Comparison of CRISPR/Cas9 and Cas12a for gene editing in Chlamydomonas reinhardtii

CRISPR/Cas-based technologies have revolutionized biology, offering a wide range of gene editing and engineering applications due to their diverse enzyme characteristics. Among the CRISPR/Cas nucleases, Cas9, and more recently, Cas12a (formerly known as Cpf1), have been employed in various gene editing applications in many eukaryotes, including the model green alga Chlamydomonas reinhardtii. To provide a comprehensive picture of their applicability in single-strand templated DNA repair and gene editing, we first mapped their targeting space by analysing their corresponding PAM frequencies, and then compared Cas9 and Cas12a activities by targeting overlapping regions at three independent loci in the Chlamydomonas genome. We identified 8 and 32 times more target sites for Cas9 compared to Cas12a within promoter regions and coding sequences, respectively. We found that Cas9 and Cas12a RNPs- co-delivered with ssODN repair templates- induced similar levels of total editing, achieving as much as 20–30 % in all viably recovered cells. Importantly, the level of precision editing was slightly higher for Cas12a. In contrast, Cas9 alone was able to induce more edits at the FKB12 locus than its Cas12a counterpart, overall making Cas9 the preferable enzyme for genome engineering among the currently available nucleases in C. reinhardtii.