| Abstract [eng] |
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. |
