CRISPR/Cas9 Induced Somatic Recombination at the CRTISO Locus in Tomato.

Ilan Ben Shlush,Cathy Melamed-Bessudo,Daniela Ben-Tov,Avraham A Levy,Shdema Filler-Hayut,Tal Dahan-Meir,Aviva Samach

doi:10.3390/genes12010059

Ilan Ben Shlush, Cathy Melamed-Bessudo + Show 5 more

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https://doi.org/10.3390/genes12010059

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Journal: Genes	Publication Date: Dec 31, 2020
Citations: 26	License type: CC BY 4.0

Affiliation: Weizmann Institute of Science

Abstract

Homologous recombination (HR) in somatic cells is not as well understood as meiotic recombination and is thought to be rare. In a previous study, we showed that Inter-Homologous Somatic Recombination (IHSR) can be achieved by targeted induction of DNA double-strand breaks (DSBs). Here, we designed a novel IHSR assay to investigate this phenomenon in greater depth. We utilized F1 hybrids from divergent parental lines, each with a different mutation at the Carotenoid isomerase (CRTISO) locus. IHSR events, namely crossover or gene conversion (GC), between the two CRTISO mutant alleles (tangerine color) can restore gene activity and be visualized as gain-of-function, wildtype (red) phenotypes. Our results show that out of four intron DSB targets tested, three showed DSB formation, as seen from non-homologous end-joining (NHEJ) footprints, but only one target generated putative IHSR events as seen by red sectors on tangerine fruits. F2 seeds were grown to test for germinal transmission of HR events. Two out of five F1 plants showing red sectors had their IHSR events germinally transmitted to F2, mainly as gene conversion. Six independent recombinant alleles were characterized: three had truncated conversion tracts with an average length of ~1 kb. Two alleles were formed by a crossover as determined by genotyping and characterized by whole genome sequencing. We discuss how IHSR can be used for future research and for the development of novel gene editing and precise breeding tools.

Highlights

Homologous recombination (HR) is the most common way to generate genetic variation in sexually reproductive organisms
A recessive mutation in the Carotenoid isomerase (CRTISO) gene from the carotenoid biosynthesis pathway in tomatoes gives rise to a tangerine phenotype [11], which causes pale yellow petals, orange fruits, and a less robust yet still visible phenotype with yellowish young leaves (Figure 1)
double-strand breaks (DSBs) induction was targeted to introns so that the recombination product can tolerate non-homologous end-joining (NHEJ) mutations without affecting which bear red fruits (WT) activity (Figure 1)

Summary

Introduction

Homologous recombination (HR) is the most common way to generate genetic variation in sexually reproductive organisms. It is achieved through the exchange of parental chromosomal segments between two homologous chromosomes followed by segregation of the new recombinant alleles into separate germinal cells [1]. This process is initiated by Spo11-mediated DNA double-strand breaks (DSBs) happening during meiosis at various locations along the chromosomes [1,2,3,4,5]. Generating genetic variation of new desirable traits in vegetatively propagated crops cannot rely on meiotic recombination

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