THIS RESEARCH ADDRESSES AREA 5A OF THE USDA BIOTECHNOLOGY RISK ASSESSMENT CALL FOR PROPOSALS, INCLUDING RESEARCH ADDRESSING THE UNINTENDED EFFECTS IN BIOENGINEERED ORGANISMS DEVELOPED USING GENOME EDITING TECHNOLOGY OR OTHER ENGINEERING TECHNIQUES.ADVANCES IN BIOLOGY AND GENE EDITING METHODS ARE ENABLING THE ADVANCED MODIFICATIONS OF GENOMES IN A WIDE VARIETY OF CROP SPECIES. GENES THAT CONTROL SHOOT DEVELOPMENT ARE POWERFUL TOOLS FOR HELPING GENE TRANSFER AND EDITING IN MANY DIFFICULT CROP VARIETIES, BUT USUALLY MUST BE REMOVED (EXCISED) FROM THE GENOME TO ENABLE NORMAL PLANT REGENERATION AND FERTILITY. IN VEGETATIVELY PROPAGATED CROPS, GENE EDITING AGENTS SUCH AS CRISPR CANNOT BE REMOVED DURING SEXUAL REPRODUCTION AS IS COMMON FOR ANNUAL CROPS, THUS ALSO BENEFIT FROM EXCISION METHODS. THE MOST COMMON TOOL FOR EXCISION IS THE CRE RECOMBINASE, HOWEVER, CRE EXPRESSION MAY IMPART UNDESIRABLE AND SUBSTANTIAL UNINTENDED GENOME MODIFICATIONS.USING POPLAR AS A TEST DICOT CROP, WE PROPOSE TO: 1) COMPLETE THE GENERATION AND TESTING OF A SYSTEM FOR CONTROLLED EXPRESSION OF CRE FOR TRANSGENE AND CRISPR-CAS REMOVAL IN CLONALLY PROPAGATED DICOT CROPS; 2) OPTIMIZE THE SYSTEM WITH RESPECT TO ITS CAPACITY TO PRODUCE AN EFFICIENT RATE OF EDITING AND EXCISION WITH MINIMAL CHIMERISM; 3) PRODUCE PROTOTYPE EDITED AND EXCISED REGENERANTS, AND ANALYZE EFFECTS OF CRE ACTIVITY ON VARIATION IN PLANT BIOMASS PRODUCTIVITY IN THE GREENHOUSE, GENOME-SCALE CHROMOSOME AND DNA BASE PAIR VARIATION, AND NEAR TO THE INSERTION/EXCISION SITE IMPACTS ON GENE EXPRESSION AND CHROMOSOME STRUCTURE. THIS WORK WILL HELP TO INFORM REGULATORS OF THE DEGREE TO WHICH THEY SHOULD BE CONCERNED WITH OFF-TARGET IMPACTS WHEN CRE RECOMBINASES ARE USED FOR GENOME EDITING.