Generation of a Collection of Mutant Tomato Lines Using Pooled CRISPR Libraries

Abstract

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 (), 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 () 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.

Revolutionize Genetic Studies and Crop Improvement with High-Throughput and Genome-Scale CRISPR/Cas9 Gene Editing Technology

“…More importantly, Jacobs et al (2017) were able to establish an association between the developmental phenotypes observed in T 0 plants and the mutations in the targeted genes. They demonstrated that mutations in a homolog of an Arabidopsis boron efflux transporter caused boron-deficiency phenotypes in tomato, defining a gene function in a single generation (Jacobs et al, 2017). Such a task would not be possible to complete in a generation using traditional mutagenesis approaches.…”

“…In a pilot study in tomato, Jacobs et al (2017) developed a CRISPR/Cas9 plasmid library that targets 54 immunity-associated leucine-rich repeat subfamily XII genes. From a single transformation with this CRISPR/Cas9 library, they were able to identify heritable mutations in 15 out of 54 genes.…”

“…From a single transformation with this CRISPR/Cas9 library, they were able to identify heritable mutations in 15 out of 54 genes. Jacobs et al (2017) generated a second CRISPR/Cas9 library, which is composed of plasmids that express three gRNAs in each plasmid. Some of the T 0 plants had mutations in multiple targeted genes.…”

“…Although still limited in scale, the studies in rice and tomato discussed here demonstrated that the CRISPR/Cas9-mediated targeted mutagenesis is reliable, efficient, and high-throughput for generating mutant collections that cover most genes in a genome (Jacobs et al, 2017; Lu et al, 2017; Meng et al, 2017). The CRISPR technology has revolutionized genetic studies in crops because of its unprecedented scale, versatility, and speed.…”

“…CRISPR technology is not only ideal for producing knockout mutants, it can also be adapted for generating gain-of-function mutant libraries if the nuclease-dead Cas9 (dCas9) fused with a transcriptional activation domain is used (Tang et al, 2017). Moreover, the technology can be used to generate multiple mutants with a single plasmid that produces several gRNAs as shown in the tomato study (Jacobs et al, 2017). Undoubtedly, more features such as fluorescence markers for transgene identification (Gao et al, 2016), different types of nucleases including Cpf1 (Tang et al, 2017), and diversified gRNA production methods (Gao and Zhao, 2014) will further improve CRISPR-mediated mutagenesis and broaden the applications of CRISPR gene editing technologies in genetic studies and crop improvement.…”