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  • Are the current gRNA ranking prediction algorithms useful for genome editing in plants?

Are the current gRNA ranking prediction algorithms useful for genome editing in plants?

PloS one (2020-01-25)
Fatima Naim, Kylie Shand, Satomi Hayashi, Martin O'Brien, James McGree, Alexander A T Johnson, Benjamin Dugdale, Peter M Waterhouse
ABSTRACT

Introducing a new trait into a crop through conventional breeding commonly takes decades, but recently developed genome sequence modification technology has the potential to accelerate this process. One of these new breeding technologies relies on an RNA-directed DNA nuclease (CRISPR/Cas9) to cut the genomic DNA, in vivo, to facilitate the deletion or insertion of sequences. This sequence specific targeting is determined by guide RNAs (gRNAs). However, choosing an optimum gRNA sequence has its challenges. Almost all current gRNA design tools for use in plants are based on data from experiments in animals, although many allow the use of plant genomes to identify potential off-target sites. Here, we examine the predictive uniformity and performance of eight different online gRNA-site tools. Unfortunately, there was little consensus among the rankings by the different algorithms, nor a statistically significant correlation between rankings and in vivo effectiveness. This suggests that important factors affecting gRNA performance and/or target site accessibility, in plants, are yet to be elucidated and incorporated into gRNA-site prediction tools.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
6-Benzylaminopurine, suitable for plant cell culture
Sigma-Aldrich
Murashige and Skoog Basal Salt Mixture (MS), powder, suitable for plant cell culture
Sigma-Aldrich
Indole-3-butyric acid, BioReagent, suitable for plant cell culture
Sigma-Aldrich
1-Naphthylacetic acid, 1 mg/mL, BioReagent, suitable for plant cell culture