PlmCas12e (CasX2) cleavage of CCR5: impact of guide RNA spacer length and PAM sequence on cleavage activity

Gene editing using CRISPR/Cas (clustered regularly interspaced palindromic repeats/CRISPR-associated) is under development as a therapeutic tool for the modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas9 systems from <i>Streptococcus pyogenes</i> and <i>Staphylococcus aureus</i>, alternative CRISPR systems have been identified from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of CRISPR/Cas enzymes. The Cas12e enzymes from non-pathogenic Deltaproteobacteria (CasX1, DpeCas12e) and Planctomycetes (CasX2, PlmCas12e) are smaller than Cas9, have a selective protospacer adjacent motif (PAM), and deliver a staggered cleavage cut with a 5–7 nucleotide overhang. We investigated the impact of guide RNA spacer length and alternative PAM sequences on cleavage activity to determine optimal conditions for PlmCas12e cleavage of the cellular gene <i>CCR5</i> (CC-Chemokine receptor-5). <i>CCR5</i> encodes the CCR5 coreceptor used by human immunodeficiency virus-type 1 (HIV-1) to infect target cells. A 32 base-pair deletion in <i>CCR5</i> (<i>CCR5-Δ32</i>) is responsible for HIV-1 resistance and reported cures following bone marrow transplantation. Consequently, <i>CCR5</i> has been an important target for gene editing utilizing CRISPR/Cas. We determined that <i>CCR5</i> cleavage activity varied with the target site, spacer length, and the fourth nucleotide in the previously described PAM sequence, TTCN. Our analyses demonstrated a PAM preference for purines (adenine, guanine) over pyrimidines (thymidine, cytosine) in the fourth position of the CasX2 PAM. This improved understanding of CasX2 cleavage requirements facilitates the development of therapeutic strategies to recreate the <i>CCR5-Δ32</i> mutation in haematopoietic stem cells.