DNA-free CRISPR-Cas9 gene editing of wild tetraploid tomato Solanum peruvianum using protoplast regeneration

第一作者：Choun-Sea Lin

第一单位：台湾"中央研究院"农业生物科技研究中心

第一通讯：Yao-Cheng Lin

 Abstarct 

背景回顾：Wild tomatoes (Solanum peruvianum) are important genomic resources for tomato research and breeding. Development of a foreign DNA-free CRISPR-Cas delivery system has potential to mitigate public concern about genetically modified organisms. 

野生番茄（Solanum peruvianum）是番茄研究和育种中的重要基因组资源。开发一套外源DNA-free的CRISPR-Cas递送系统有助于缓解公众对于转基因生物的担忧。

主要研究：Here, we established a DNA-free CRISPR-Cas9 genome editing system based on an optimized protoplast regeneration protocol of S. peruvianum, an important resource for tomato introgression breeding. 

本文中，作者基于野生番茄最优原生质体再生流程，建立了一套DNA-free的CRISPR-Cas9基因组编辑系统。

结果1-突变体：We generated mutants for genes involved in small interfering RNAs (siRNA) biogenesis, RNA-DEPENDENT RNA POLYMERASE 6 (SpRDR6) and SUPPRESSOR OF GENE SILENCING 3 (SpSGS3); pathogen-related peptide precursors, PATHOGENESIS-RELATED PROTEIN-1 (SpPR-1) and PROSYSTEMIN (SpProSys); and fungal resistance (MILDEW RESISTANT LOCUS O, SpMlo1) using diploid or tetraploid protoplasts derived from in vitro-grown shoots.

作者利用离体培养的Shoots制备了二倍体或四倍体原生质体，从而构建了两个参与siRNA生物发生基因的突变体，分别是SpRDR6和SpSGS3基因；两个病原相关多肽前体的突变体，分别是SpPR-1和SpProSys基因；以及真菌抗性相关基因SpMlo1。

结果2-倍性影响：The ploidy level of these regenerants was not affected by PEG-Ca²⁺-mediated transfection, CRISPR reagents, or the target genes. 

这些再生植株的倍性水平并不受到PEG-Ca2+介导的转染、CRISPR试剂以及靶基因的影响。

结果3-脱靶检测：By karyotyping and whole genome sequencing analysis, we confirmed that CRISPR-Cas9 editing did not introduce chromosomal changes or unintended genome editing sites. 

通过核型分析和全基因组测序分析，作者确定了CRISPR-Cas9编辑并不会引入染色体的改变或者非靶点的基因组编辑位点。

结果4-稳定遗传：All mutated genes in both diploid and tetraploid regenerants were heritable in the next generation. 

所有在二倍体和四倍体再生植株中突变的基因都遗传到了下一代植株中。

结果5-突变体表型及拯救：spsgs3 null T₀ regenerants and sprdr6 null T₁ progeny had wiry, sterile phenotypes in both diploid and tetraploid lines. The sterility of the spsgs3 null mutant was partially rescued, and fruits were obtained by grafting to wild-type stock and pollination with wild-type pollen. The resulting seeds contained the mutated alleles. 

spsgs3无义突变T0代再生植株和sprdr6无义突变T1子代在二倍体和四倍体株系中，都具有结实和不育的表型。通过将突变体嫁接到野生型砧木上，能够部分拯救spsgs3无义突变的不育表型，利用野生型花粉授粉，能够成功获得果实。获得的种子包含被突变的等位基因。

结果6-突变体抗病性：Tomato yellow leaf curl virus proliferated at higher levels in spsgs3 and sprdr6 mutants than in the wild type.

番茄黄化曲叶病毒在spsgs3和sprdr6突变体中的增殖水平要高于野生型。

结论：Therefore, this protoplast regeneration technique should greatly facilitate tomato polyploidization and enable the use of CRISPR-Cas for S. peruvianum domestication and tomato breeding.

因此，该原生质体再生技术将极大地促进番茄多倍化，在此基础上开发的CRISPR-Cas技术有助于野生番茄的驯化和番茄育种。

doi: https://doi.org/10.1093/plphys/kiac022

Journal: Plant Physiology

Published date: January 28, 2022