CAS12e (CASX2) CLEAVAGE OF CCR5: IMPACT OF GUIDE RNA LENGTH AND PAM SEQUENCE ON CLEAVAGE ACTIVITY

Abstract

ABSTRACTCRISPR/Cas is under development as a therapeutic tool for the cleavage, excision, and/or modification of genes in eukaryotic cells. While much effort has focused on CRISPR/Cas fromStreptococcus pyogenes(SpCas9) andStaphylococcus aureus(SaCas9), alternative CRISPR systems have been identified using metagenomic datasets from non-pathogenic microbes, including previously unknown class 2 systems, adding to a diverse toolbox of gene editors. The Cas12e (CasX1, CasX2) endonucleases from non-pathogenic Deltaproteobacteria (DpeCas12e) and Planctomycetes (PlmCas12e) are more compact than SpCas9, have a more selective protospacer adjacent motif (PAM) requirement, and deliver a staggered cleavage cut with 5-7 base overhangs. We investigated varying guide RNA (spacer) lengths and alternative PAM sequences to determine optimal conditions for PlmCas12e cleavage of the cellular geneCCR5(CC-Chemokine receptor-5).CCR5encodes one of two chemokine coreceptors required by HIV-1 to infect target cells, and a mutation ofCCR5(delta-32) is responsible for HIV-1 resistance and reported cures following bone marrow transplantation. Consequently,CCR5has been an important target for gene editing utilizing CRISPR, TALENs, and ZFNs. We determined thatCCR5cleavage activity varied with the target site, guide RNA length, and the terminal nucleotide in the PAM sequence. Our analyses demonstrated a PlmCas12e PAM preference for purines (A, G) over pyrimidines (T, C) in the fourth position of the CasX2 PAM (TTCN). These analyses have contributed to a better understanding of CasX2 cleavage requirements and will position us more favorably to develop a therapeutic that creates the delta-32 mutation in theCCR5gene in hematopoietic stem cells.