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A miniature alternative to Cas9 and Cas12: Transposon-associated TnpB mediates targeted genome editing in plants
Friday, 2024/08/02 | 08:23:10
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Subhasis Karmakar, Debasmita Panda, Sonali Panda, Manaswini Dash, Romio Saha, Priya Das, S.P. Avinash, Justin Shih, Yinong Yang, A. K. Nayak, Mirza J. Baig, Kutubuddin A. Molla Plant Biotechnology Journal; 28 June 2024; https://doi.org/10.1111/pbi.14416
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).
See https://onlinelibrary.wiley.com/doi/full/10.1111/pbi.14416
Fig.1: TnpB system developed as a hypercompact plant genome editor. (a) Schematic of the IsDra2TnpB attached with target dsDNA. TAM, Transposon associated motif; reRNA, right end RNA. (b) Plant protoplast assay system workflow. (c) Schematic of the TnpB1 vector. HDV, hepatitis delta virus ribozyme; NLS, nuclear localization signal. (d) TnpB-mediated indel efficiency in six rice loci. (e) TnpB activity in targets with noncanonical TAM (TCGAT). (f, g) Multiple reRNA-guide expression system and indel generation through multiplexing. (h) TnpB vector systems for Arabidopsis genome editing. (i) Indel efficiency in the target sites in Arabidopsis with TnpB-D1 and TnpB-D2. (j) Schematic of TnpB2, TnpB3 and TnpB4. HH, Hammerhead ribozyme. (k–p) Comparison of indel generation efficiency of TnpB vectors across six rice genomic loci. Protoplast transfections were done simultaneously for this comparative analysis. (q) Representative mutation spectrum generated by TnpB2 in OsHMBPP site. (r) TnpB binary vector used for Agrobacterium-mediated transformation. (s) Albino mutants showing loss-of-function phenotypes for OsSLA4. Sanger chromatogram showing the causal 53 bp deletion. (t) T1 plants from T1 seeds collected from T0 plant with HMBPP monoallelic edit. Albino mutants are due to complete loss-of-function of OsHMBPP. Representative chromatogram showing biallelic homozygous editing. All data represented as the mean of three biologically independent experiments (shown in dots) ± SEM.
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