Abstract
IscB has a similar domain organization to Cas9, but the small size of IscB is better suited for delivery by adeno-associated virus. To improve the low editing efficiency of OgeuIscB (IscB from human gut metagenome) in mammalian cells, we developed high-efficiency miniature base editors by engineering OgeuIscB nickase and its cognate ωRNA, termed IminiBEs. We demonstrated the robust editing efficiency of IminiCBE (67% on average) or IminiABE (52% on average). Fusing non-specific DNA-binding protein Sso7d to IminiBEs increased the editing efficiency of low-efficiency sites by around two- to threefold, and we termed it SIminiBEs. In addition, IminiCBE and SIminiCBE recognize NNRR, NNRY and NNYR target-adjacent motifs, which broaden the canonical NWRRNA target-adjacent motif sites for the wild-type IscB nickase. Overall, IminiBEs and SIminiBEs are efficient miniature base editors for site-specific genomic mutations.
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Data availability
All deep sequencing data are available at the National Center for Biotechnology Information (NCBI) Sequence Read Archive database under the BioProject accession code PRJNA1005862. Other relevant methods and plasmids are available from the corresponding author upon reasonable request.
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Acknowledgements
We thank W. Qi Lab for the gift of HEK293FT cells. We thank P. W. Zhang and L. S. Zhang for their help with cell sorting. FACS was provided by Shanghai Institute for Advanced Immunochemical Studies (SIAIS) at ShanghaiTech University. We are grateful to J. Chen Lab for the gifts of pCMV-BEACON1, miniABEmax and pCMV_BE4max plasmids. This work was funded by the National Natural Science Foundation of China (no. 31970591 to H. Ma) and the Shanghai Science and Technology Innovation Action Plan (21JC1404800 to H. Ma).
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Authors and Affiliations
Contributions
H.M. conceived the project. L.H., Y.H. and H.M. designed the experiments. H.Y., F.B. and Y.S. performed the structural analysis for IscB. L.H. and Y.H. performed the experiments for IscB and ωRNA engineering and BE assays. Q.M. and L.H. analyzed the next-generation sequencing data. L.H. and F.G. performed TAM Definition assay. Q.M. analyzed the library sequencing data. L.H. and H.M. interpreted the data. L.H., Y.H. and H.M. wrote the paper with input from all the authors.
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Competing interests
H.M., L.H. and Y.H. are inventors on a US provisional patent application on IminiBEs and SIminiBEs. The provisional patent number is PCT/CN2024/080287. The other authors declare no competing interests.
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Nature Chemical Biology thanks Hiroshi Nishimasu and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
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Extended data
Extended Data Fig. 1 Comparison of editing efficiencies of IscBn variants-mediated CBE at DYRK1A site.
a, Comparison of C-to-T editing efficiencies induced by IscBn variants at the DYRK1A site in HEK293FT cells. b, Comparison of the efficiencies of C-to-T editing mediated by the combinations of D97K with D90R, F138N, S387R, S431K or S457R at the DYRK1A site respectively. c, Comparison of the efficiencies of C-to-T editing mediated by the combinations of D97K-S457R with F138N, S431K or F138N/S431K at the DYRK1A site respectively. WT is the wild-type IscB nickase (D61A). The most efficient position C6 of the DYRK1A site was chosen for the comparison in a-c. All values and error bars represent means ± s.d., n = 3 independent biological replicates.
Extended Data Fig. 2 Comparison of editing efficiencies of IscBn-CBE and IminiCBE.
IscBn-CBE and IminiCBE induced C-to-T efficiencies at every single cytosine in the indicated spacer region of 16 target sites in HEK293FT cells. Data are means ± s.d. (n = 3 independent experiments).
Extended Data Fig. 3 Comparison of editing efficiencies of IscBn-ABE and IminiABE.
IscBn-ABE and IminiABE induced A-to-G efficiencies at every single adenine in the indicated spacer region of 20 endogenous sites in HEK293FT cells. Data are means ± s.d. (n = 3 independent experiments).
Extended Data Fig. 4 Comparison of editing efficiencies of IminiCGBE1, IminiCGBE2 and IminiCGBE3.
a. Schematic of three C-to-G base editors (IminiCGBE) A1 (R33A): rat APOBEC1 (R33A); UNG1: uracil DNA N-glycosylase 1 from S. cerevisiae; A1: rat APOBEC1; UdgX: UNG ortholog from Mycobacterium smegmatis; eA3A: human APOBEC3A (N57G). b. Comparison of the C-to-G editing efficiencies of IminiCGBE1, IminiCGBE2 and IminiCGBE3 at different sites. The most efficient position of each site was chosen for the comparison. All data are means ± s.d. (n = 3 independent experiments).
Extended Data Fig. 5 Comparison of editing efficiencies among IminiCBE, SIminiCBE, enIscBn-CBE and SpGn-CBE.
a, Diagram of IminiCBE, SIminiCBE, enIscBn-CBE and SpGn-CBE. EnIscBn is an IscB nickase variant containing D61A, E85R, H369R, S387R and S457R mutations. IscBnQM is an IscB nickase variant containing D61A, D97K, F138N, S431K and S457R mutations. SpGn is an SpCas9 nickase variant containing D10A, D1135L, S1136W, G1218K, E1219Q, R1335Q and T1337R mutations. b, Diagram of the TAM/PAM matched sites for IminiCBE, SIminiCBE, enIscBn-CBE and SpGn-CBE. c, Comparison of C-to-T editing efficiencies among IminiCBE, SIminiCBE, enIscBn-CBE and SpGn-CBE at 9 target sites in HEK293FT cells. All data are means ± s.d. (n = 3 independent experiments).
Extended Data Fig. 6 Comparison of editing efficiencies among IminiABE, SIminiABE, enIscBn-ABE and SpGn-ABE.
a, Diagram of IminiABE, SIminiABE, enIscBn-ABE and SpGn-ABE. EnIscBn is an IscB nickase variant containing D61A, E85R, H369R, S387R and S457R mutations. IscBnQM is an IscB nickase variant containing D61A, D97K, F138N, S431K and S457R mutations. SpG is an SpCas9 nickase variant containing D10A, D1135L, S1136W, G1218K, E1219Q, R1335Q and T1337R mutations. b, Diagram of the TAM/PAM matched sites for IminiABE, SIminiABE, enIscBn-ABE and SpGn-ABE. c, Comparison of A-to-T editing efficiencies among IminiABE, SIminiABE, enIscBn-ABEs and SpGn-ABE at 9 target sites in HEK293FT cells. All data are means ± s.d. (n = 3 independent experiments).
Extended Data Fig. 7 The nuclease-dependent off-target analysis of IminiBEs, SIminiBEs and SpGn-BEs at DNMT1-6 site.
a, On-target and off-target efficiencies of IminiCBE, SIminiCBE and SpGn-CBE at the DNMT1-6 sites in HEK293FT cells. b, On-target and off-target efficiencies of IminiABE, SIminiABE and SpGn-ABE at the DNMT1-6 sites in HEK293FT cells. All data are means ± s.d. (n = 3 independent experiments).
Extended Data Fig. 8 The nuclease-independent off-target analysis of IminiBEs, SIminiBEs and SpGn-BEs.
a, Nuclease-independent DNA off-target analysis of C-to-T editing efficiencies induced by IminiCBE, SIminiCBE and SpGn-CBE using the R-loop assay by dSaCas9. b, Nuclease-independent DNA off-target analysis of A-to-G editing efficiencies induced by IminiABE, SIminiABE and SpGn-ABE using the R-loop assay by dSaCas9. All data are means ± s.d. (n = 3 independent experiments).
Extended Data Fig. 9 TAM profiling of IminiCBE and enIscBn-CBE.
a, Sequence logos of IminiCBE or enIscBn-CBE by Weblogo shows the TAM preferences from position +1 to +6. b, Percentage of edited targets with different TAM sequences from total edited reads. A total of 16 TAMs with different nucleotides in +3 and +4 were used to statistically analyze the TAM preference by IminiCBE or enIscBn-CBE. c, Editing efficiencies with different TAM sequences. A total of 64 TAMs with different nucleotides in +2, +3, and +4 were used to statistically analyze the PAM preference by IminiCBE or enIscBn-CBE. Editing efficiency represents the percentage of the edited targets with a TAM sequence from total reads with the same TAM.
Extended Data Fig. 10 Comparison of mutant residues of OgeuIscBQM and enIscB.
OgeuIscBQM variant contains D97K, F138N, S431K and S457R mutations (left panel), while enIscB variant contains E85R, H369R, S387R and S457R mutations (right panel).
Supplementary information
Supplementary Information
Supplementary Fig. 1 and Tables 1–6.
Supplementary Data 1
The predicted off-target sites of IscB and SpG.
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Han, L., Hu, Y., Mo, Q. et al. Engineering miniature IscB nickase for robust base editing with broad targeting range. Nat Chem Biol (2024). https://doi.org/10.1038/s41589-024-01670-w
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DOI: https://doi.org/10.1038/s41589-024-01670-w
