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[Preprint]. 2023 Jan 2:2023.01.02.522476.
doi: 10.1101/2023.01.02.522476.

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

David A Armstrong  1   2   3 , Taylor R Hudson  1 , Christine A Hodge  2   3 , Thomas H Hampton  4 , Alexandra L Howell  1   5   4 , Matthew S Hayden  2   3
Affiliations

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

David A Armstrong et al. bioRxiv. .

Update in

Abstract

CRISPR/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 from Streptococcus pyogenes (SpCas9) and Staphylococcus 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 gene CCR5 (CC-Chemokine receptor-5). CCR5 encodes one of two chemokine coreceptors required by HIV-1 to infect target cells, and a mutation of CCR5 (delta-32) is responsible for HIV-1 resistance and reported cures following bone marrow transplantation. Consequently, CCR5 has been an important target for gene editing utilizing CRISPR, TALENs, and ZFNs. We determined that CCR5 cleavage 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 the CCR5 gene in hematopoietic stem cells.

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Conflict of interest statement

Disclosure statement

No potential conflict of interest was reported by the authors.

Figures

Figure 1.
Figure 1.. Target sequences for sgRNAs within the human CCR5 gene.
Schematic of the relative locations of the target sequences for sgRNAs (red rectangles) relative to the location of the region that would be deleted in the Δ−32 mutation (blue), within a 1,000 base pair segment of exon 2 of the human CCR5 gene located on chromosome 3.
Figure 2.
Figure 2.. Cleavage activity of Cas12e is spacer length and target location dependent.
A) Agarose gel separation was used to visualize Cas12e cleavage products after in vitro cleavage. A CCR5 gene target sequence of 2,812 nucleotides (nt) was used to examine the effect of spacer length on DNA cleavage. A representative gel for sgRNA 7 is depicted as an example from which densitometry measurements were derived. The sgRNA 7 generated cleavage products of 1,368 nt and 1,444 nt in length. B) Seven different spacer lengths from 17 nt to 23 nt for each of ten different sgRNAs were analyzed for in vitro cleavage activity of the 2,812 nt CCR5 DNA target by agarose gel electrophoresis. The densitometry of the cleavage products was analyzed for each sgRNA at each spacer length, and the median percent cleavage of the target plotted in a dot blot using Prism Soft Inc. All experiments were run in triplicate.
Figure 3.
Figure 3.. Location of nine terminal PAM bases that were changed to assess CasX2 PAM preference.
Four different CCR5 gene fragments of 1,114 bp (gBlocks) each were synthesized to include nine of the ten gRNA target regions. The terminal PAM base for each sgRNA was changed to either an A, C, G or T in each gBlock. Each of the four gene fragments were separately cloned into the pcDNA3.1 vector and then restriction enzyme digested with NheI and XhoI to yield a 1,074 bp target. A yellow highlighted N (N) indicates a terminal base on the (+) strand, and a grey highlighted N (N) indicates a terminal base on the (−) strand. The blue highlighted region is the wild-type sequence of CCR5 that would be deleted in the Δ−32 mutation. The sgRNAs 1, 2, 3 and 5 bind upstream of the Δ−32 region and sgRNAs 6, 7, 8, 9 and 10 bind downstream.
Figure 4.
Figure 4.. The terminal PAM base influences Cas12e cleavage activity.
A) CCR5 gene fragments (1,074 nt) were generated with different terminal PAM bases of either A, C, G or T. These four DNA targets were assessed for cleavage activity by sgRNA 7 at a spacer length of 18 nt. Cleavage products were then run on a 1% agarose gel so that differences in the cleavage activity for each target could be quantified. A representative experiment is shown in panel A. In triplicate experiments, cleavage activities for sgRNA 7 at spacer length of 18 nt, were highest for the terminal “G” (median cleavage 88.5%), followed by the terminal “A” (66.3%), terminal “C” (59.4%), and terminal “T” (43.6%). B) Cleavage activity for each terminal PAM base substitution was determined by gel densitometry and normalized to the “G” target. Results from four different sgRNAs across multiple spacer sizes shows purines A and G as the terminal PAM base demonstrated greater cleavage activity compared to C and T. Linear modeling reveals that for percent cleavage as a function of guide, guide length and base, both C and T are significantly less than G (**p < 0.01, ***p < 0.001).
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