas12a proteins (1300 – 1500 amino acids) remains one of the biggest obstacles limiting their cellular delivery. Therefore, recently discovered Cas12f nucleases, which are half the size of Cas9 and Cas12a, could provide an attractive alternative in these applications. This dissertation presents a collection of 10 exceptionally compact (422–603 amino acids) CRISPR–Cas12f nucleases. In this work, we show for the first time that Cas12f proteins cleave dsDNA in the protospacer adjacent motif (PAM) dependent manner. Additionally, we demonstrated that CRISPR-Cas12f1 systems from Syntrophomonas palmitatica (Sp) and Acidibacillus sulfuroxidans (As) provide robust dsDNA interference activity in heterologous Escherichia coli cells. Therefore, SpCas12f1 and AsCas12f1 nucleases were selected for thorough biochemical characterization which allowed us to demonstrate that SpCas12f1 is able to cleave genomic DNA in human and plant cells. Altogether, these findings pave the way for the development of miniature Cas12f-based genome editing tools.

Class 2 CRISPR systems are exceptionally diverse, nevertheless, all share a single effector protein that contains a conserved RuvC-like nuclease domain. Interestingly, the size of these CRISPR-associated (Cas) nucleases ranges from >1000 amino acids (aa) for Cas9/Cas12a to as small as 400-600 aa for Cas12f. For in vivo genome editing applications, compact RNA-guided nucleases are desirable and would streamline cellular delivery approaches. Although miniature Cas12f effectors have been shown to cleave double-stranded DNA, targeted DNA modification in eukaryotic cells has yet to be demonstrated. Here, we biochemically characterize two miniature type V-F Cas nucleases, SpCas12f1 (497 aa) and AsCas12f1 (422 aa), and show that SpCas12f1 functions in both plant and human cells to produce targeted modifications with outcomes in plants being enhanced with short heat pulses. Our findings pave the way for the development of miniature Cas12f1-based genome editing tools.

Transposition has a key role in reshaping genomes of all living organisms¹. Insertion sequences of IS200/IS605 and IS607 families² are among the simplest mobile genetic elements and contain only the genes that are required for their transposition and its regulation. These elements encode tnpA transposase, which is essential for mobilization, and often carry an accessory tnpB gene, which is dispensable for transposition. Although the role of TnpA in transposon mobilization of IS200/IS605 is well documented, the function of TnpB has remained largely unknown. It had been suggested that TnpB has a role in the regulation of transposition, although no mechanism for this has been established^3–5. A bioinformatic analysis indicated that TnpB might be a predecessor of the CRISPR–Cas9/Cas12 nucleases^6–8. However, no biochemical activities have been ascribed to TnpB. Here we show that TnpB of Deinococcus radiodurans ISDra2 is an RNA-directed nuclease that is guided by an RNA, derived from the right-end element of a transposon, to cleave DNA next to the 5′-TTGAT transposon-associated motif. We also show that TnpB could be reprogrammed to cleave DNA target sites in human cells. Together, this study expands our understanding of transposition mechanisms by highlighting the role of TnpB in transposition, experimentally confirms that TnpB is a functional progenitor of CRISPR–Cas nucleases and establishes TnpB as a prototype of a new system for genome editing.