Efficient targeted DNA editing and replacement in Chlamydomonas reinhardtii using Cpf1 ribonucleoproteins and single-stranded DNA

Aron Ferenczi, Douglas Euan Pyott, Andromachi Xipnitou, Attila Molnar

Research output: Contribution to journalArticlepeer-review

Abstract

The green alga Chlamydomonas reinhardtii is an invaluable reference organism to research fields including algal, plant, and ciliary biology. Accordingly, decades-long standing inefficiencies in targeted nuclear gene editing broadly hinder Chlamydomonas research. Here we report that single-step codelivery of CRISPR/Cpf1 ribonucleoproteins with single-stranded DNA repair templates results in precise and targeted DNA replacement with as much as ∼10% efficiency in C. reinhardtii. We demonstrate its use in transgene- and selection-free generation of sequence-specific mutations and epitope tagging at an endogenous locus. As the direct delivery of gene-editing reagents bypasses the use of transgenes, this method is potentially applicable to a wider range of species without the need to develop methods for stable transformation.

Original languageEnglish
Pages (from-to)13567-13572
Number of pages6
JournalProceedings of the National Academy of Sciences
Volume114
Issue number51
Early online date5 Dec 2017
DOIs
Publication statusPublished - 19 Dec 2017

Keywords

  • Chlamydomonas reinhardtii
  • CRISPR/Cpf1
  • RNP
  • editing
  • ssODN

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