TY - JOUR
T1 - Crispr/cas9 induced somatic recombination at the crtiso locus in tomato
AU - Shlush, Ilan Ben
AU - Samach, Aviva
AU - Melamed-Bessudo, Cathy
AU - Ben-Tov, Daniela
AU - Dahan-Meir, Tal
AU - Filler-Hayut, Shdema
AU - Levy, Avraham A.
N1 - The authors would like to thank Tal Dahan-Meir for the CRTISO 18-3 Micro-Tom mutant; Joseph Hirschberg and Dani Zamir for the CRTISO M82 e3406 mutant plant materials; Ziva Amsellem for guidance in tissue culture; and the Weizmann Institute greenhouse staff for the plants’ maintenance. The authors would like to thank all the Levy lab members for fruitful discussions and assistance during the course of this study. This work was funded by Israel Science Foundation and Minerva grants to A.A.L. Funding - This research and the APC were funded by Israel Science Foundation, grant number 1027/14, and by Minerva Foundation, grant number 2016. I.B.S. performed most of the experimental work as part of his Master thesis. A.S. performed part of the experimental work and wrote most of the manuscript. C.M.-B. performed part of the experimental work. D.B.-T., T.D.-M. and S.F.-H. advised the experimental design and analysis. A.A.L. was involved in the planning and supervising of the work and writing of the manuscript. All authors have read and agreed to the published version of the manuscript.
PY - 2021/1
Y1 - 2021/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=85098785570&partnerID=8YFLogxK
U2 - 10.3390/genes12010059
DO - 10.3390/genes12010059
M3 - مقالة
SN - 2073-4425
VL - 12
SP - 1
EP - 12
JO - Genes
JF - Genes
IS - 1
M1 - 59
ER -