CRISPR/CasΦ2‐mediated gene editing in wheat and rye

The recent advancements in developing the CRISPR/Cas9 system and various derivative tools (e.g., base editors) have accelerated basic plant science research and crop improvement by creating multiple types of genetic variations (Li et al., 2023a). However, the use of Cas9 protein is frequently limited by the requirement of G/C-rich protospacer-adjacent motif (PAM) sequences, especially in triticeae plants, many of which are important food and forage crops carrying large and complex genomes. The CRISPR/CasΦ (CRISPR/Cas12j) system has recently been discovered from bacteriophages, and prefers 5′-TBN-3′ PAMs suitable for specific biological and therapeutic applications (Pausch et al., 2020). With a smaller size (700–800 aa) than Cas12a (1,100 aa) and SpCas9 (1,300 aa), it is valuable for DNA editing where protein or nucleic acid size is a limiting factor (Zhan et al., 2021). Lately, CRISPR/CasΦ2 has been demonstrated useful for gene editing by transient or transgenic experiments in Arabidopsis, tomato, rice, and maize (Liu et al., 2022; Li et al., 2023b). However, it remains unclear whether the system may function in triticeae plants. More importantly, it is worth exploring if this hypercompact system may be adopted for precise base editing in plants (Figure 1A). Improving CRISPR/CasΦ2 for gene and base editing in plants (A) CRISPR/CasΦ2-meadited toolkits. (B) Comparison of editing frequency among pBlunt-CasΦ2Ta-V1/V2/V3/V4 at TaGW2 target sites (TBN PAMs) with TaU3-tRNA-crRNA in wheat protoplasts. Representative deletion locations were summarized using TaGW2-crRNA-TTN/pBlunt-CasΦ2Ta-V3 data. (C) Assessment of editing frequencies of CRISPR/CasΦ2Ta-V3 and CRISPR/VCasΦ2Ta-V3 at TaGW2-crRNA-TTN target site in transgenic wheat plants. (D) Test of editing frequency of CRISPR/VCasΦ2Ta-V3 at TaGW2/ScPhyA-crRNA-TTN target sites using paired opposite crRNAs in wheat or rye protoplasts. (E) Diagram of CasΦ2/dCasΦ2 or VCasΦ2/dVCasΦ2-based CBEs. (F) Frequencies and locations of C-to-T editing and indels at TaGW2-crRNA-TTN target site obtained by four CBEs in wheat protoplasts. (G) Assessment of C-to-T editing and indels frequencies, and editing window at five different target sites obtained by pBlunt-dCasΦ2Ta-CBE in wheat and rye protoplasts. (H) Diagram of dCasΦ2-based adenine base editor (ABE). (I) Assessment of A-to-G editing and indels frequencies, and editing window inducing by dCasΦ2-based ABE at four target sites in wheat or rye protoplasts. (J) Frequencies of CasΦ2/dCasΦ2-derived cytosine adenine base editors (CBEs) at TaGW2/TaPIN-crRNA-TTN target sites and dCasΦ2-derived ABE at TaALS-crRNA-TTN target site in transgenic wheat plants. All values are mean ± s.e.m. *P < 0.05, **P < 0.01; ns, no significant difference by two-tailed Student's t test. To address the above questions, we first synthesized wheat codon-optimized CasΦ2 and constructed the UBQ::CasΦ2Ta with the TaU3::crRNA cassette placed into the pBlunt vector (Figure S1A). We chose four genes for editing: two from wheat (TaGW2 and TaPIN) and two from rye (ScPhyA and ScPhyB). As CasΦ2 prefers 5′-TBN-3′ PAMs where B is G, T or C, we designed three target sites for each gene with TTN, TGN, or TCN PAM (creating 12 target sites), covering similar crRNA binding sites. When testing in protoplasts-based transient systems, no obvious editing was detected by PCR/RE assays in any target sites (Figure S1B). This is in line with the very low efficiency (<1%) of genome editing by CasΦ2 in Arabidopsis (Pausch et al., 2020). Consequently, we set out to improve the editing efficiency of CRISPR/CasΦ2. First, we changed the way of crRNA processing and tested various nuclear localization signals (NLSs) for CasΦ2. By combining TaU3 promoter-driven polycistronic-tRNA-crRNA cassette with two NLSs (SV40 and nucleoplasmin long NLS) fused to CasΦ2Ta, we generated three different versions of CRISPR/CasΦ2Ta (Figure 1B, V2, V3, and V4). By testing in wheat protoplasts at TaGW2 sites, we found that pBlunt-TaU3-tRNA-crRNA/pBlunt-CasΦ2Ta-V3, with one and three NLSs fused to the N- and C-termini of CasΦ2Ta, respectively, could most efficiently improve gene editing from near-background level up to 3.2% in the target site of TaGW2-crRNA-TTN (Figure 1B). Considering that the CasΦ variants NCasΦ and VCasΦ cleave substrate DNA faster than CasΦ (Pausch et al., 2021), we next prepared two new constructs expressing NCasΦ2Ta-V3 and VCasΦ2Ta-V3 and tested their editing efficiencies at the TaGW2 or ScPhyA target sites in wheat or rye protoplasts, combining with appropriate pBlunt-TaU3-tRNA-crRNA (Figure S2A). PCR/RE assays showed both NCasΦ2Ta-V3 and VCasΦ2Ta-V3 improved the editing efficiency compared with CasΦTa-V3, with VCasΦ2Ta-V3 exhibiting an overall higher efficiency (2.5–6.0-fold of increase, Figure S2B, C). Hence we prepared two T-DNA constructs, pLH-CasΦ2Ta-V3 and pLH-VCasΦ2Ta-V3, and compared their editing efficiency at the TaGW2-crRNA-TTN target site using the TaU3::tRNA-crRNA design in transgenic wheat (Figure S3A). PCR/RE and Sanger sequencing analysis indicated that pLH-VCasΦ2Ta-V3 induced indels in 30% (6/20) T0 plants, higher than pLH-CasΦ2Ta-V3 did (12.5%, 2/16) (Figure 1C), with the mutations being 3–27 bp deletions (Figure S3B). Analysis of T1 plants verified the inheritance of the indel mutations induced by pLH-VCasΦ2Ta-V3-TaGW2-crRNA-TTN in T0 generation (Table S3). Interestingly, we observed that paired opposite crRNAs, arranged as 5′-TTN-N18-spacing-N18-YAA-3′, further improved the editing efficiency of pBlunt-VCasΦ2Ta-V3 in transient assays. The tested sites were TaGW2-crRNA-TTN and ScPhyA-crRNA-TTN, with ~30 and ~60-bp spacing sequences in each site (Figure 1D). The editing efficiency using paired crRNAs (~30 bp spacing sequence) was ~1.5-fold higher than that produced with a single crRNA (Figure 1D). Altogether, the above results suggest that combining VCasΦ2Ta-V3 with TaU3::tRNA-crRNA enables genome editing in wheat and rye, with potential further improvement using paired crRNAs. Furthermore, we endeavored to develop cytosine and adenine base editors (CBE and ABE) using CasΦ2Ta-V3. We created catalytically inactive dCasΦ2Ta-V3 by mutating the active sites of the RuvC domain (D394, E606, and D695) (Pausch et al., 2021). Then we fused human APOBEC3A to CasΦ2Ta-V3 or dCasΦ2Ta-V3 (Zong et al., 2018), and created pBlunt-CasΦ2Ta-CBE or pBlunt-dCasΦ2Ta-CBE (Figure 1E). To investigate if VCasΦ2Ta may influence CBE, we also constructed the pBlunt-VCasΦ2Ta-CBE and pBlunt-dVCasΦ2Ta-CBE vectors, with dVCasΦ2Ta-V3 developed similarly to dCasΦ2Ta-V3. All constructs were tested in wheat protoplasts using the TaGW2-crRNA-TTN target site. Deep sequencing results demonstrated that pBlunt-dCasΦ2Ta-CBE and pBlunt-dVCasΦ2Ta-CBE showed similar C-to-T base editing efficiencies (up to ~4%) with very low levels of indels (Figure 1F). As expected, pBlunt-CasΦ2Ta-CBE and pBlunt-VCasΦ2Ta-CBE exhibited high efficiency of indels (Figure 1F). Thus, another five target sites, four from wheat and one from rye, were employed to test the efficiency of pBlunt-dCasΦ2Ta-CBE in protoplasts. In these assays, the CBE efficiencies ranged from 1.9% to 5.5%, with the editing window spanning from C2 to C17 in the protospacers (Figure 1G), which is wider than that reported for Cas9-based CBE (Zong et al., 2018). Therefore, two T-DNA vectors, pLH-dCasΦ2Ta-CBE and pLH-CasΦ2Ta-CBE (Figure S5A), were tested in transgenic wheat using TaGW2-crRNA-TTN and TaPIN-crRNA-TTN as target sites. In the T0 transgenic wheat obtained with pLH-dCasΦ2Ta-CBE, CBE activities were detected in 9.1% of plants for TaGW2-crRNA-TTN and 6.9% for TaPIN-crRNA-TTN, with only C substitution found in the target site (Figure 1J). In contrast, among the T0 mutants obtained with pLH-CasΦ2Ta-CBE-TaGW2-crRNA-TTN, 80% showed deletions (Figure 1J). The C-to-T mutations induced by pLH-dCasΦ2Ta-CBE at the tested sites were mostly transmitted to T1 plants (Table S3). For evaluating the usefulness of dCasΦ2-ABE, we fused wheat codon-optimized TadA8e to dCasΦ2Ta-V3 with XTEN linker (Yan et al., 2021), thus generating pBlunt-dCasΦ2Ta-ABE (Figure 1H). When investigated in wheat or rye protoplasts, A-to-G editing was observed for all four targets (in TaALS, TaNAC2, ScPhyB, and ScPhyC, respectively), with the efficiencies ranging from 0.8% to 3.0% (Figure 1I). The deamination window spanned from protospacer positions A9–A11, with very low levels of unwanted indels (Figure 1I). Hence, we constructed the T-DNA vector pLH-dCasΦ2Ta-ABE (Figure S6A) to examine ABE activity in transgenic wheat using the TaALS-crRNA-TTN target site. A-to-G editing was detected in 6% T0 transgenic plants (Figures 1J, S6B), which were transmitted to T1 plants (Table S3). Collectively, these results illustrated the feasibility of dCasΦ2-derived CBE and ABE in wheat and rye. Finally, we examined potential off-targeting in CRISPR/VCasΦ2Ta-V3, CRISPR/dCasΦ2Ta-CBE or CRISPR/dCasΦ2Ta-ABE mediated editing in T0 mutants. We used TaGW2-crRNA-TTN or TaALS-crRNA-TTN for the analysis, with eight similar sites (1–4 bp mismatches; Table S4) identified in the common wheat genome. Deep sequencing revealed no significant off-target events in all eight sites (Figure S7A, B), indicating high specificity in the editing activity of CRISPR/VCasΦ2Ta-V3, CRISPR/dCasΦ2Ta-CBE and CRISPR/dCasΦ2Ta-ABE in wheat. In summary, we demonstrated that the efficiency of CRISPR/CasΦ2 could be improved by changes in crRNA expression, NLS incorporation, and CasΦ2 protein variants, resulting in successful genome editing of triticeae crops by CasΦ2 mediated gene knockout and base editing with high specificity. We proved for the first time that CRISPR/dCasΦ2-CBE and CRISPR/dCasΦ2-ABE are functional in plants. With its unique properties, i.e., efficient use of TTN PAM and alternative base editing window, CRISPR/CasΦ2 provides a complementary genome engineering tool, which may find wide applications in future research on CRISPR/CasΦ2-mediated genome modifications. This work was supported by the National Key Research and Development Program of China (2021YFF1000203) and the National Natural Science Foundation of China (32000286 and 32370432). The authors declare no conflict of interest. S.Z., X.H., Y.Z., Y.H., H.Liu., Z.C., H.Li., Dan.W., C.T., Y.Y., and Y.G. designed and performed the experiments. X.J., Dao.W., and X.S. conceived the project and wrote the manuscript. All authors approved the final manuscript. Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.13624/suppinfo Figure S1. Diagram of pBlunt-TaU3-crRNA and pBlunt-CasΦ2Ta-V1 vectors and their editing frequencies in wheat or rye protoplasts-based transient system Figure S2. Diagram of pBlunt-CasΦ2Ta-V3, pBlunt-NCasΦ2Ta-V3 and pBlunt-VCasΦ2Ta-V3 vectors and their efficiency assessment in wheat or rye protoplasts Figure S3. Diagram of pLH-CasΦ2Ta-V3 and pLH-VCasΦ2Ta-V3 vectors and representative genotypes of gene editing T0 mutants Figure S4. The efficiency assessment of paired crRNA strategy for increasing pBlunt-VCasΦ2Ta-V3-mediated editing frequencies in (A) wheat or (B) rye protoplasts by PCR/RE assay Figure S5. The diagram of pLH-CasΦ2Ta-CBE and pLH-dCasΦ2Ta-CBE vectors and representative genotypes of cytosine base editing T0 mutants Figure S6. The diagram of pLH-dCasΦ2Ta-ABE vector and representative genotypes of adenine base editing T0 mutants Figure S7. Assessment of potential off-targeting by CRISPR/VCasΦ2Ta-V3-TaGW2-crRNA-TTN, CRISPR/dCasΦ2Ta-CBE-TaGW2-crRNA-TTN or CRISPR/dCasΦ2Ta-ABE-TaALS-crRNA-TTN in the T0 mutants with indel, C-to-T or A-to-G editing Table S1. The primer sequences for constructing the vectors used in this study Table S2. The primers used for preparing target crRNAs Table S3. Analysis of mutation transmission from 12 T0 plants derived from pLH-VCasΦ2Ta-V3, pLH-CasΦ2Ta-CBE, pLH-dCasΦ2Ta-CBE, or pLH-dCasΦ2Ta-ABE to the T1 progenies Table S4. Potential off-target sites for TaGW2-crRNA-TTN and TaALS-crRNA-TTN protospacer in common wheat Table S5. The primers with barcodes for deep sequencing Table S6. Other primer sequences used in this study Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.