A CRISPR-Library Approach for Targeted Knockout of the LRR-RLK XII Gene Family in Tomato

Large collections of knockout mutant lines are broadly useful for functional genomics studies. However, in tomato, there is no such large-scale collection. The CRISPR system is a powerful and efficient tool for inducing targeted DNA deletions in plant genomes. The ease with which new targeting vectors can be generated, and the overall efficiency of mutagenesis, enables high-throughput plant transformation schemes. Here, we report the development of a transformation method using a library of CRISPR vectors to efficiently generate a collection of knockout mutants. For proof-of-concept, a CRISPR library was constructed to target the 55-member LRR-RLK XII gene family in tomato. Three gRNAs were designed for each gene, for a total of 165 individual targeting vectors. The targets were synthesized and pooled into a single cloning reaction to generate the library. The library was transformed en masse into tomato cotyledons. The incorporated targets were identified by Sanger sequencing. Typically, each transgenic event contains a single, unique targeting vector. Genome modifications were confirmed in approximately 66% of the T₀ events. From a single transformation experiment, ~15 genes from the LRR-RLK XII gene family were modified. This efficient targeted-mutagenesis approach will enable the generation of large collections of mutant tomato lines.