CRISPR-Cas is currently the most widely used gene-editing tool, and has broad applications in both basic scientific research and clinical applications1. However, the off-targets lead to gene mutations and even cancers, which seriously hinder the utilities of SpCas92. To solve the problem, several strategies in recent years have focused on the protein engineering of SpCas9 to improve its cutting specificity3, 4. However, although the off-target effect of the engineered SpCas9 was significantly reduced, its on-target cutting activity was also affected5.
Our team has been working in the field of HPV targeted therapy for more than ten years6, 7, 8. We are actively seeking a new gene-editing tool with superior cutting efficiency and good fidelity. And we hope to develop a novel gene therapy to eliminate HPV infections.
After multiple rounds of screening and comparison, we finally found that the FrCas9 from Faecalibaculum rodentium is with high cutting efficiency and low off-target effect6. FrCas9 belongs to Type II-A CRISPR (Fig.1a) with a size of 1372 amino acids (Fig.1b). Through a depletion assay and plasmid interference assay, we found that FrCas9 has gene editing ability at both prokaryotic and eukaryotic cells, and determined its PAM sequence to be 5′-NNTA-3′ (Fig.1c). Further, we selected 11 human endogenous loci with 5′-GGTA-3′ PAM and compared the cutting efficiency and off-target effect of FrCas9 and SpCas9 using GUIDE-seq (Fig.1d). The results showed that FrCas9 had comparable cutting efficiency to SpCas9 but with lower off-target effect (Fig.1e).
Interestingly, we found that the PAM of FrCas9 is a palindromic sequence. The unique structure of the PAM of FrCas9 can widen the targeting scope of the FrCas9 gene editor and increase the target distribution and density of FrCas9 sgRNAs. By simultaneously modifying two close editing windows, FrCas9 has great potential for both base editing and prime editing.
Since the PAM of FrCas9 is NNTA, it can perfectly match the TATA box sequence which is the transcription start point of most eukaryotic genes. Therefore, FrCas9 can directly target the TATA region of the gene and can be applied to the following scenarios: 1) gene transcription inhibition. By binding to TATA-box, FrCas9 reduced the expression of ABCA1, UCP3 and RANKL by 61.67%, 45.61% and 42.60%, respectively (Fig.1f); 2) Gene transcription activation. dFrCas9-VP64 can effectively activate transcription, and the activation efficiency in ABCA1, GH1 and MBL2 is higher than that of dSpCas9-VP64 (Fig.1g).
Taken together, our findings broaden the understanding of CRISPR/Cas-mediated genetic engineering9. We envision that this CRISPR system will not only benefit biological and clinical research in this field, but will also provide a deeper understanding towards the mechanisms of CRISPR-mediated gene editing.
Fig. 1. (a) The phylogenetic tree of FrCas9 and 13 active Cas9 orthologs. (b) The schematic of Faecalibaculum rodentium systems. Insert above displayed the domains of FrCas9 with active residues indicated with asterisks. I, II, III represented three RuvC domains. Insert below showed expressed crRNA and tracrRNA from small RNA-seq of E.coli harboring pET-28A plasmid with simplified FrCas9 locus. (c) The schematic of depletion assay and web-logo results for SpCas9 and FrCas9. (d) Summary of GUIDE-seq on-target reads of SpCas9 and FrCas9 at the above 11 sites. (e) The off-targets of SpCas9 and FrCas9 for 11 sites, generated by GUIDE-seq in HEK293T cells. The sgRNA and PAM ranges of SpCas9 (20 nt sgRNA and 3 nt PAM) and FrCas9 (22 nt sgRNA and 4 nt PAM) were marked in rose and lavender, respectively. GUIDE-seq read counts of each site were shown on the right side. CRISPRi (f) and CRISPRa (g) of FrCas9 and SpCas9 by targeting TATA-boxes. The experiments were conducted in HEK293T cells and expression was quantified by qPCR. Error bars indicated S.D. (n = 3 per group, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001, Student's t-test).
- Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell157, 1262-1278 (2014).
- Tsai SQ, et al.GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR- Cas nucleases. Nat Biotechnol33, 187-197 (2015).
- Hsu PD, et al.DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol31, 827-832 (2013).
- Doench JG, et al.Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol34, 184-191 (2016).
- Murugan K, Suresh SK, Seetharam AS, Severin AJ, Sashital DG. Systematic in vitro specificity profiling reveals nicking defects in natural and engineered CRISPR–Cas9 variants. Nucleic acids research49, 4037-4053 (2021).
- Cui Z, et al.FrCas9 is a CRISPR/Cas9 system with high editing efficiency and fidelity. Nature Communications13, 1-12 (2022).
- Fan W, et al.Non-homologous dsODN increases the mutagenic effects of CRISPR-Cas9 to disrupt oncogene E7 in HPV positive cells. Cancer Gene Ther, (2021).
- Cui Z, et al.The comparison of ZFNs, TALENs, and SpCas9 by GUIDE-seq in HPV-targeted gene therapy. Mol Ther Nucleic Acids26, 1466-1478 (2021).
- Makarova KS, et al.Evolutionary classification of CRISPR–Cas systems: a burst of class 2 and derived variants. Nature Reviews Microbiology18, 67-83 (2020).