Abstract
As a miniature RNA-guided endonuclease, IscB is presumed to be the ancestor of Cas9 and to share similar functions. IscB is less than half the size of Cas9 and thus more suitable for in vivo delivery. However, the poor editing efficiency of IscB in eukaryotic cells limits its in vivo applications. Here we describe the engineering of OgeuIscB and its corresponding ωRNA to develop an IscB system that is highly efficient in mammalian systems, named enIscB. By fusing enIscB with T5 exonuclease (T5E), we found enIscB-T5E exhibited comparable targeting efficiency to SpG Cas9 while showing reduced chromosome translocation effects in human cells. Furthermore, by fusing cytosine or adenosine deaminase with enIscB nickase, we generated miniature IscB-derived base editors (miBEs), exhibiting robust editing efficiency (up to 92%) to induce DNA base conversions. Overall, our work establishes enIscB-T5E and miBEs as versatile tools for genome editing.
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Data availability
All the PEM-seq data have been deposited in the National Center for Biotechnology Information Sequence Read Archive under project accession number PRJNA889595 and the Sequence Read Archive numbers are provided in Supplementary Table 1. The related plasmids have been deposited to Addgene. All materials are available upon reasonable request. Source data are provided with this paper.
Code availability
Bioinformatics codes were deposited in GitHub repository (https://github.com/yszhou2016/IscB).
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Acknowledgements
This work was supported by HUIDAGENE Therapeutics Inc., HUIEDIT Therapeutics Inc.
Author information
Authors and Affiliations
Contributions
Y.Z. and H.Y. jointly conceived the project. D.H. and Q.X. jointly designed experiments. D.H. and Y.W. performed ωRNA and protein engineering and endogenous sites cleavage assay. Q.X. conducted base editors-related experiments, including engineering IscB nickases, base editors efficiency comparison and off-target analysis. D.H. performed off-target assays. H.Z., W.Z., J.Z. and L.B. performed PEM-seq experiments. Y.Z. performed bioinformatics analysis. X.D., G.L., X.K., S.W., J.S., Y.Y., N.Z. and L.S. assisted with experiments. H.Y. and Y.Z. supervised the whole project. Y.Z. and H.Y. wrote the manuscript with data contributed by all authors who participated in the project.
Corresponding authors
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Competing interests
Y.Z., D.H. and Q.X. have filed patent applications related to this work through HUIDAGENE and CAS. H.Y. and L.S. are cofounders of HUIDAGENE Therapeutics. H.Y. and Y.Z. are cofounders of HUIEDIT Therapeutics. The remaining authors declare no competing interests.
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Nature Methods thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editor: Lei Tang and Rita Strack, in collaboration with the Nature Methods team.
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Extended data
Extended Data Fig. 1 Effect of truncation of R1, R2, R3 and R4 regions of ωRNA with 5-nt.
Each point represents the editing efficiency of independent biological replicate by FACS analysis. Data are presented as means ± s.d., n = 3 independent biological replicates.
Extended Data Fig. 2 Structure-based engineering of IscB at ten amino acid sites.
Wild type ωRNA was used in this experiment. Each point represents the editing efficiency of independent biological replicate by FACS analysis. Data are presented as means ± s.d., n = 3. P-values determined by Sidak’s multiple comparisons test following ordinary one-way analysis of variance.
Extended Data Fig. 3 TAM profiling of IscB-ωRNA, IscB*-ωRNA, IscB-ωRNA* and IscB*-ωRNA*.
The panels from left to right are 5′-NWGGNA-3′, 5′-NWAGNA-3′, 5′-NWGANA-3′ and 5′-NWAANA-3′ TAMs profiling of IscB-ωRNA, IscB*-ωRNA, IscB-ωRNA* and IscB*-ωRNA* in HEK293T cells with the GFxxFP fluorescence reporter systems, which contain different TAMs. Colors reflect the mean of three independent biological replicates.
Extended Data Fig. 4 Comparison of cleavage efficiency of the wild-type IscB after fusing T5 exonuclease at N- and C-terminals.
IscB-T5E and T5E-IscB represent fusing T5 exonuclease at C-terminal and N-terminal respectively. IscB represents WT-IscB without T5E. NT, non-targeting gRNA. Each point represents the editing efficiency of independent biological replicate by FACS analysis. Data are presented as means ± s.d., n = 3 independent biological replicates.
Extended Data Fig. 5 Comparison the indel features of enIscB-T5E, enIscB and SpG.
a. The cleavage patterns of small deletions and insertions generated by enIscB-T5E, enIscB and SpG in HEK-293T cells at EMX1-sg1 site. b. Length distribution of deletions and insertions generated by enIscB-T5E, enIscB and SpG in HEK-293T cells at EMX1-sg1 site.
Extended Data Fig. 6 Off-target efficiency of enIscB, enIscB-T5E and SpG at in-silico predicted off-target sites of EMX1, ALDH1A3 and VEGFA genes.
b. off-target effects of enIscB and enIscB-T5E (a) and SpG (b) targeting ALDH1A3 gene respectively. c-d. off-target effects of enIscB and enIscB-T5E (c) and SpG (d) targeting EMX1 gene respectively. e-f. off-target effects of enIscB and enIscB-T5E (e) and SpG (f) targeting VEGFA gene respectively. The underlined sequences represent the on-target or predicted off-target sites. The mismatches were labeled in red and lower case. Each point represents the editing efficiency of independent biological replicate by deep sequencing analysis. Data are presented as means ± s.d., n = 3 independent biological replicates.
Extended Data Fig. 7 Base editing activity window plots showing mean A-to-G and C-to-T editing at all tested target positions.
Top represents ABEs and bottom represents CBEs. Each point represents the average editing efficiency of three independent biological replicates measured at each endogenous locus. Data are presented as means ± SEM.
Extended Data Fig. 8 Indel frequency of base editors.
Left represents ABEs and right represents CBEs. Data are presented as means ± s.d., n = 3 independent biological replicates.
Extended Data Fig. 9 The gRNA-dependent off-target levels of CBEs at the in-silico predicted off-target sites.
The left and right panel is off-target effects of miCBE and SpG-CBE targeting EMX1, VEGFA and PCSK9 genes respectively. The underlined sequences represent the on-target or predicted off-target sites. The mismatches were labeled in red and lower case. Each point represents the editing efficiency of independent biological replicates by deep sequencing analysis. Data are presented as means ± s.d., n = 3 independent biological replicates.
Extended Data Fig. 10 The gRNA-dependent off-target levels of ABEs at the in-silico predicted off-target sites.
The left and right panel is off-target effects of miABE and SpG-ABE targeting EMX1, PCSK9 and TTR genes respectively. The underlined sequences represent the on-target or predicted off-target sites. The mismatches were labeled in red and lower case. Each point represents the editing efficiency of independent biological replicate by deep sequencing analysis. Data are presented as means ± s.d., n = 3 independent biological replicates.
Supplementary information
Supplementary Information
Supplementary Figs. 1–5.
Supplementary Table 1
Primers and sequences.
Supplementary Table 2
Predicted off-target sites from Cas-OFFinder.
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Han, D., Xiao, Q., Wang, Y. et al. Development of miniature base editors using engineered IscB nickase. Nat Methods (2023). https://doi.org/10.1038/s41592-023-01898-9
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DOI: https://doi.org/10.1038/s41592-023-01898-9
