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Utilization of nicking properties of CRISPR-Cas12a effector for genome editing – Scientific Reports

Production of the en-AsCas12a (R1226A) variant and identification of the nicking property

The CRISPR-Cas12a system consists of a single CRISPR RNA (crRNA) and Cas12a endonuclease that induces target DNA recognition15,16. The Cas12a effector recognizes a T-rich protospacer adjacent motif (PAM) among target sequences (Fig. 1a, left inset), crRNA and target DNA hybridization induces an R-loop, and finally the active site of the RuvC domain (Fig. 1a, right inset) induces DNA cleavage17. The recently reported enhanced AsCas12a (en-AsCas12a) system shows that engineered amino acids near the PAM sequence (TTTN) dramatically enhances target DNA recognition, thereby increasing genome editing efficiency18. We optimized a nickase effector based on the en-AsCas12a (E174R, S542R, K548R) system, hereafter en-AsCas12a (R1226A), to effectively induce target-specific gene mutations with single- or dual mode of DNA targeting. To confirm the nicking property at the in-vitro level, a cleavage assay was performed by targeting the plasmid containing the target (EMX1, CCR5) nucleotide sequence (Fig. 1b,c, Supplementary Supplementary Fig. 1a, Tables 1, 2). As previously reported11,12, the wild-type (WT) en-AsCas12a effector showed a minor nicking property while inducing DNA double strand break (Fig. 1c, Supplementary Fig. 1b). On the other hand, the en-AsCas12a (R1226A) form showed a typical activity as a DNA nickase by mainly inducing the open circular form of the plasmid (Fig. 1c). After confirming the nicking property of the en-Cas12a (R1226A) variant, we tried to determine whether en-Cas12a (R1226A) cleaves the target-strand or non-target strand on the target DNA. To this end, we co-treated the Cas12a (R1226A) variant with SpCas9 (D10A) or SpCas9 (H840A) nickases, whose cleavage points have already been identified, and the cleaved strand of target DNA by Cas12a (R1226A) variant was identified by the formation of double strand breaks (Fig. 1d). As a result, we found that when Cas12a (R1226A) variant, SpCas9 (D10A), and SpCas9 (H840A) nickases were simultaneously treated (Fig. 1e), double strand breaks were mainly formed in case of the Cas12a (R1226A) variant and SpCas9 (D10A) nickase were co-treated (Fig. 1f). Furthermore, we analyzed the product after cleavage using the Cas12a (R1226A) variant and compared it to Cas9 (D10A or H840A) and found that a cleavage was formed at a distance of 15–18 bp from the PAM sequence (TTTN), depending on the target sequence site (Supplementary Fig. 1c). Through these results, it can be seen that, like the wild-type Cas12a nickase, the en-Cas12a (R1226A) variant also mainly cleaves the non-target strand and induces nick formation on the target DNA (Supplementary Fig. 1d).

Figure 1
figure 1

Confirmation of the nicking activity of en-AsCas12a (R1226A) module. (a) Structure of the AsCas12a-crRNA-target DNA complex (PDB: 5B43). Left inset: substituted amino acid residues in the AsCas12a protein around the PAM sequence (TTTN) (E174, S542, K548). Right inset: Active residues in the nuclease domain required for target DNA cleavage. (b, c) Sanger sequencing data of the plasmid containing the target nucleotide sequence (CCR5) (b). PAM and target sequences are shown in cyon (SpCas9) and yellow (AsCas12a) and dark blue (SpCas9) and gray (AsCas12a), respectively. Plasmid cleavage assay using recombinant proteins (en-AsCas12a, en-AsCas12a (R1226A), SpCas9 (D10A)) (c). NC: negative control, HindIII: Treated by restriction enzyme (HindIII), OC: open circular form, L: linear form, SC: super-coiled form. (–) guide RNA: only en-AsCas12a or en-AsCas12a (R1226A) protein treated, ( +) guide RNA: en-AsCas12a or en-AsCas12a (R1226A) protein and target-specific crRNA were co-treated. (df) Schematics depicting a method for identification of the cleaved strand formed by en-AsCas12a (R1226A) on target DNA (d). NTS and TS indicates non-target strand and target strand in target DNA, respectively. The red arrowhead indicates the nick formed by en-Cas12a (R1226A). Blue arrows indicate nicks formed by SpCas9 (D10A) or SpCas9 (H840A), respectively. Sanger sequencing data of the plasmid containing the target nucleotide sequence (CCR5) (e). In-vitro DNA cleavage assay for cleavage strand identification using en-AsCas12a (R1226A), SpCas9 (D10A), and SpCas9 (H840A) (f). Red asterisk indicates a cleaved DNA fragments. RE: restriction enzyme, NC: negative control, SpCas9 (D10A) or SpCas9 (H840A): SpCas9 nickases, en-AsCas12a (R1226A): en-AsCas12a nickase.

Optimization of the distance and direction between dual Cas12a (R1226A) modules to effectively induce mutations in target DNA

Based on these cleavage test results, target-specific mutations were induced on the target gene in a human-derived cell line (HeLa) using the dual-nickase-type AsCas12a (R1226A) or SpCas9 (D10A) effector (Fig. 2, Supplementary Fig. 2). Since en-AsCas12a, an improved form of wt-AsCas12a, has shown overall superior gene editing properties in human-derived cell lines in previous studies18, all subsequent experiments testing Cas12a nickase-related activity were based on the en-AsCas12a effector. To confirm the directionality issue of the CRISPR nickases, the mutation induction efficiency (%) was compared for the different combination according to the directionality of each nickase (Fig. 2, Supplementary Fig. 2a,b). For each combination of SpCas9 (D10A) and en-AsCas12a (R1226A) nickase, the indel frequency (%) in the target genes (EMX1, CCR5), which is induced in a PAM-out (Fig. 2a,c,e, Supplementary Fig. 2c) and PAM-in (Fig. 2b,d,f, Supplementary Fig. 2c) fashion, was analyzed by targeted amplicon sequencing (Supplementary Figs. 3, 6, Table 3). Due to the limited issue of PAM (TTTN) recognition when applying en-AsCas12a (R1226A)-based dual nickase at each gene site, we compared the gene editing efficiency at different locations. Consequently, the indel frequency (%) induced in the direction of the PAM-out fashion (mean 12.4% for EMX1 and 5.8% for CCR5), (Fig. 2a,c,e) was higher than that in the PAM-in fashion (mean 0.9% for EMX1 and 1.0% for CCR5), (Fig. 2b,d,f) for all nickase combinations. Particularly, as the distance between the PAMs of nickase combinations increased, the overall indel frequency (%) decreased; a significant difference was observed depending on the nucleotide sequence in the target gene (EMX1, CCR5) and orientation between nickases (Fig. 2, Supplementary Fig. 2c). Most of the induced mutation pattern shows large deletions, which is possibly generated by tandem nicking of sequential nickase binding (Supplementary Figs. 3, 6).

Figure 2
figure 2

Optimization of cellular gene mutation by en-AsCas12a (R1226A). (af) Comparison of mutation induction efficiency on target genes (EMX1, CCR5) in human derived cell line (HeLa) by using SpCas9 (D10A) or en-AsCas12a nickase (R1226A) with PAM orientation dependency. (a, c, e) Comparison of mutation induction efficiency for target genes (EMX1 and CCR5) using the dual nickase (dual SpCas9 (D10A) nickase (a), dual SpCas9 (D10A)-en-AsCas12a (R1226A) nickase combination (c), dual en-AsCas12a (R1226A) nickase (e)) method of PAM-out direction. (b, d, f) Comparison of mutation induction efficiency for target genes (EMX1 and CCR5) using the dual nickase (dual SpCas9 (D10A) nickase (b), dual SpCas9 (D10A)-en-AsCas12a (R1226A) nickase combination (d), dual en-AsCas12a (R1226A) nickase (f)) method of PAM-in direction. Information about the targeted gene sites is given in parentheses under each gene name with the guide RNA number, and the corresponding sequences are given in Supplementary Tables 1–3. NC: negative control, X: end-to-end distance (bp) between protospacers for PAM-out orientation and between PAMs for PAM-in orientation, respectively. Negative value (-X) indicates overlapping between two targets. PAM sequences are indicated in yellow color. Protospacers for SpCas9 (D10A) and en-AsCas12a (R1226A) are indicated in blue and red, respectively. Each histogram was plotted by applying standard error of the mean values to three repeated experimental values (n = 3). SpCas9 (D10A): SpCas9 nickase, en-AsCas12a (R1226A): en-AsCas12a based nickase.

Comparison of the efficiency of mutation induction at target sites between dual- and single-Cas12a (R1226A) variants

The indel frequency (%) induced by the combination of dual nickases, each optimized with respect to PAM orientation, was compared with that induced by a single nickase or WT effector (Fig. 3). Most of the mutations formed by treatment with the single/dual nickase or WT effector were deletions (Supplementary Figs. 4, 6). The mutation frequency was highest for wt-enCas12a (mean 37.6% for EMX1 and 10.8% for CCR5), followed by dual-Cas12a (R1226A) (mean 6.6% for EMX1 and 3.5% for CCR5) and single-Cas12a (R1226A) (mean 2.1% for EMX1 and 0.6% for CCR5) (Fig. 3, Supplementary Figs. 3, 4, 6). As shown in Fig. 2, mutations were only effectively induced when binding in the PAM-out configuration was adopted using dual nickases (Fig. 3d–f,j, top). Notably, the targeted mutations formed by a single nickase were also observed for the dual nickase (Fig. 3, Supplementary Figs. 3, 4). The tendency of single nickase targeted mutation appears to be increased by the treatment of dual nickase rather than only single nickase treated (Fig. 3, Supplementary Figs. 3, 4, 6). Notably, when using the en-AsCas12a (R1226A) effector in a single nickase fashion, the editing efficiency (1.4–3.9%) was similar to that of the previously reported SpCas9 (D10A) nickase (0.4–2.2%), (Fig. 3d–f,j–l middle). Although the indel ratio (%) was lower than that for dual nickase, which is guided by two crRNAs, substantial indels (mean 2.1% for EMX1 and 0.6% for CCR5) were induced by intracellular delivery with the single nickase en-AsCas12a (R1226A) using one crRNA.

Figure 3
figure 3

Direct comparison of mutagenic property in cell line for mono- or dual-nicking methods based on en-AsCas12a (R1226A) module. (al) Comparison of mutation induction efficiency of EMX and CCR5 in cell line (HeLa) with different combinations of SpCas9 (D10A) or en-AsCas12a nickase (R1226A). (ac) Schematic diagram of the target nucleotide sequence when mutations in the target gene (EMX1) are induced using the mono- or dual-nickase (SpCas9 (D10A) nickase only (a), SpCas9 (D10A) or en-AsCas12a (R1226A) nickase (b), en-AsCas12a (R1226A) nickase only (c)) method. Targeted positions are indicated in red arrowhead (Supplementary Tables 1–3), and discriminated by blue (SpCas9 (D10A), S1-S2, A1-A4) and orange (en-AsCas12a (R1226A), S3-S6, A5-A8) number, respectively. S1/A1-4, S3/A1-4, S5/A5-8: PAM-out orientation, S2/A1-4, S4/A1-4, S6/A5-8: PAM-in orientation. (df) Comparison of intracellular gene mutagenesis efficiencies (df) corresponding to mono- or dual-nickase targeting (ac). (g–i) A schematic diagram of the target nucleotide sequence when mutations in the target gene (CCR5) are induced using the mono- or dual-nickase method [SpCas9 (D10A) nickase only (g), SpCas9 (D10A) or en-AsCas12a (R1226A) nickase (h), en-AsCas12a (R1226A) nickase only (i)]. Targeted positions are indicated in red arrowhead, and discriminated by blue (SpCas9 (D10A), S7-S8, A9-A12) and orange (en-AsCas12a (R1226A), S9-S12, A13-A16) number, respectively. S7/A9-12, S9/A9-12, S11/A13-16: PAM-out orientation, S8/A9-12, S10/A9-12, S12/A13-16: PAM-in orientation. (j–l) Comparison of intracellular gene mutagenesis efficiencies (jl) corresponding to mono- or dual-nickase targeting (gi). Indel frequency (%) from dual nickase (SpCas9 (D10A), dual nickase (SpCas9 (D10A) and en-AsCas12a (R1226A), dual nickase (en-AsCas12a (R1226A), single nickase and wild-type nuclease are indicated by brown, blue, red, peach, and orange color, respectively. NC: negative control. Each histogram was plotted by applying standard error of the mean values to three repeated experimental values (n = 3). SpCas9 (D10A): SpCas9 nickase, en-AsCas12a (R1226A): en-AsCas12a based nickase.

Target-specific genome editing with decreased off-target activity using the dual-en-AsCas12a (R1226A) variant

To determine whether unintended mutations are induced (i.e., off-target effects) by the dual- and single-en-Cas12a (R1226A) variants, on-/off-target indel ratio (%) was investigated for five target loci (DNMT1, POLQ, SIRPa, AAVS1, and CCR5) (Fig. 4, Supplementary Figs. 5, 7). First, off-target candidates with up to 3 mismatched sequences compare to those of each target gene were selected based on an in-silico analysis19, and targeted amplicons were prepared for each predicted off-target site (Fig. 4a–e, left). Then, the mutation frequency at each off-target site (%) was investigated by targeted amplicon sequencing (see Methods section, Supplementary Table 3). The indel mutations generated by dual en-Cas12a (R1226A) variants for each on-target and predicted off-target sites were compared with that of the wild-type en-Cas12a effector, and histograms were obtained (Fig. 4a–e, right). In a comparison of five target loci, the dual en-Cas12a (R1226A) nickase showed an editing efficiency of 27.7%, on average, compared with estimates of 64.2% for the wild-type en-Cas12a effector (WT). In comparison, for the selected off-target site, indel frequencies of 0.26% was induced by dual en-Cas12a (R1226A) variants, compared to a frequency of 9.14% for the wild-type en-Cas12a effector (WT) (Fig. 4a–e, right).

Figure 4
figure 4

Comparison of on/off-target mutation induction between mono- or dual-en-AsCas12a (R1226A) and wild-type en-AsCas12a. (ae) Left: Off-target candidate sequences selected in-silico for each target sequence (DNMT1 (a), POLQ (b), SIRPa (c), AAVS1 (d), and CCR5 (e)). The PAM sequence and mismatched bases between the target and off-target sequences are shown in cyon and red, respectively. Middle and Right: Histogram showing each indel frequency (%) induced for target sequences and corresponding off-target sites by wild-type en-AsCas12a (WT) and dual-en-AsCas12a (R1226A). On and OT1-10 indicates the on- / off-target sites for each targeted locus (DNMT1 (a), POLQ (b), SIRPa (c), AAVS1 (d), and CCR5 (e)). The middle and right histograms show the on- and off-target indel ratio (%) values of wild-type en-AsCas12a and dual nickase en-AsCas12a for the target genes in log-scale and actual values, respectively. Information about the targeted gene sites is given in parentheses under each gene name with the guide RNA number, and the corresponding sequences are given in Supplementary Tables 1–3. Each histogram was plotted by applying standard error of the mean values to three repeated experimental values (n = 3).