Precise, predictable multi-nucleotide deletions in rice and wheat using APOBEC–Cas9

Abstract

Short insertions and deletions can be produced in plant genomes using CRISPR–Cas editors, but reliable production of larger deletions in specific target sites has proven difficult to achieve. We report the development of a series of APOBEC–Cas9 fusion-induced deletion systems (AFIDs) that combine Cas9 with human APOBEC3A (A3A), uracil DNA-glucosidase and apurinic or apyrimidinic site lyase. In rice and wheat, AFID-3 generated deletions from 5′-deaminated C bases to the Cas9-cleavage site. Approximately one-third of deletions produced using AFID-3 in rice and wheat protoplasts (30.2%) and regenerated plants (34.8%) were predictable. We show that eAFID-3, in which the A3A in AFID-3 is replaced with truncated APOBEC3B (A3Bctd), produced more uniform deletions from the preferred TC motif to the double-strand break. AFIDs could be applied to study regulatory regions and protein domains to improve crop plants.

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Fig. 1: Characterization of the AFID system.
Fig. 2: AFID-3 induces predictable, multi-nucleotide-targeted deletions in protoplasts and regenerated plants.
Fig. 3: A3Bctd allows AFIDs to generate more uniform precise deletions within the protospacer.

Data availability

NGS data have been deposited in the NCBI Sequence Read Archive database (accession no. PRJNA630559). Two plasmids encoding AFID-3 and eAFID-3 in the present study will be available through Addgene. All data supporting the findings of the present study are available in the article and its supplementary figures and tables, or from the corresponding author on request. For sequence data, OsAAT (LOC_Os01g55540), OsACC (LOC_Os05g22940), OsCDC48 (LOC_Os03g05730), OsEV (LOC_Os02g11010), OsNRT1.1B (LOC_Os10g40600), OsSPL14/OsIPA1 (LOC_Os08g39890) and OsSWEET14 (LOC_Os11g31190) are from Rice Genome Annotation Project (http://rice.plantbiology.msu.edu); TaMYB10 (AB191458.1, AB191459.1, AB191460.1) and TaVRN1 (AY747603.1, AY747604.1, AY747605.1) are from the NCBI (https://www.ncbi.nlm.nih.gov); TaF3H (TraesCS2A02G493500, TraesCS2B02G521500, TraesCS2D02G493400), TaGASR6 (TraesCS1A02G270100, TraesCS1D02G270100), TaPDS (TraesCS4A02G004900, TraesCS4B02G300100, TraesCS4D02G299000) and TaPMK (TraesCS5A02G449000, TraesCS5B02G453800, TraesCS5D02G455500) are from the International Wheat Genome Sequencing Consortium (https://urgi.versailles.inra.fr); and tae-Pri-miR160, tae-Pri-miR319, tae-Pri-miR396 and tae-Pri-miR444a are from miRBase (http://www.mirbase.org).

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Acknowledgements

We thank Professor G. Chen (Shanghai Jiao Tong University) for providing the PH strain containing the TAL effector AvaXa7 for evaluation of bacterial blight resistance. This work was supported by grants from the Strategic Priority Research Program of the Chinese Academy of Sciences (Precision Seed Design and Breeding, grant no. XDA24020100), the National Transgenic Science and Technology Program (grant no. 2016ZX08010002), the National Natural Science Foundation of China (grant no. 31788103, 31971370), the National Key Research and Development Program of China (grant no. 2016YFD0101804), the Chinese Academy of Sciences (grant no. QYZDY-SSW-SMC030) and the Postdoctoral Innovative Talent Support Program of China (grant no. BX20190365).

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S.W., H.Z. and C.G. designed the project. S.W., Y.Z., Q.L., H.Z., Z.C., K.C. and D.Z. performed the experiments. C.G. supervised the project. S.W., Y.Z., Q.L., H.Z, J.-L.Q. and C.G. wrote the manuscript.

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Correspondence to Caixia Gao.

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Supplementary Figs. 1–9, Tables 1 and 2 and Sequences 1 and 2.

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Wang, S., Zong, Y., Lin, Q. et al. Precise, predictable multi-nucleotide deletions in rice and wheat using APOBEC–Cas9. Nat Biotechnol (2020). https://doi.org/10.1038/s41587-020-0566-4

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