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A cyclical marker system enables indefinite series of oligonucleotide-directed gene editing in Chlamydomonas reinhardtii.
Ross, Ian L; Le, Hong Phuong; Budiman, Sabar; Xiong, Dake; Hemker, Fritz; Millen, Elizabeth A; Oey, Melanie; Hankamer, Ben.
Afiliación
  • Ross IL; Institute for Molecular Bioscience (IMB), The University of Queensland, Brisbane, Queensland 4072, Australia.
  • Le HP; Institute for Molecular Bioscience (IMB), The University of Queensland, Brisbane, Queensland 4072, Australia.
  • Budiman S; Institute for Molecular Bioscience (IMB), The University of Queensland, Brisbane, Queensland 4072, Australia.
  • Xiong D; Institute for Molecular Bioscience (IMB), The University of Queensland, Brisbane, Queensland 4072, Australia.
  • Hemker F; Institute for Molecular Bioscience (IMB), The University of Queensland, Brisbane, Queensland 4072, Australia.
  • Millen EA; Institute for Molecular Bioscience (IMB), The University of Queensland, Brisbane, Queensland 4072, Australia.
  • Oey M; Institute for Molecular Bioscience (IMB), The University of Queensland, Brisbane, Queensland 4072, Australia.
  • Hankamer B; Institute for Molecular Bioscience (IMB), The University of Queensland, Brisbane, Queensland 4072, Australia.
Plant Physiol ; 2024 Aug 23.
Article en En | MEDLINE | ID: mdl-39179421
ABSTRACT
CRISPR/Cas9 gene editing in the model green alga Chlamydomonas reinhardtii relies on the use of selective marker genes to enrich for non-selectable target mutations. This becomes challenging when many sequential modifications are required in a single cell line, as useful markers are limited. Here, we demonstrate a cyclical selection process which only requires a single marker gene to identify an almost infinite sequential series of CRISPR-based target gene modifications. We used the NIA1 (Nit1, NR; nitrate reductase) gene as the selectable marker in this study. In the forward stage of the cycle, a stop codon was engineered into the NIA1 gene at the CRISPR target location. Cells retaining the wild-type NIA1 gene were killed by chlorate, while NIA1 knockout mutants survived. In the reverse phase of the cycle, the stop codon engineered into the NIA1 gene during the forward phase was edited back to the wild-type sequence. Using nitrate as the sole nitrogen source, only the reverted wild-type cells survived. By using CRISPR to specifically deactivate and reactivate the NIA1 gene, a marker system was established that flipped back and forth between chlorate- and auxotrophic (nitrate)-based selection. This provided a scarless cyclical marker system that enabled an indefinite series of CRISPR edits in other, non-selectable genes. We demonstrate that this 'Sequential CRISPR via Recycling Endogenous Auxotrophic Markers (SCREAM)' technology enables an essentially limitless series of genetic modifications to be introduced into a single cell lineage of C. reinhardtii in a fast and efficient manner to complete complex genetic engineering.
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Plant Physiol Año: 2024 Tipo del documento: Article País de afiliación: Australia Pais de publicación: Estados Unidos
Texto completo
Añadir a Mi BVS
Imprimir
XML
PubMed Links
Buscar en Google

Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Idioma: En Revista: Plant Physiol Año: 2024 Tipo del documento: Article País de afiliación: Australia Pais de publicación: Estados Unidos