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** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** ADVANCES IN BIOLOGY ARE ENABLING MORE EFFICIENT GENETIC TRANSFORMATION AND EDITING, INCLUDING FOR BIOSAFETY-PROMOTING TRAITS SUCH AS FOR GENETIC CONTAINMENT. MANY ASEXUALLY PROPAGATED SPECIES, SUCH AS THE FOREST TREES WE PROPOSE FOR STUDY, ARE NOTABLY RECALCITRANT TO TRANSFORMATION YET IN NEED OF BIOSAFETY-PROMOTING TRANSGENES OR EDITS DUE TO THEIR POTENTIAL LARGE ECOLOGICAL IMPACTS AND PROPENSITY FOR LONG DISTANCE GENE FLOW. HOWEVER, THE GENES EMPLOYED (SUCH AS CRISPR/CAS9) OFTEN NEED TO BE EXCISED (REMOVED) TO FACILITATE REGULATORY APPROVAL OR ENSURE PLANT HEALTH--PARTICULARLY IN THESE SPECIES FOR WHICH SEXUAL SEGREGATION IS DIFFICULT OR IMPOSSIBLE. THE MOST COMMON TOOLS FOR EXCISION ARE THE CRE AND FLP RECOMBINASES, HOWEVER, THEIR ACTIVITY IS CONSTRAINED BY TARGET-SITE DNA METHYLATION IN PLANTS, MAKING THEIR USE INEFFICIENT. VARIOUS MEANS FOR DEMETHYLATION ARE KNOWN TO ENHANCE EFFICIENCY, BUT HAVE NOT BEEN COMPARED OR OPTIMIZED, NOR HAVE THEIR OFF-TARGET IMPACTS EXAMINED AT THE LOCUS, GENOME, OR PHENOTYPIC LEVELS. USING TOBACCO, ARABIDOPSIS, AND POPLAR AS STUDY SYSTEMS, WE WILL: 1) DEVELOP NOVEL RECOMBINASE TARGET SITES THAT ARE DEVOID OF POTENTIALLY METHYLATED CYTOSINES BUT ARE ACTIVE IN SUPPORTING EXCISION; 2) TEST A VARIETY OF CONSTITUTIVE, INDUCIBLE, AND SITE-SPECIFIC METHYLATION INHIBITORS FOR ENHANCEMENT OF TRANSGENE EXCISION; AND 3) EXAMINE LOCAL, GENOME-SCALE AND PHENOTYPIC IMPACTS OF THE MOST EFFECTIVE METHYLATION CONTROL SYSTEMS. THIS WORK WILL HELP ENHANCE GENOME TRANSFORMATION AND ENGINEERING TOOLS RELEVANT TO BIOSAFETY TECHNOLOGY APPLICATIONS AND INFORM REGULATORS OF THE DEGREE TO WHICH THEY SHOULD BE CONCERNED WITH OFF-TARGET IMPACTS WHEN RECOMBINASES ARE USED FOR GENOME MODIFICATION.

$649,916FY2023National Institute of Food and AgricultureUSDA

Oregon State University, Corvallis OR

Investigators

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** AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** ADVANCES IN BIOLOGY ARE ENABLING MORE EFFICIENT GENETIC TRANSFORMATION AND EDITING, INCLUDING FOR BIOSAFETY-PROMOTING TRAITS SUCH AS FOR GENETIC CONTAINMENT. MANY ASEXUALLY PROPAGATED SPECIES, SUCH AS THE FOREST TREES WE PROPOSE FOR STUDY, ARE NOTABLY RECALCITRANT TO TRANSFORMATION YET IN NEED OF BIOSAFETY-PROMOTING TRANSGENES OR EDITS DUE TO THEIR POTENTIAL LARGE ECOLOGICAL IMPACTS AND PROPENSITY FOR LONG DISTANCE GENE FLOW. HOWEVER, THE GENES EMPLOYED (SUCH AS CRISPR/CAS9) OFTEN NEED TO BE EXCISED (REMOVED) TO FACILITATE REGULATORY APPROVAL OR ENSURE PLANT HEALTH--PARTICULARLY IN THESE SPECIES FOR WHICH SEXUAL SEGREGATION IS DIFFICULT OR IMPOSSIBLE. THE MOST COMMON TOOLS FOR EXCISION ARE THE CRE AND FLP RECOMBINASES, HOWEVER, THEIR ACTIVITY IS CONSTRAINED BY TARGET-SITE DNA METHYLATION IN PLANTS, MAKING THEIR USE INEFFICIENT. VARIOUS MEANS FOR DEMETHYLATION ARE KNOWN TO ENHANCE EFFICIENCY, BUT HAVE NOT BEEN COMPARED OR OPTIMIZED, NOR HAVE THEIR OFF-TARGET IMPACTS EXAMINED AT THE LOCUS, GENOME, OR PHENOTYPIC LEVELS. USING TOBACCO, ARABIDOPSIS, AND POPLAR AS STUDY SYSTEMS, WE WILL: 1) DEVELOP NOVEL RECOMBINASE TARGET SITES THAT ARE DEVOID OF POTENTIALLY METHYLATED CYTOSINES BUT ARE ACTIVE IN SUPPORTING EXCISION; 2) TEST A VARIETY OF CONSTITUTIVE, INDUCIBLE, AND SITE-SPECIFIC METHYLATION INHIBITORS FOR ENHANCEMENT OF TRANSGENE EXCISION; AND 3) EXAMINE LOCAL, GENOME-SCALE AND PHENOTYPIC IMPACTS OF THE MOST EFFECTIVE METHYLATION CONTROL SYSTEMS. THIS WORK WILL HELP ENHANCE GENOME TRANSFORMATION AND ENGINEERING TOOLS RELEVANT TO BIOSAFETY TECHNOLOGY APPLICATIONS AND INFORM REGULATORS OF THE DEGREE TO WHICH THEY SHOULD BE CONCERNED WITH OFF-TARGET IMPACTS WHEN RECOMBINASES ARE USED FOR GENOME MODIFICATION. · GrantIndex