基于荧光标记双向筛选的拟南芥CRISPR基因编辑突变体库构建

Construction of CRISPR-edited Mutant Library in Arabidopsis with Fluorescence-based Bidirectional Selection

突变体库是研究基因功能的重要工具。拟南芥种质资源库(ABRC)储藏了几乎涵盖所有基因的突变体材料,其中T-DNA插入突变体占绝大多数。然而,当在T-DNA插入突变体中进行遗传转化或与其他转基因报告系统进行杂交时容易引起转基因沉默,阻碍了相关研究的开展,甚至产生错误结论。甲基磺酸乙酯(Ethyl Methane Sulfonate,EMS)诱变和快中子诱变技术可获得非转基因突变体,但这两种方法均为随机诱变技术,需要通过基因定位来确认突变位点,无法实现基因靶向敲除。CRISPR/Cas9可以对目的基因进行靶向编辑,获得不携带转基因的突变体。拟南芥中尚无利用CRISPR/Cas9进行高通量突变体库构建的报道。本研究共设计900条sgRNAs靶向300个RNA通路相关基因,通过合成sgRNA混池文库、引入DsRed2荧光筛选标记、将DNA条形码和二代测序技术相结合,实现了对转基因阳性苗的高通量鉴定和分型,并对T2代具有发育缺陷的无荧光植株进行负向筛选,成功鉴定到了不含转基因的smd3-b和rlua4突变体。

Mutant libraries are essential for functional genomics.The Arabidopsis Biological Resource Center(ABRC)has a collection of mutant lines covering almost all genes,with most of them being T-DNA insertional lines.However,introducing additional transgenes into T-DNA mutants through genetic transformation or cross often cause unwanted transgene silencing,limiting its application in such experiments and sometimes even leading to wrong conclusions.Non-transgenic mutants can be obtained by Ethyl Methane Sulfonate(EMS)or fast neutron mutagenesis,but both methods are random mutagenetic techniques,which require further genetic mapping to characterize the causal mutation.CRISPR/Cas can generate transgene-free plants with specific and heritable mutations.There is no report on the high-throughput construction of mutant libraries using CRISPR/cas9 in Arabidopsis.In this study,we designed 900 sgRNAs targeting 300 RNA pathway-related genes.We achieved high-throughput characterization of sgRNA-indexed transgenic individuals by synthesizing sgRNA arrays,bulk sub-cloning and transformation,fluorescence-based screening,and multiplexed barcoded next-generation sequencing of sgRNAs.By counterselection of non-fluorescent plants with developmental defects,we identified transgene-free smd3-b and rlua4 mutants in the T2 generation successfully.