An engineered hypercompact CRISPR-Cas12f system with boosted gene-editing activity

Abstract

Compact CRISPR-Cas systems offer versatile treatment options for genetic disorders, but their application is often limited by modest gene-editing activity. Here we present enAsCas12f, an engineered RNA-guided DNA endonuclease up to 11.3-fold more potent than its parent protein, AsCas12f, and one-third of the size of SpCas9. enAsCas12f shows higher DNA cleavage activity than wild-type AsCas12f in vitro and functions broadly in human cells, delivering up to 69.8% insertions and deletions at user-specified genomic loci. Minimal off-target editing is observed with enAsCas12f, suggesting that boosted on-target activity does not impair genome-wide specificity. We determine the cryo-electron microscopy (cryo-EM) structure of the AsCas12f-sgRNA-DNA complex at a resolution of 2.9 Å, which reveals dimerization-mediated substrate recognition and cleavage. Structure-guided single guide RNA (sgRNA) engineering leads to sgRNA-v2, which is 33% shorter than the full-length sgRNA, but with on par activity. Together, the engineered hypercompact AsCas12f system enables robust and faithful gene editing in mammalian cells.

Fig. 1 |. Engineering AsCas12f for increased genome-editing efficiency.

a, Domain organization of AsCas12f compared with SpCas9, AsCas12a, DpbCasX, and UnCas12f. HNH, REC, and RuvC domains are indicated. Protein lengths are drawn to scale. aa: amino acid. b, Sequence alignment of AsCas12f and its homologous proteins. Representative regions are shown, with candidates for mutagenesis highlighted in red boxes. c, Workflow to determine the cellular activity of AsCas12f and its variants. d, e, Indel levels at TP53-1 (d) and HEXA (e) loci generated by AsCas12f variants that bear one, two, three, four, or five single-point mutations. A list of mutations included in each AsCas12f variant is provided in Supplementary Fig. 4. Two independent replicates were carried out in HEK293T cells. f, g, Time-course in vitro DNA cleavage by wild-type AsCas12f and enAsCas12f at 37 °C (f) and 50 °C (g). Data points were fitted to one-phase exponential association curves. Two independent replicates were carried out. Gel images are provided in Supplementary Fig. 5.

Fig. 2 |. Genome editing facilitated by engineered AsCas12f systems.

a, Indel frequencies mediated by wild-type and engineered AsCas12f, in comparison to wild-type UnCas12f and CasMINI with an engineered UnCas12f sgRNA (ge4.1), in HEK293T cells. b, Box-and-whisker plot of indel frequencies delivered by AsCas12f and UnCas12f systems shown in a. c, Indel frequencies mediated by wild-type AsCas12f, enAsCas12f, and CasMINI-ge4.1 in HCT116 (left) and HeLa (right) cells. d, Box-and-whisker plot of indel frequencies delivered by enAsCas12f and AsCas12a. e, Box-and-whisker plot of indel frequencies delivered by enAsCas12f and SpCas9. Two independent replicates were carried out in a and c. For b, d, and e, all data points (n = 17 target sites in b and d, n = 8 target sites in e) were plotted, with the centerline representing the median and the whiskers showing the minimum to the maximum. The boundaries of the box indicate the first and third quantiles. P values were determined by two-tailed paired Student’s t-test.

Fig. 3 |. Cryo-EM structure of the AsCas12f-sgRNA-DNA complex.

a, Domain structure of AsCas12f. The N-lobe contains a wedge (WED) domain and a recognition (REC) domain. The C-lobe includes a RuvC nuclease domain and a zinc finger (ZF) motif. b, Unsharpened cryo-EM map for the AsCas12f-gRNA-DNA complex (contoured at a level of 0.020). c, Top: atomic model of the AsCas12f-gRNA-DNA complex. Bottom: close-up views of residues mutated in enAsCas12f (D196K, N199K, G276R). Note that these residues are close to the backbone of the sgRNA. In b and c, AsCas12f domains are colored as shown in a.

Fig. 4 |. Structure-guided engineering of the AsCas12f gRNA.

a, Indel frequencies mediated by enAsCas12f with engineered AsCas12f gRNAs at HEXA and PDCD1 loci. The structures of engineered gRNAs are shown in Supplementary Fig. 14. b, Structure of sgRNA-v2. c, Time-course in vitro DNA cleavage using full-length sgRNA and sgRNA-v2. The assay was conducted using enAsCas12f at 37 °C. Data points were fitted to one-phase exponential association curves. Gel images are provided in Supplementary Fig. 16. d, Indel frequencies mediated by the full-length sgRNA and sgRNA-v2 in complex with enAsCas12f at denoted genomic loci in HEK293T cells. e, Box-and-whisker plot of indel frequencies mediated by the full-length sgRNA or sgRNA-v2 in complex with enAsCas12f. All data points (n = 8 target sites) were plotted, with the centerline showing the median and the whiskers showing the minimum to the maximum. The boundaries of the box indicate the first and third quantiles. P values were determined by two-tailed paired Student’s t-test. f, Relative abundance of full-length sgRNA and sgRNA-v2 targeting HEXA and PDCD1 loci in HEK293T cells. Two independent replicates were carried out in a, c, and d.

Fig. 5 |. Genome-wide specificity of wild-type and engineered AsCas12f.

a, On-target indel frequencies in GUIDE-seq samples. b-f, Off-target editing sites for wild-type AsCas12f, AsCas12f-v4.1, and enAsCas12f with sgRNAs targeting HEXA (b), TP53-2 (c), PDCD1 (d), APOB (e), and MRPL39 (f) loci reported by GUIDE-seq in HEK293T cells. Mismatch positions are highlighted in colors. GUIDE-seq experiments were performed in duplicates, with the read counts of one replicate shown to the right of the corresponding sequences. Results from the other replicate are shown in Supplementary Fig. 17. Full-length sgRNAs were used in all GUIDE-seq experiments.