Co-targeting strategy for precise, scarless gene editing with CRISPR/Cas9 and donor ssODNs in Chlamydomonas

Authors

Soujanya Akella, University of Nebraska–Lincoln
Xinrong Ma, University of Nebraska–Lincoln
Romana Bacova, Mendel University in Brno
Zachary P. Harmer, University of Nebraska–Lincoln
Martina Kolackova, Mendel University in Brno
Xiaoxue Wen, University of Nebraska–Lincoln
David A. Wright, Iowa State University
Martin H. Spalding, Iowa State University
Donald P. Weeks, University of Nebraska-Lincoln
Heriberto Cerutti, University of Nebraska–LincolnFollow

Date of this Version

2021

Citation

doi:10.1093/plphys/kiab418 PLANT PHYSIOLOGY 2021: 187: 2637–2655

Comments

VC The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial reuse, please contact journals.permissions@oup.com

Abstract

Programmable site-specific nucleases, such as the clustered regularly interspaced short palindromic repeat (CRISPR)/ CRISPR-associated protein 9 (Cas9) ribonucleoproteins (RNPs), have allowed creation of valuable knockout mutations and targeted gene modifications in Chlamydomonas (Chlamydomonas reinhardtii).

However, in walled strains, present methods for editing genes lacking a selectable phenotype involve co-transfection of RNPs and exogenous doublestranded DNA (dsDNA) encoding a selectable marker gene. Repair of the dsDNA breaks induced by the RNPs is usually accompanied by genomic insertion of exogenous dsDNA fragments, hindering the recovery of precise, scarless mutations in target genes of interest. Here, we tested whether co-targeting two genes by electroporation of pairs of CRISPR/Cas9 RNPs and single-stranded oligodeoxynucleotides (ssODNs) would facilitate the recovery of precise edits in a gene of interest (lacking a selectable phenotype) by selection for precise editing of another gene (creating a selectable marker)— in a process completely lacking exogenous dsDNA. We used PPX1 (encoding protoporphyrinogen IX oxidase) as the generated selectable marker, conferring resistance to oxyfluorfen, and identified precise edits in the homolog of bacterial ftsY or the WD and TetratriCopeptide repeats protein 1 genes in ~1% of the oxyfluorfen resistant colonies. Analysis of the target site sequences in edited mutants suggested that ssODNs were used as templates for DNA synthesis during homology directed repair, a process prone to replicative errors. The Chlamydomonasacetolactate synthase gene could also be efficiently edited to serve as an alternative selectable marker. This transgene-free strategy may allow creation of individual strains containing precise mutations in multiple target genes, to study complex cellular processes, pathways, or structures.