DataSheet1_CRISPR/Cas9 mutagenesis of the Arabidopsis GROWTH-REGULATING FACTOR (GRF) gene family.PDF

Genome editing in plants typically relies on T-DNA plasmids that are mobilized by Agrobacterium-mediated transformation to deliver the CRISPR/Cas machinery. Here, we introduce a series of CRISPR/Cas9 T-DNA vectors for minimal settings, such as teaching labs. Gene-specific targeting sequences can be inserted as annealed short oligonucleotides in a single straightforward cloning step. Fluorescent markers expressed in mature seeds enable reliable selection of transgenic or transgene-free individuals using a combination of inexpensive LED lamps and colored-glass alternative filters. Testing these tools on the Arabidopsis GROWTH-REGULATING FACTOR (GRF) genes, we were able to create a collection of predicted null mutations in all nine family members with little effort. We then explored the effects of simultaneously targeting two, four and eight GRF genes on the rate of induced mutations at each target locus. In our hands, multiplexing was associated with pronounced disparities: while mutation rates at some loci remained consistently high, mutation rates at other loci dropped dramatically with increasing number of single guide RNA species, thereby preventing a systematic mutagenesis of the family.

植物基因组编辑通常依赖于通过农杆菌介导转化进行转移的T-DNA质粒（T-DNA plasmid），以递送CRISPR/Cas系统（CRISPR/Cas machinery）。本研究构建了一系列适用于极简场景（如教学实验室）的CRISPR/Cas9 T-DNA载体。基因特异性靶向序列可通过退火短寡核苷酸（short oligonucleotides），经单次简便克隆步骤插入载体。成熟种子中表达的荧光标记，可结合廉价LED灯与有色玻璃滤光片的组合，实现对转基因或无转基因个体的高效筛选。我们以拟南芥（Arabidopsis）的生长调节因子（GROWTH-REGULATING FACTOR, GRF）基因家族为测试对象，仅通过少量工作即可获得该家族全部9个成员的预测性无效突变体集合。随后我们探究了同时靶向2个、4个及8个GRF基因时，各靶位点的诱导突变率变化。实验发现，多重靶向存在显著差异：部分位点的突变率始终维持在较高水平，而另一些位点的突变率则随单引导RNA（single guide RNA）数量增加出现大幅下降，从而无法实现该基因家族的系统性诱变。