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

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.

Use of CRISPR-Cas9 To Target Homologous Recombination Limits Transformation-Induced Genomic Changes in Candida albicans

“…In other organisms, it has been shown that CRISPR-Cas9 can induce off-target DNA DSB, which may have grave implications ( 23 , 24 ). To the best of our knowledge, this has not been studied in C. albicans .…”

“…To limit these effects, a transient system has been developed in C. albicans circumventing constitutive Cas9 and sgRNA expression ( 21 ). Another issue is raised by reports of off-target DSB by Cas9 in mammalian cells ( 22 – 24 ). Overall, our understanding of the effects of CRISPR-Cas9 technology in C. albicans is limited, especially in terms of its impact on the integrity of the genome.…”

Applying gene editing to tailor precise genetic modifications in plants

“…To investigate the effects of delivery methods on DNA-free editing, Banakar et al (59) compared three approaches, biolistic RNP/DNA co-delivery, biolistic delivery of CRISPR/Cas reagents as plasmid DNA, and Agrobacterium-mediated delivery of CRISPR/Cas reagents into mature-seed derived rice embryos that were cultured on medium for 7 days. All three approaches were successful at achieving the intended edits, however, both biolistic delivery approaches resulted in more than 14% random plasmid or chromosomal DNA fragment insertion at the intended target sites.…”

An improved biolistic delivery and analysis method for evaluation of DNA and CRISPR-Cas delivery efficacy in plant tissue

“…5). The CRISPR plasmid pTF6005 [28] (Fig. 5A) has the Cas9 gene driven by the maize ubiquitin promoter and an OsPDS gRNA (sgRNA1) driven by the rice U6 promoter.…”