Efficient large fragment deletion in plants: double pairs of sgRNAs are better than dual sgRNAs

Acknowledgements

We thank Professor Pengcheng Wei of Anhui Agricultural University for his guidance on the experimental methods. This work was supported by the National Key Research and Development Program of China (Grant No. 2022YFD2100101), the Joint NSFC-ISF Research Program (Grant No. 32061143022), the 2115 Talent Development Program of China Agricultural University (Grant No. 1061-00109017) to HZ and the National Natural Science Foundation of China (Grant No. 3217180159).

Author contributions

H.Z. and G.Z. designed the studies. G.Z. and H.Z. performed the experiments. G.Z., Q.L., K.W. and H.Z. analysed the data. L.M., J.L., L.Z., G.Q, B.Z., D.F., Y.L. and H.Z. provided lab support. H.Z. and G.Z. wrote the paper.

Data availability

The Sanger sequencing chromatogram which was named ‘chromatogram of Sanger sequencing.rar’ is available in the supplementary material.

Conflict of interest

The authors declare no conflict of interest.

References

[1] Lowder LG, Zhang D, Baltes NJ et al. A CRISPR/Cas9 toolbox for multiplexed plant genome editing and transcriptional regulation. Plant Physiol. 2015; 169: 971-85

[2] Qing R, Tao F, Zhang S et al. Non-full-length water-soluble CXCR4 QTY and CCR5 QTY chemokine receptors: implication for overlooked truncated but functional membrane receptors . iScience. 2020; 23.

[3] Wang Y, Geng L, Yuan M et al. Deletion of a target gene in Indica rice via CRISPR/Cas9. Plant Cell Rep. 2017; 36: 1333-43

[4] Cai Y, Chen L, Sun S et al. CRISPR/Cas9-mediated deletion of large genomic fragments in soybean. Int J Mol Sci. 2018; 19.

[5] Abdelrahman M, Wei Z, Rohila JS et al. Multiplex genome-editing technologies for revolutionizing plant biology and crop improvement. Front Plant Sci. 2021; 12.

[6] Tan J, Zhao Y, Wang B et al. Efficient CRISPR/Cas9-based plant genomic fragment deletions by microhomology-mediated end joining. Plant Biotechnol J. 2020; 18: 2161-3

[7] Seol J, Shim EY, Lee SE . Microhomology-mediated end joining: good, bad and ugly. Mutat Res. 2018; 809: 81-7

[8] Giménez E, Dominguez E, Pineda B et al. Transcriptional activity of the MADS box ARLEQUIN/TOMATO AGAMOUS-LIKE1 gene is required for cuticle development of tomato fruit. Plant Physiol. 2015; 168: 1036-48

[9] Yu T, Tzeng DTW, Li R et al. Genome-wide identification of long non-coding RNA targets of the tomato MADS box transcription factor RIN and function analysis. Ann Bot. 2019; 123: 469-82

[10] Wu R, Lucke M, Jang YT et al. An efficient CRISPR vector toolbox for engineering large deletions in Arabidopsis thaliana. Plant Methods. 2018; 14: 65-5