CRISPR–Cas (Clustered Regularly Interspaced Short Palindromic Repeats – CRISPR associated protein) – prokaryotic defence systems, which provide resistance against foreign mobile genetic elements and have a wide range of genome editing and other biotechnological applications. Due to their ease of programmability, Cas9, and Cas12a, representative effector proteins of class 2 CRISPR-Cas systems, became the most commonly used CRISPR-Cas molecular tools in genome engineering to date [1]. Guided by short and easily interchangeable RNA molecules, these proteins can be programmed to target any DNA sequence of need. However, in in vivo therapy, the delivery of such nucleases to cells is limited by the capacity of adeno-associated viruses (AAVs) and the packaging of large Cas12a (850-1400 aa) or Cas9 (1000-1600 aa) nuclease-coding genes into these carriers remains complicated [2]. As a result, recent efforts have focused on expanding the number and characterizing new CRISPR-Cas systems to identify nucleases with novel properties. Smaller CRISPR-Cas nucleases in particular are being sought to treat hereditary genetic disorders in in vivo using AAV although the functional diversity of these compact nucleases remains poorly explored.
Here, we describe a selection of novel Cas12 protein candidates, ranging from 500-700 aa in size. Using a selection of biochemical approaches, we characterize key elements of such Cas12 effectors complexes and their activities on nucleic acid targets. Altogether, this study expands the CRISPR-Cas toolbox and provides a thorough characterization of DNA target elements, needed for successful RNA-guided interference.