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

PLoS One. 2020 Jan 24;15(1):e0227994. doi: 10.1371/journal.pone.0227994. eCollection 2020.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms*
  • Base Sequence
  • CRISPR-Associated Protein 9 / metabolism
  • Gene Editing*
  • Genome, Plant*
  • Nicotiana / genetics
  • Plant Leaves / genetics
  • Plants / genetics*
  • Plants, Genetically Modified
  • RNA, Guide, CRISPR-Cas Systems / genetics*
  • Transgenes

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • CRISPR-Associated Protein 9

Grants and funding

FL160100155 awarded to PMW funded by Australian Research Council, The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.