Highly efficient genome editing in plant protoplasts by Ribonucleoprotein delivery of CRISPR-Cas12a nucleases

Authors: ZHANG, YINGXIAO - UNIVERSITY OF MARYLAND; CHENG, YANHAO - UNIVERSITY OF MARYLAND; FANG, HONG - UNIVERSITY OF MARYLAND; ROBERTS, NATHANIEL - INTEGRATED DNA TECHNOLOGIES; ZHANG, LIYANG - INTEGRATED DNA TECHNOLOGIES; VAKULSKAS, CHRISTOPHER - INTEGRATED DNA TECHNOLOGIES; Niedz, Randall; CULVER, JAMES - UNIVERSITY OF MARYLAND; QI, YIPING - UNIVERSITY OF MARYLAND

Submitted to: Frontiers in Genome Editing
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/22/2022
Publication Date: 1/31/2022
Citation: Zhang, Y., Cheng, Y., Fang, H., Roberts, N., Zhang, L., Vakulskas, C.A., Niedz, R.P., Culver, J.N., Qi, Y. 2022. Highly efficient genome editing in plant protoplasts by Ribonucleoprotein delivery of CRISPR-Cas12a nucleases. Frontiers in Genome Editing. 4: Article 780238. https://doi.org/10.3389/fgeed.2022.780238.
DOI: https://doi.org/10.3389/fgeed.2022.780238

Interpretive Summary: Gene editing is the modification of a gene that is already present in an organism. This contrasts with conventional GMO technology that typically adds genes that are not present in the organism. The distinction is important for public discussion of the use of crops that have been altered through gene editing or GMO technologies. The basic difference is that GMOs are created by the introduction of foreign DNA, whereas gene edited crops are generated by changes made to the organism’s native DNA. For example, flower color mutations change the color of a flower by mutating the genes for flower color that are already present in the plant. Gene editing can make the exact same changes – no foreign DNA is added. One significant challenge to using gene editing technology in plants is that many plants are difficult to culture in vitro. Gene editing requires efficient in vitro cell and tissue culture systems because gene editing is done at the cell or tissue level, and then the edited plants are regenerated from cultured cells and tissues. The objective of this study was to increase the efficiency of the gene editing technology using a deliver method called ribonucleoprotein (RNP) delivery of CRISPR-Cas. The study showed that RNP delivery resulted in much better gene editing in the two crops tested – rice and citrus. RNP delivery will help make gene editing useable in plants that are difficult to culture in vitro.

Technical Abstract: Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) mediated genome editing is a powerful approach for crop improvement. Traditional transformation methods based on plasmid delivery pose concerns of transgene integration and off-target effects. CRISPR reagents delivered as ribonucleoproteins (RNPs) can prevent exogenous DNA integration, minimize off-target effects and cellular toxicity. Although CRISPR genome editing using RNP delivery has been demonstrated in many plant species, optimization for high editing efficiencies has not been thoroughly investigated. Based on a rice protoplast system, we demonstrated highly efficient genome editing using Cas12a delivered as RNPs. Four Cas12a variants, including LbCas12a, LbCas12a-E795L, AsCas12a V3 and AsCas12a Ultra, were used for genome editing. Nearly 100% editing efficiency was observed for three out of four target sites by LbCas12a, LbCas12a-E795L and AsCas12a Ultra, as measured by restriction fragment length polymorphism (RFLP). High efficiency editing was further verified by next generation sequencing of PCR amplicons. RNP delivery resulted in higher editing efficiencies than plasmid delivery at 32C and 25C. LbCas12a and LbCas12a-E795L showed higher editing efficiencies than AsCas12a V3 and AsCas12a Ultra, especially when used at lower concentrations. In addition, we discovered that 1:1 Cas12a:crRNA ratio is sufficient for efficient genome editing. Nuclear localization signals (NLSs) are essential for efficient RNP based genome editing. However, the different crRNA modifications tested did not significantly improve genome editing efficiency. Finally, we applied the Cas12a RNP system in citrus protoplasts and obtained high editing efficiency at the target site. Our study provides a comprehensive guideline for Cas12a-mediated genome editing using RNP delivery in plant cells, setting the foundation for the generation of transgene-free genome edited plants.