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MODIFYING THE PHYSICAL CHARACTERISTICS OR TRAITS OF PLANTS, BOTH FOR BASIC RESEARCH AND FOR CROP IMPROVEMENT, HAS LONG INVOLVED THE INTRODUCTION OF NEW GENETIC VARIATION. ONCE INTRODUCED, THE NEW VARIATION IS SELECTED UPON TO IDENTIFY INDIVIDUALS THAT CARRY THE DESIRED TRAIT WHILE MINIMIZING OTHER PHYSICAL CHANGES. HISTORICALLY NEW VARIATION HAS BEEN INTRODUCED THROUGH CROSSING SCHEMES INVOLVING PARENTS WITH DESIRED TRAITS OR THROUGH THE INTENTIONAL INDUCTION OF NEW MUTATIONS. BOTH OF THESE APPROACHES CREATE GENETIC CHANGES THAT EXTEND BEYOND ANY SINGLE LOCATION IN THE GENOME TARGETED FOR TRAIT MODIFICATION. EMERGING APPROACHES FOR GENETIC MODIFICATION ARE DESIGNED TO CREATE DESIRABLE CHANGES IN PLANT TRAITS WHILE MINIMIZING CHANGES ELSEWHERE IN THE GENOME. THE MOST POPULAR APPROACH, KNOWN AS CRISPR/CAS9 MODIFICATION, HAS THE POTENTIAL TO ALTER SINGLE DNA POSITIONS WITHOUT MAKING CHANGES ELSEWHERE IN THE GENOME AND WITHOUT LEAVING BEHIND THE MOLECULAR MACHINERY NECESSARY FOR MAKING THE CHANGE. HOWEVER, THERE IS AN ONGOING DEBATE IN THE SCIENTIFIC COMMUNITY ABOUT THE POTENTIAL FOR CRISPR/CAS9 MODIFICATIONS TO CLEAVE OR ALTER DNA AT UNINTENDED LOCATIONS, A PROCESSED REFERRED TO AS "OFF-TARGET" MUTAGENESIS. FURTHERMORE, THERE IS DEBATE REGARDING THE POSSIBILITY THAT THE OFF-TARGET MUTATIONS, WHEN UNDETECTED, MAY BE HARMFUL TO PLANT OR EVEN HUMAN HEALTH.TO DETERMINE THE PREVALENCE AND EFFECTS OF OFF-TARGET CHANGES DUE TO CRISPR/CAS9 MODIFICATION IT IS NECESSARY TO DISTINGUISH CRISPR/CAS9-INDUCED CHANGES FROM EXISTING (STANDING) GENETIC VARIATION AND DNA CHANGES ARISING FROM PLANT REGENERATION AND TISSUE CULTURE. WE WILL TEST FOR OFF-TARGET CHANGES OVER FOUR GENERATIONS IN 15 FAMILIES OF PLANTS THAT DIFFER IN THE PRESENCE OR ABSENCE OF THE CRISPR/CAS9 MACHINERY (A TRANSGENE). WE WILL USE TWO SOYBEAN CULTIVARS THAT ARE SUBJECT TO DIFFERENT TISSUE CULTURE TECHNIQUES. WE WILL USE A SIMPLE ASSAY TO SCREEN FOR THE PRESENCE OF THE CRISPR/CAS9 TRANSGENE. BECAUSE ALL PLANTS WILL BE SUBJECT TO THE SAME REGENERATION TECHNIQUES AND HAVE THE SAME GENETIC BACKGROUND, THIS EXPERIMENT WILL ISOLATE THE EFFECTS OF CRISPR/CAS9 IN INDUCING GENETIC CHANGES. THE ISOLATION OF CRISPR/CAS9 OFF-TARGET CHANGES RELATIVE TO OTHER SOURCES SUCH AS TISSUE CULTURE OR GENETIC HETEROGENEITY SHOULD HELP PLANT BREEDERS, GENETIC RESEARCHERS, AND REGULATORY AGENCIES IN MAKING SCIENCE-BASED DECISIONS REGARDING APPLICATIONS AND POLICY APPLIED TO THE GENETIC EFFECTS OF TARGETED MODIFICATION.

$460,764FY2019National Institute of Food and AgricultureUSDA

Regents Of The University Of Minnesota

Investigators

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MODIFYING THE PHYSICAL CHARACTERISTICS OR TRAITS OF PLANTS, BOTH FOR BASIC RESEARCH AND FOR CROP IMPROVEMENT, HAS LONG INVOLVED THE INTRODUCTION OF NEW GENETIC VARIATION. ONCE INTRODUCED, THE NEW VARIATION IS SELECTED UPON TO IDENTIFY INDIVIDUALS THAT CARRY THE DESIRED TRAIT WHILE MINIMIZING OTHER PHYSICAL CHANGES. HISTORICALLY NEW VARIATION HAS BEEN INTRODUCED THROUGH CROSSING SCHEMES INVOLVING PARENTS WITH DESIRED TRAITS OR THROUGH THE INTENTIONAL INDUCTION OF NEW MUTATIONS. BOTH OF THESE APPROACHES CREATE GENETIC CHANGES THAT EXTEND BEYOND ANY SINGLE LOCATION IN THE GENOME TARGETED FOR TRAIT MODIFICATION. EMERGING APPROACHES FOR GENETIC MODIFICATION ARE DESIGNED TO CREATE DESIRABLE CHANGES IN PLANT TRAITS WHILE MINIMIZING CHANGES ELSEWHERE IN THE GENOME. THE MOST POPULAR APPROACH, KNOWN AS CRISPR/CAS9 MODIFICATION, HAS THE POTENTIAL TO ALTER SINGLE DNA POSITIONS WITHOUT MAKING CHANGES ELSEWHERE IN THE GENOME AND WITHOUT LEAVING BEHIND THE MOLECULAR MACHINERY NECESSARY FOR MAKING THE CHANGE. HOWEVER, THERE IS AN ONGOING DEBATE IN THE SCIENTIFIC COMMUNITY ABOUT THE POTENTIAL FOR CRISPR/CAS9 MODIFICATIONS TO CLEAVE OR ALTER DNA AT UNINTENDED LOCATIONS, A PROCESSED REFERRED TO AS "OFF-TARGET" MUTAGENESIS. FURTHERMORE, THERE IS DEBATE REGARDING THE POSSIBILITY THAT THE OFF-TARGET MUTATIONS, WHEN UNDETECTED, MAY BE HARMFUL TO PLANT OR EVEN HUMAN HEALTH.TO DETERMINE THE PREVALENCE AND EFFECTS OF OFF-TARGET CHANGES DUE TO CRISPR/CAS9 MODIFICATION IT IS NECESSARY TO DISTINGUISH CRISPR/CAS9-INDUCED CHANGES FROM EXISTING (STANDING) GENETIC VARIATION AND DNA CHANGES ARISING FROM PLANT REGENERATION AND TISSUE CULTURE. WE WILL TEST FOR OFF-TARGET CHANGES OVER FOUR GENERATIONS IN 15 FAMILIES OF PLANTS THAT DIFFER IN THE PRESENCE OR ABSENCE OF THE CRISPR/CAS9 MACHINERY (A TRANSGENE). WE WILL USE TWO SOYBEAN CULTIVARS THAT ARE SUBJECT TO DIFFERENT TISSUE CULTURE TECHNIQUES. WE WILL USE A SIMPLE ASSAY TO SCREEN FOR THE PRESENCE OF THE CRISPR/CAS9 TRANSGENE. BECAUSE ALL PLANTS WILL BE SUBJECT TO THE SAME REGENERATION TECHNIQUES AND HAVE THE SAME GENETIC BACKGROUND, THIS EXPERIMENT WILL ISOLATE THE EFFECTS OF CRISPR/CAS9 IN INDUCING GENETIC CHANGES. THE ISOLATION OF CRISPR/CAS9 OFF-TARGET CHANGES RELATIVE TO OTHER SOURCES SUCH AS TISSUE CULTURE OR GENETIC HETEROGENEITY SHOULD HELP PLANT BREEDERS, GENETIC RESEARCHERS, AND REGULATORY AGENCIES IN MAKING SCIENCE-BASED DECISIONS REGARDING APPLICATIONS AND POLICY APPLIED TO THE GENETIC EFFECTS OF TARGETED MODIFICATION. · GrantIndex