Homology-directed transgene-free gene editing in Chlamydomonas reinhardtii

Aron Ferenczi, Attila Molnar

Research output: Chapter in Book/Report/Conference proceedingChapter (peer-reviewed)peer-review

Abstract

Chlamydomonas reinhardtii is a microalgal model organism with a suite of molecular and genetic techniques, but routine editing of its nuclear genome is yet to be realised. DNA-based transformation techniques are prohibitively inefficient and lead to predominantly non-homologous (i.e. off-target) integration. Standard CRISPR-based gene editing protocols have proved too ineffective to enable routine application. We have found that the use of CRISPR/Cpf1 in conjunction with single-stranded DNA (ssODN) repair templates achieves nuclear gene editing efficiencies as high as 30% [1]. This produces edits with predictable outcomes in a transgene- and selection marker-free manner. The possibility to purchase all necessary reagents commercially with no preparation time (besides design) facilitates rapid and routine genetic engineering in this organism. Here we describe the use of this technique to knockout locus FKB12, which leads to rapamycin resistance and lends itself to an easy assay when adopting this gene-editing protocol.
Original languageEnglish
Title of host publicationCRISPR-Cas Methods
PublisherSpringer International Publishing
Chapter14
Pages237-252
ISBN (Electronic)978-1-0716-0616-2
ISBN (Print)978-1-0716-0615-5
DOIs
Publication statusE-pub ahead of print - 1 Jul 2020

Publication series

NameSpringer Protocols Handbooks

Keywords

  • CRISPR
  • Cpf1
  • Chlamydomonas reinhardtii
  • ssODN
  • homology-directed repair (HDR)

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