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Genetic dissection of mutagenic repair and T-DNA capture at CRISPR-induced DNA breaks in Arabidopsis thaliana.
Kamoen, Lycka; Kralemann, Lejon E M; van Schendel, Robin; van Tol, Niels; Hooykaas, Paul J J; de Pater, Sylvia; Tijsterman, Marcel.
Afiliación
  • Kamoen L; Department of Plant Sciences, Institute of Biology Leiden, Leiden University, Leiden 2333 BE, The Netherlands.
  • Kralemann LEM; Department of Plant Sciences, Institute of Biology Leiden, Leiden University, Leiden 2333 BE, The Netherlands.
  • van Schendel R; Department of Human Genetics, Leiden University Medical Center, Leiden 2300 RC, The Netherlands.
  • van Tol N; Department of Human Genetics, Leiden University Medical Center, Leiden 2300 RC, The Netherlands.
  • Hooykaas PJJ; Department of Plant Sciences, Institute of Biology Leiden, Leiden University, Leiden 2333 BE, The Netherlands.
  • de Pater S; Department of Human Genetics, Leiden University Medical Center, Leiden 2300 RC, The Netherlands.
  • Tijsterman M; Department of Plant Sciences, Institute of Biology Leiden, Leiden University, Leiden 2333 BE, The Netherlands.
PNAS Nexus ; 3(3): pgae094, 2024 Mar.
Article en En | MEDLINE | ID: mdl-38463035
ABSTRACT
A practical and powerful approach for genome editing in plants is delivery of CRISPR reagents via Agrobacterium tumefaciens transformation. The double-strand break (DSB)-inducing enzyme is expressed from a transferred segment of bacterial DNA, the T-DNA, which upon transformation integrates at random locations into the host genome or is captured at the self-inflicted DSB site. To develop efficient strategies for precise genome editing, it is thus important to define the mechanisms that repair CRISPR-induced DSBs, as well as those that govern random and targeted integration of T-DNA. In this study, we present a detailed and comprehensive genetic analysis of Cas9-induced DSB repair and T-DNA capture in the model plant Arabidopsis thaliana. We found that classical nonhomologous end joining (cNHEJ) and polymerase theta-mediated end joining (TMEJ) are both, and in part redundantly, acting on CRISPR-induced DSBs to produce very different mutational outcomes. We used newly developed CISGUIDE technology to establish that 8% of mutant alleles have captured T-DNA at the induced break site. In addition, we find T-DNA shards within genomic DSB repair sites indicative of frequent temporary interactions during TMEJ. Analysis of thousands of plant genome-T-DNA junctions, followed up by genetic dissection, further reveals that TMEJ is responsible for attaching the 3' end of T-DNA to a CRISPR-induced DSB, while the 5' end can be attached via TMEJ as well as cNHEJ. By identifying the mechanisms that act to connect recombinogenic ends of DNA molecules at chromosomal breaks, and quantifying their contributions, our study supports the development of tailor-made strategies toward predictable engineering of crop plants.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: PNAS Nexus Año: 2024 Tipo del documento: Article País de afiliación: Países Bajos Pais de publicación: Reino Unido
Texto completo
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Imprimir
XML
PubMed Links
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: PNAS Nexus Año: 2024 Tipo del documento: Article País de afiliación: Países Bajos Pais de publicación: Reino Unido