Abstract

The two popular genome editor nucleases, Cas9 and Cas12a, hypothetically evolved from IscB and TnpB, respectively (Altae-Tran et al., 2021). Recent reports showed that TnpBs also function as RNA-guided nucleases in human cells (Karvelis et al., 2021). TnpB proteins are much smaller (~400 aa) than Cas9 (~1000–1400 aa) and Cas12a (~1300 aa). The large cargo size of Cas9 and Cas12a hinders their delivery into cells, particularly through viral vectors. Hence, TnpB offers an attractive candidate that can be adopted as a new type of genome editing tool for eukaryotes. However, it is unknown whether TnpB can mediate genome editing in plant systems. In this study, we developed and optimized hypercompact genome editor based on TnpB protein from Deinococcus radiodurans ISDra2 and achieved editing efficiency as high as 33.58% on average in the plant genome. To develop a TnpB genome editing system in plants, we first codon optimized the ISDra2TnpB and cloned it under the OsUbi10 promoter. The right end element (reRNA), which forms an RNP complex with TnpB protein, is required for target DNA recognition and cleavage (Karvelis et al., 2021; Figure 1a). We used a protoplast system workflow for evaluating TnpB-mediated editing (Figure 1b; Panda et al., 2024). We cloned the reRNA component under the OsU3 promoter to construct pK-TnpB1 (Figure 1c; Figure S1). Analogous to the PAM requirement of Cas12, TnpB cleavage is dependent on the presence of transposon-associated motif (TAM) 5′ to the target sequence. For ISDra2TnpB (only TnpB from this point onward), the TAM sequence is 5′-TTGAT-3′. Genome-wide analysis revealed a 0.35% TTGAT TAM coverage in rice, highlighting TnpB's unique targetability to regions not accessible by Cas9 or Cas12a. TnpB cleaves targets at 15–21 bp from TAM, generating staggered patterns (Karvelis et al., 2021; Figure 1a). We have designed guide RNAs for six rice genomic loci, with five containing a recognition sequence for a specific restriction enzyme at the expected cleavage site. To assess effectiveness, we transfected rice protoplasts with these six constructs and cloned amplified target loci into pGEM-T-Easy vector. We digested the colony PCR products with target-specific restriction endonucleases (REs). On average, screening 100 colonies per guide revealed 1.5–7.15% of undigested bands due to disruption of RE sites. Sanger sequencing of the undigested bands confirmed the result. We observed mostly deletions ranging 7–53 bp across the targets (Figure S1). To assess editing efficiency in the whole protoplasts population, we repeated transfection and performed targeted deep amplicon sequencing. pK-TnpB1 induced mutations at all target loci, exhibiting the highest indel efficiency (average 14.84 ± 4.88%) at the HMBPP locus (Figure 1d). To verify TAM specificity, we assessed TnpB activity in two loci with the noncanonical TCGAT TAM. TnpB caused 90 bp across the sites (Figures S7–S12). Taken together, on average across the six loci, TnpB2 outperformed the other three versions of TnpB systems (Figure S13). Finally, to evaluate TnpB's ability to perform genome editing in regenerated rice plants, we constructed a binary pKb-TnpB2 vector (Figure 1r). We strategically chose the HMBPP and SLA4 genes, pivotal for chloroplast development, as their disruption yields readily observable albino phenotypes (Liu et al., 2020; Wang et al., 2018). Agrobacterium-mediated transformation generated 53 and 65 T0 plants for HMBPP and SLA4g2 targets, respectively. Locus-specific genotyping, through Sanger sequencing, revealed both monoallelic and biallelic editing. In the case of SLA4g2, 12 T0 plants displayed biallelic editing, resulting in albino phenotypes (Figure 1s; Tables S1 and S2). The majority of albino plants exhibited a 53-bp deletion in the SLA4g2 site. However, for the HMBPP locus, we achieved monoallelic editing in T0 plants with an efficiency of 38% (20 plants out of 53) (Tables S1 and S2). Subsequently, T1 plants were generated from the T1 seeds obtained from T0 plants, yielding hmbpp homozygous and albino mutants in the T1 generation with high efficiency (Figure 1t). Sanger sequencing unveiled a 23 bp deletion in majority of the hmbpp mutants. In conclusion, our study pioneers the use of TnpB as a compact genome editor in both monocot and dicot plant species. By optimizing the expression system, we successfully increased editing efficiency. Repurposing dTnpB for transcriptional activation and base editing showcases TnpB's versatile potential. Future endeavours will refine reRNA structure and length, and explore TnpB's natural diversity. Our findings position TnpB as a versatile and promising tool for plant genome engineering. S.K. acknowledges the support received from a Science and Engineering Research Board (SERB)-National Post-Doctoral Fellowship. D.P. and S.P.A. express gratitude for the support provided through the University Grants Commission (UGC), Government of India-JRF programme. S.P. is backed by the Department of Science and Technology (DST), Government of India-INSPIRE programme. P.D. acknowledges the fellowship from the Department of Biotechnology (DBT), Government of India-JRF programme. JS has been supported by a pre-doctoral fellowship from USDA/NIFA (2020-67034-31727). Y.Y. is supported by the USDA National Institute of Food and Agriculture and Hatch Appropriations under Project #PEN04842 and Accession #7005064. M.J.B, K.M. and M.D. acknowledge funding from the Indian Council of Agricultural Research (ICAR), New Delhi, under the Plan Scheme—'Incentivizing Research in Agriculture' project, along with the support from the Director, NRRI. K.M and M.J.B acknowledge the funding from the National Agricultural Science Fund (NASF), New Delhi. K.M. and R.S. appreciate the funding received from Ignite Life Science Foundation, Bangalore, India. Special thanks are extended to Chandana Ghosh, Asif Ali V.K., Chinmayee Mohanty and Akash P. for their assistance in various experiments and analyses. The Indian Council of Agricultural Research (ICAR), New Delhi, filed a patent application related to this study. K.M. and M.J.B. conceived and designed the study and supervised the entire project. K.M. and S.K. planned the experiments. S.K., D.P., M.D., S.P., R.S. and S.P.A. performed experiments and collected data. D.P., S.K., K.M., S.P., R.S., P.D., S.P.A. and J.S. analysed the data. Y.Y. provided advice on various experiments. K.M., S.K. and D.P. wrote the manuscript. Y.Y., J.S., A.K.N. and M.J.B. edited the manuscript. Indian Council of Agricultural Research (ICAR), New Delhi, in the form of the Plan Scheme—'Incentivizing Research in Agriculture' project. All data supporting the findings of this study are accessible within the article and its supplemental file. The sequencing data for all target loci have been deposited in the National Center for Biotechnology Information (NCBI) under the Sequence Read Archive (SRA) with the BIOPROJECT ID: PRJNA1066105. Plasmids, pKb-TnpB1 (plasmid #215333) and pKb-TnpB2 (plasmid #215334), will be available through Addgene. Figure S1 Schematic diagram of pK-TnpB1 vector and genome editing in rice. Figure S2 pK-TnpB multiplexing vector and frequency of deletion types in different loci in rice. Figure S3 pK-TnpB1 mediated genome editing in Arabidopsis (as a model dicot) and frequency of deletion types for different target genes. Figure S4 pK-dTnpB-ABE8e mediated base editing and indel status at different target loci in rice. Figure S5 dTnpB mediated gene activation in rice. Figure S6 Schematic diagrams of different versions of TnpB constructs used for genome editing in rice. Figure S7 Rice HMBPP (OsHMBPP) gene-targeted with different versions of the TnpB vectors. Figure S8 Rice SLA4 (OsSLA4) gene-targeted with different versions of the TnpB vectors with guide sequence (1). Figure S9 Rice SLA4 (OsSLA4) gene-targeted with different versions of the TnpB vectors with guide sequence (2). Figure S10 Rice Pi21 (OsPi21) gene-targeted with different versions of the TnpB vectors. Figure S11 Rice CKX2 (OsCKX2) gene-targeted with different versions of the TnpB vectors. Figure S12 Rice CAF2 (OsCAF2) gene-targeted with different versions of the TnpB vectors. Figure S13 Indel efficiency comparison of TnpB systems across six loci. Table S1 Details of type of mutations in T0 plants. Table S2 Details of the type of mutations in T1 plants. Table S3 Different vectors, polynucleotide and polypeptide sequences, and their sequence IDs. Table S4 Target genes with locus IDs and guide sequences. Table S5 List of primers used for making different TnpB constructs. Table S6 List of primers used for cloning different guides. Table S7 List of primers used for screening of mutants and qRT-PCR analysis. Table S8 List of primers used for deep amplicon sequencing (NGS). 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.