Generation of a Collection of Mutant Tomato Lines Using Pooled CRISPR Libraries
Generation of a Collection of Mutant Tomato Lines Using Pooled CRISPR Libraries
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DOI：
10.1104/pp.17.00489
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发表时间：
2017-08-01
期刊：
影响因子：
7.4
通讯作者：
Martin, Gregory B.
中科院分区：
文献类型：
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作者：
Jacobs, Thomas B.;Zhang, Ning;Martin, Gregory B.
成簇的规则间隔短回文重复序列（CRISPR）介导的诱变在植物中的高效率使得能够开发高通量诱变策略。通过将合并的CRISPR文库转化到番茄（Solanum lycopersicum）中，以最少的转化尝试并且在相对短的时间段内产生突变株系的集合。通过对初级转基因事件中掺入的指导RNA进行测序，容易地确定靶基因的鉴定。从使用靶向免疫相关的富含亮氨酸重复序列亚家族XII基因的CRISPR文库的单次转化中，在靶向的54个基因中的15个中回收了可遗传的突变。为了增加通量，制备了第二个CRISPR文库，每个构建体含有三个向导RNA以靶向18个推定的转运蛋白基因。这导致18个靶基因中的15个稳定突变，其中一些初级转基因植物具有多达5个突变基因。此外，该植物集合中的冗余允许异常T0表型与潜在靶基因的关联。拟南芥（拟南芥）硼外排转运蛋白的同源物突变的植物显示缺硼表型。本文所述的策略提供了一种技术上简单但高通量的方法，用于产生具有靶向突变的系的集合，并且应该适用于任何植物转化系统。
The high efficiency of clustered regularly interspaced short palindromic repeats (CRISPR)-mediated mutagenesis in plants enables the development of high-throughput mutagenesis strategies. By transforming pooled CRISPR libraries into tomato (Solanum lycopersicum), collections of mutant lines were generated with minimal transformation attempts and in a relatively short period of time. Identification of the targeted gene(s) was easily determined by sequencing the incorporated guide RNA(s) in the primary transgenic events. From a single transformation with a CRISPR library targeting the immunity-associated leucine-rich repeat subfamily XII genes, heritable mutations were recovered in 15 of the 54 genes targeted. To increase throughput, a second CRISPR library was made containing three guide RNAs per construct to target 18 putative transporter genes. This resulted in stable mutations in 15 of the 18 targeted genes, with some primary transgenic plants having as many as five mutated genes. Furthermore, the redundancy in this collection of plants allowed for the association of aberrant T0 phenotypes with the underlying targeted genes. Plants with mutations in a homolog of an Arabidopsis (Arabidopsis thaliana) boron efflux transporter displayed boron deficiency phenotypes. The strategy described here provides a technically simple yet high-throughput approach for generating a collection of lines with targeted mutations and should be applicable to any plant transformation system.