CRISPR Genome Editing in Outcrossing Woody Perennials: Living with SNPs

The CRISPR/Cas9 technology is revolutionizing all facets of biology from medicine to agriculture, owing to its precision, efficiency, versatility and ease of adoption.  We recently reported the first application of CRISPR/Cas9 in stably transformed Populus, extending the species range of this powerful technology to woody perennials.  We achieved 100% mutational efficiency in two 4-coumarate:CoA ligase (4CL) genes tested—all stably transformed plants contain bi-allelic DNA modifications.  In both cases, the primary transformants exhibited strikingly uniform phenotypes among independent transgenic lines, a consistency that is unmatched by previous gene silencing methods.  An underappreciated obstacle in genome editing of outcrossing species is the frequent occurrence of sequence polymorphisms that can render CRISPR/Cas9 unproductive.  However, current genome data mining tools and resources do not support exploration of sequence variations.  We present experimental evidence as well as genome-wide computational analysis to demonstrate the sensitivity of CRISPR/Cas9 to allelic heterozygosity, and discuss tools and strategies that can help deal with such sequence polymorphisms.  With its specificity, CRISPR/Cas9 offers a less equivocal means than previous approaches for discerning functional redundancy of paralogous genes that are prevalent in plant genomes.  Continuing improvements of the CRISPR/Cas9 system for multiplex genome engineering should facilitate these efforts.  For woody perennials such as forest trees, fruit/nut trees and woody ornamentals with long generation times, CRISPR/Cas9 affords a facile means for precision gene editing to accelerate not only basic research but also crop improvement.