Establishment of an efficient seed fluorescence reporter-assisted CRISPR/Cas9 gene editing in maize

文献类型： 外文期刊

第一作者： Yan, Yuanyuan

作者： Yan, Yuanyuan;Zhu, Jinjie;Qi, Xiantao;Liu, Changlin;Xie, Chuanxiao;Yan, Yuanyuan;Cheng, Beijiu

作者机构：

关键词： CRISPR; Cas9; embryo‐ specific reporter; endosperm‐ specific reporter; genome editing; maize; seed fluorescence reporter

期刊名称：JOURNAL OF INTEGRATIVE PLANT BIOLOGY （ 影响因子：4.885； 五年影响因子：4.586 ）

ISSN： 1672-9072

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收录情况： SCI

摘要： Genome editing by clustered regularly interspaced short palindromic sequences (CRISPR)/CRISPR-associated protein 9 (Cas9) has revolutionized functional gene analysis and genetic improvement. While reporter-assisted CRISPR/Cas systems can greatly facilitate the selection of genome-edited plants produced via stable transformation, this approach has not been well established in seed crops. Here, we established the seed fluorescence reporter (SFR)-assisted CRISPR/Cas9 systems in maize (Zea mays L.), using the red fluorescent DsRED protein expressed in the endosperm (En-SFR/Cas9), embryos (Em-SFR/Cas9), or both tissues (Em/En-SFR/Cas9). All three SFRs showed distinct fluorescent patterns in the seed endosperm and embryo that allowed the selection of seeds carrying the transgene of having segregated the transgene out. We describe several case studies of the implementation of En-SFR/Cas9, Em-SFR/Cas9, and Em/En- SFR/Cas9 to identify plants not harboring the genome-editing cassette but carrying the desired mutations at target genes in single genes or in small-scale mutant libraries, and report on the successful generation of single-target mutants and/or mutant libraries with En-SFR/Cas9, Em-SFR/Cas9, and Em/En-SFR/Cas9. SFR-assisted genome editing may have particular value for application scenarios with a low transformation frequency and may be extended to other important monocot seed crops.

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