High-frequency random DNA insertions upon co-delivery of CRISPR-Cas9 ribonucleoprotein and selectable marker plasmid in rice

Raviraj Banakar,Kan Wang,Scott Zarecor,Keunsub Lee,David A Wright,Alan L Eggenberger,Carolyn J Lawrence-Dill,Dipali G Sashital,Karthik Murugan

doi:10.1038/s41598-019-55681-y

Raviraj Banakar, Kan Wang + Show 7 more

Open Access

https://doi.org/10.1038/s41598-019-55681-y

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Journal: Scientific Reports	Publication Date: Dec 1, 2019
Citations: 73	License type: open-access

Affiliation: Iowa State University, University of Minnesota

Abstract

An important advantage of delivering CRISPR reagents into cells as a ribonucleoprotein (RNP) complex is the ability to edit genes without reagents being integrated into the genome. Transient presence of RNP molecules in cells can reduce undesirable off-target effects. One method for RNP delivery into plant cells is the use of a biolistic gun. To facilitate selection of transformed cells during RNP delivery, a plasmid carrying a selectable marker gene can be co-delivered with the RNP to enrich for transformed/edited cells. In this work, we compare targeted mutagenesis in rice using three different delivery platforms: biolistic RNP/DNA co-delivery; biolistic DNA delivery; and Agrobacterium-mediated delivery. All three platforms were successful in generating desired mutations at the target sites. However, we observed a high frequency (over 14%) of random plasmid or chromosomal DNA fragment insertion at the target sites in transgenic events generated from both biolistic delivery platforms. In contrast, integration of random DNA fragments was not observed in transgenic events generated from the Agrobacterium-mediated method. These data reveal important insights that must be considered when selecting the method for genome-editing reagent delivery in plants, and emphasize the importance of employing appropriate molecular screening methods to detect unintended alterations following genome engineering.

Highlights

The CRISPR-Cas system has been a method of choice for precise genome editing in many organisms, including plants
Biolistic co-delivery of CRISPR-Cas[9] RNP and selectable marker plasmid DNA leads to integration of DNA fragments at target site
We reasoned that the use of a visual phenotypic alteration in plants would be useful to track the progress of our genome editing experiments

Summary

Introduction

The CRISPR-Cas (clustered regularly interspaced short palindromic repeat/CRISPR-associated) system has been a method of choice for precise genome editing in many organisms, including plants. CRISPR reagents are generally introduced into plants cells by biolistic- and Agrobacterium-mediated plant transformation[12]. In the case of Agrobacterium-mediated transformation, CRISPR reagents are introduced as DNA molecules by placing Cas[9] and gRNA expression cassettes within the transfer DNA (T-DNA)[13]. Once Agrobacterium delivers the T-DNA into the plant cell, the expression of the CRISPR reagents are enabled, resulting in genome editing[14]. In contrast to Agrobacterium-mediated transformation, biolistics utilize physical force to introduce DNA molecules into plant cells[21]. It is not limited by complex interactions with the host plant required for Agrobacterium-mediated transformation. The selectable marker transgene can be segregated from the edited locus through segregation in subsequent generations

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Conclusion