In Chlamydomonas reinhardtii, the model organism for eukaryotic green algae and plants, the processes of nuclear transformation and genome editing in particular are still marked by a low level of efficiency, and so intensive work is required in order to create and identify mutants for the investigation of basic physiological processes, as well as the implementation of biotechnological applications. In this work, we show that cell synchronization during the stages of the cell cycle, obtained from long-term cultivation under specific growth conditions, greatly enhances the efficiency of transformation and allows the identification of DNA repair mechanisms that occur preferentially at different stages of the cell cycle. We demonstrate that the transformation of synchronized cells at different times was differentially associated with nonhomologous end joining (NHEJ) and/or homologous recombination (FIR), and makes it possible to knock-in specific foreign DNA at the genomic nuclear location desired by exploiting HR This optimization greatly reduces the overall complexity of the genome editing procedure and creates new opportunities for altering genes and their products.

Cell Synchronization Enhances Nuclear Transformation and Genome Editing via Cas9 Enabling Homologous Recombination in Chlamydomonas reinhardtii

Max Angstenberger
Investigation
;
Francesco de Signori
Investigation
;
Valeria Vecchi
Resources
;
Luca Dall'Osto
Writing – Review & Editing
;
Roberto Bassi
Conceptualization
2020-01-01

Abstract

In Chlamydomonas reinhardtii, the model organism for eukaryotic green algae and plants, the processes of nuclear transformation and genome editing in particular are still marked by a low level of efficiency, and so intensive work is required in order to create and identify mutants for the investigation of basic physiological processes, as well as the implementation of biotechnological applications. In this work, we show that cell synchronization during the stages of the cell cycle, obtained from long-term cultivation under specific growth conditions, greatly enhances the efficiency of transformation and allows the identification of DNA repair mechanisms that occur preferentially at different stages of the cell cycle. We demonstrate that the transformation of synchronized cells at different times was differentially associated with nonhomologous end joining (NHEJ) and/or homologous recombination (FIR), and makes it possible to knock-in specific foreign DNA at the genomic nuclear location desired by exploiting HR This optimization greatly reduces the overall complexity of the genome editing procedure and creates new opportunities for altering genes and their products.
Chlamydomonas reinhardtii
genome editing
CRESPR/Cas9
nonhomologous end joining
homologous recombination
cpftsy
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11562/1032781
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