Carl S. Mendoza, Annika Findlay, Howard S. Judelson

A Variant of LbCas12a and Elevated Incubation Temperatures Enhance the Rate of Gene Editing in the Oomycete Phytophthora infestans

  • Agronomy and Crop Science
  • General Medicine
  • Physiology

CRISPR-Cas editing systems have proved to be powerful tools for functional genomics research, but their effectiveness in many non-model species remains limited. In the potato and tomato pathogen Phytophthora infestans, an editing system was previously developed that expresses the Lachnospiracae bacterium Cas12a endonuclease (LbCas12a) and guide RNA from a DNA vector. However, the method works at low efficiency. Based on a hypothesis that editing is constrained by a mismatch between the optimal temperatures for P. infestans growth and endonuclease catalysis, we tested two strategies that increased the frequency of editing of two target genes by about 10-fold. First, we found that editing was boosted by a mutation in LbCas12a (D156R) that had been reported to expand its catalytic activity over a broader temperature range. Second, we observed that editing was enhanced by transiently incubating transformed tissue at a higher temperature. These modifications should make CRISPR-Cas12a more useful for interrogating gene and protein function in P. infestans and its relatives, especially species that grow optimally at lower temperatures. [Formula: see text] Copyright © 2023 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

Need a simple solution for managing your BibTeX entries? Explore CiteDrive!

  • Web-based, modern reference management
  • Collaborate and share with fellow researchers
  • Integration with Overleaf
  • Comprehensive BibTeX/BibLaTeX support
  • Save articles and websites directly from your browser
  • Search for new articles from a database of tens of millions of references
Try out CiteDrive

More from our Archive