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  • An LbCas12a variant and elevated incubation temperatures enhance the rate of gene editing in the oomycete Phytophthora infestans
Title: An LbCas12a variant and elevated incubation temperatures enhance the rate of gene editing in the oomycete Phytophthora infestans
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 ten-fold. First, we found that editing was boosted by a mutation in LbCas12a (D156R), which 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.  more » « less
Award ID(s):
1753749 2143897 2051131
PAR ID:
10454910
Author(s) / Creator(s):
; ;
Date Published:
Journal Name:
Molecular Plant-Microbe Interactions®
ISSN:
0894-0282
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
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