清脆的
Cas9
基因组编辑
胚乳
生物
转化(遗传学)
突变体
基因
转基因
报告基因
遗传学
转基因作物
引导RNA
计算生物学
绿色荧光蛋白
基因组
转基因生物
基因表达
作者
Yuanyuan Yan,Jinjie Zhu,Xiantao Qi,Beijiu Cheng,Changlin Liu,Chuanxiao Xie
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.
科研通智能强力驱动
Strongly Powered by AbleSci AI