| Abstract [eng] |
Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats – CRISPR associated) systems provide adaptive immunity to bacteria and archaea against foreign nucleic acids. Cas proteins are specific nucleases, which can be guided to any DNA target by changing the sequence of small RNA molecules which form complexes with these enzymes. This enabled the use of Cas nucleases in genome editing editing applications. However, the actual targeting space of Cas proteins is limited by the requirement of the recognition of a short nucleic acid sequence near the target, termed the PAM (protospacer adjacent motif), which is inherent to each individual Cas nuclease. Furthermore, the relatively large size of the most used genome editors CRISPR-Cas9 and Cas12a (~1300 amino acids) make the delivery of these systems into cells a challenge. We sought to harness the natural diversity of CRISPR-Cas systems to search for novel Cas nucleases which could help circumvent these issues. This thesis presents a collection of 79 type II CRISPR-Cas9 nucleases which recognize diverse PAM sequences, as well as exhibit varied physical and biochemical properties. Also, we discovered and evaluated experimentally a new family of compact (~860 amino acids) type V CRISPR-Cas12l effectors and their nuclease activity. In summary, the nucleases identified and characterized in this work expand on the diversity of potential genome editors. |
