ABSTRACT
As the focus for CRISPR edited plants moves from proof-of-concept to real world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one gRNA per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in Populus tremula × P. alba INRA 717-1B4. Unexpected duplications of MYB186 and MYB138 resulted in a total of eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and PCR analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the non-glandular trichomes of poplar.
ONE SENTENCE SUMMARY Targeting conserved sequences with a single gRNA allowed efficient mutagenesis of a multigene family and the recovery of trichomeless and triterpene-free poplar mutants.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors is Chung-Jui Tsai (cjtsai{at}uga.edu).
FUNDING The work was funded in part by The Center for Bioenergy Innovation, a U.S. Department of Energy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science, the Division of Integrative Organismal Systems (grant nos. IOS-1546867) of the National Science Foundation, the Community Science Program of the Joint Genome Institute, a DOE Office of Science User Facility. The work conducted by the U.S. Department of Energy Joint Genome Institute is supported by the Office of Science of the U.S. Department of Energy under Contract No DE-AC02-05CH11231.