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Microinjection of the CRISPR/Cas9 editing system through the germ pore of a wheat microspore induces mutations in the target Ms2 gene

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Abstract

Background

Microinjection is a direct procedure for delivering various compounds via micropipette into individual cells. Combined with the CRISPR/Cas9 editing technology, it has been used to produce genetically engineered animal cells. However, genetic micromanipulation of intact plant cells has been a relatively unexplored area of research, partly due to the cytological characteristics of these cells. This study aimed to gain insight into the genetic micromanipulation of wheat microspores using microinjection procedures combined with the CRISPR/Cas9 editing system targeting the Ms2 gene.

Methods and results

Microspores were first reprogrammed by starvation and heat shock treatment to make them structurally suitable for microinjection. The large central vacuole was fragmented and the nucleus with cytoplasm was positioned in the center of the cell. This step and an additional maltose gradient provided an adequate source of intact single cells in the three wheat genotypes. The microcapillary was inserted into the cell through the germ pore to deliver a working solution with a fluorescent marker. This procedure was much more efficient and less harmful to the microspore than inserting the microcapillary through the cell wall. The CRISPR/Cas9 binary vectors injected into reprogrammed microspores induced mutations in the target Ms2 gene with deletions ranging from 1 to 16 bp.

Conclusions

This is the first report of successful genome editing in an intact microspore/wheat cell using the microinjection technique and the CRISPR/Cas9 editing system. The study presented offers a range of molecular and cellular biology tools that can aid in genetic micromanipulation and single-cell analysis.

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Data availability

No datasets were generated or analysed during the current study.

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Acknowledgements

The authors are grateful to Prof. Paweł Sowiński, Warsaw University, for a lease of the Narishige MMO-202ND micromanipulator and Prof. Wacław Orczyk, Institute of Plant Breeding, Radzików, for a gift of the pBract211-Cas9 vector. The authors thank Agata Michalak and Stefan Smolenski for their contributions to plant maintenance and technical assistance in the growth chamber, respectively.

Funding

This work was supported by the NCBR Grant BIOSTRATEG3/343665/6/NCBR/2017.

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BMS and LAŁ designed the CRISPR/Cas9 vectors for wheat genome editing. BMS, MŚ, and LAŁ prepared the material, collected the data, and conducted the analysis. LAŁ wrote the first draft of the manuscript. BMS revised the draft version. All authors approved the final version of the manuscript.

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Correspondence to Bartosz M. Szabała.

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Szabała, B., Święcicka, M. & Łyżnik, L. Microinjection of the CRISPR/Cas9 editing system through the germ pore of a wheat microspore induces mutations in the target Ms2 gene. Mol Biol Rep 51, 706 (2024). https://doi.org/10.1007/s11033-024-09644-w

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