ABSTRACT
The clustered regularly interspaced short palindromic repeats (CRISPR) technology is revolutionizing biology and paving the way for the future of agriculture and forestry. For many crops including perennial trees, those limitations include recalcitrance to tissue culture regeneration and ineffective genetic transformation methods. This chapter discusses additional challenges facing woody perennial trees, namely the heterozygosity of outcrossing species and limited genomic resources. Gene knockouts (KO) represent the predominant applications of the CRISPR technology in woody perennial crops to date. Fortunately, the issue of sequence polymorphisms can be proactively addressed by incorporating additional considerations during gRNA design. We have addressed those concerns by amplicon deep sequencing of both on-target and off-target sites in a suite of transgenic poplars after multiple years of vegetative propagation or repeated cycles of cutback-and-sprouting. CRISPR genome editing is rapidly leveling the playing field, providing those traditionally non-model species with a powerful platform to accelerate genetic improvement.
