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

Figures & data

Table 1. CCR5 sgRNA position on chromosome 3 and specificity score.

RNA Guide	Position	Strand	Specificity Score	PAM
1	46372957	(-)	93.552	TTCA
2	46373065	(-)	92.825	TTCA
3	46373164	(+)	93.068	TTCT
4	46373172	(-)	98.547	TTCT
5	46373178	(+)	98.657	TTCC
6	46373664	(+)	97.692	TTCT
7	46373680	(-)	95.179	TTCA
8	46373897	(+)	96.788	TTCT
9	46373904	(+)	98.825	TTCC
10	46373921	(-)	96.577	TTCC

Figure 1. Target sequences for gRNAs within the human CCR5 gene. Schematic of the locations of the target sequences for gRNAs (20 base protospacer sequence; yellow highlighting) and PAM sequences (light blue highlighting) relative to the location of the region that would be deleted in the $\mathrm{\Delta }$32 mutation (purple highlighting), within a 1,000 base pair segment of exon 2 of the human CCR5 gene located on chromosome 3.

Figure 2. Cleavage activity of PlmCas12e is dependent on both gRNA length and target location.

Note: (A) Agarose gel separation was used to visualize PlmCas12e cleavage products after cell free in vitro cleavage. A CCR5 target sequence of 2,812 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 cleavage generated 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 gRNAs were analysed for in vitro cleavage activity of the 2,812 nt CCR5 DNA target by agarose gel electrophoresis. Molar ratios (MR) of RNP:target of 11.6:1 were used for sg5, MR of 16.5: for sg9, MR of 19:1 for sg1,3,4,7,8 and 10, MR of 21.5:1: for sg6, and MR of 35:1 for sg2. The densitometry of the cleavage products was analysed for each gRNA at spacer lengths ranging from 17 to 23 nts, 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. Location of nine terminal PAM bases that were changed to assess CasX2 PAM preference. Four different CCR5 gene fragments of 1,114 bp (gBlocks) were synthesized to include nine of the ten gRNA target regions. The terminal PAM base for each gRNA 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. In the CCR5 gene region of interest, four PAM regions were located on the (+) strand and five PAM regions were located on the (-) strand. For clarity, only the (+) strand (5’→ 3”) of the CCR5 sequence is illustrated here, where a yellow highlighted N indicates a PAM terminal base on the (+) strand, and a grey highlighted N indicates a PAM terminal base on the (-) strand. The blue highlighted region is the wild-type sequence of CCR5 that would be deleted in the CCR5-$\mathrm{\Delta }$32 mutation. The sgRNAs 1, 2, 3 and 5 bind upstream of the CCR5-$\mathrm{\Delta }$32 region and sgRNAs 6, 7, 8, 9 and 10 bind downstream.

Figure 4. The terminal PAM base influences PlmCas12e 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 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 length of 18 nt, were highest for targets with a terminal ‘G’ (median cleavage 88.5%), followed by those with a terminal ‘A’ (66.3%), a terminal ‘C’ (59.4%), and a terminal ‘T’ (43.6%).

B). Cleavage activity for each terminal PAM base substitution was determined by gel densitometry and normalized to the ‘G’ target. Normalization to the TTCG target was performed to allow comparisons for each sgRNA for all targets. Results from four different sgRNAs across multiple spacer lengths show that purines A and G as the terminal PAM base demonstrated greater cleavage activity compared to pyrimidines C and T. Linear modelling reveals that for percent cleavage as a function of gRNA, spacer length and terminal PAM base, both C and T are significantly less than G (**p < 0.01, ***p < 0.001).

Data availability statement

The data that support the findings of this study are included in the manuscript.