title: An Effective Strategy for Reliably Isolating Heritable and Cas9-Free Arabidopsis Mutants Generated by CRISPR/Cas9-Mediated Genome Editing.
abstract: Mutations generated by CRISPR/Cas9 in Arabidopsis (Arabidopsis thaliana) are often somatic and are rarely heritable. Isolation of mutations in Cas9-free Arabidopsis plants can ensure the stable transmission of the identified mutations to next generations, but the process is laborious and inefficient. Here, we present a simple visual screen for Cas9-free T2 seeds, allowing us to quickly obtain Cas9-free Arabidopsis mutants in the T2 generation. To demonstrate this in principle, we targeted two sites in the AUXIN-BINDING PROTEIN1 (ABP1) gene, whose function as a membrane-associated auxin receptor has been challenged recently. We obtained many T1 plants with detectable mutations near the target sites, but only a small fraction of T1 plants yielded Cas9-free abp1 mutations in the T2 generation. Moreover, the mutations did not segregate in Mendelian fashion in the T2 generation. However, mutations identified in the Cas9-free T2 plants were stably transmitted to the T3 generation following Mendelian genetics. To further simplify the screening procedure, we simultaneously targeted two sites in ABP1 to generate large deletions, which can be easily identified by PCR. We successfully generated two abp1 alleles that contained 1,141- and 711-bp deletions in the ABP1 gene. All of the Cas9-free abp1 alleles we generated were stable and heritable. The method described here allows for effectively isolating Cas9-free heritable CRISPR mutants in Arabidopsis.
authors.affiliation: Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California 92093-0116 (X.G., J.C., X.D., D.Z., Y.Z.);Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (X.G.);College of Life Sciences, Xiamen University, Xiamen 361005, China (J.C.); andCollege of Life Sciences, Northeast Agricultural University, Harbin 150030, China (D.Z.).
authors.forename: Xiuhua
authors.initials: X
authors.lastname: Gao
authors.affiliation: Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California 92093-0116 (X.G., J.C., X.D., D.Z., Y.Z.);Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (X.G.);College of Life Sciences, Xiamen University, Xiamen 361005, China (J.C.); andCollege of Life Sciences, Northeast Agricultural University, Harbin 150030, China (D.Z.).
authors.forename: Jilin
authors.initials: J
authors.lastname: Chen
authors.affiliation: Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California 92093-0116 (X.G., J.C., X.D., D.Z., Y.Z.);Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (X.G.);College of Life Sciences, Xiamen University, Xiamen 361005, China (J.C.); andCollege of Life Sciences, Northeast Agricultural University, Harbin 150030, China (D.Z.).
authors.forename: Xinhua
authors.initials: X
authors.lastname: Dai
authors.affiliation: Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California 92093-0116 (X.G., J.C., X.D., D.Z., Y.Z.);Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (X.G.);College of Life Sciences, Xiamen University, Xiamen 361005, China (J.C.); andCollege of Life Sciences, Northeast Agricultural University, Harbin 150030, China (D.Z.).
authors.forename: Da
authors.initials: D
authors.lastname: Zhang
authors.affiliation: Section of Cell and Developmental Biology, University of California San Diego, La Jolla, California 92093-0116 (X.G., J.C., X.D., D.Z., Y.Z.);Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China (X.G.);College of Life Sciences, Xiamen University, Xiamen 361005, China (J.C.); andCollege of Life Sciences, Northeast Agricultural University, Harbin 150030, China (D.Z.) yundezhao@ucsd.edu.
authors.forename: Yunde
authors.identifier: 0000-0002-7224-8449
authors.initials: Y
authors.lastname: Zhao
mesh_terms: D017360:Arabidopsis / Q000235:genetics*; D064113:CRISPR-Cas Systems; D000072669:Gene Editing; D008164:Luminescent Proteins / Q000235:genetics; D009154:Mutation; D010940:Plant Proteins / Q000235:genetics; D030821:Plants, Genetically Modified; D000094704:RNA, Guide, CRISPR-Cas Systems; D011956:Receptors, Cell Surface / Q000235:genetics; D017384:Sequence Deletion; D000097573:Red Fluorescent Protein
chemical_list: D008164:Luminescent Proteins; D010940:Plant Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D011956:Receptors, Cell Surface; D000097573:Red Fluorescent Protein; C083846:auxin-binding protein 1