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

Author:

Akella Soujanya1ORCID,Ma Xinrong1,Bacova Romana2ORCID,Harmer Zachary P1ORCID,Kolackova Martina2ORCID,Wen Xiaoxue1,Wright David A3ORCID,Spalding Martin H3ORCID,Weeks Donald P4,Cerutti Heriberto1ORCID

Affiliation:

1. School of Biological Sciences and Center for Plant Science Innovation, University of Nebraska–Lincoln, Lincoln, Nebraska 68588, USA

2. Department of Chemistry and Biochemistry, Mendel University in Brno, Zemedelska 1, CZ-613 00, Brno, Czech Republic

3. Department of Genetics, Development and Cell Biology, Iowa State University, Ames, Iowa 50011, USA

4. Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska 68588, USA

Abstract

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 double-stranded 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 Chlamydomonas acetolactate 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.

Funder

National Science Foundation

Gordon and Betty Moore Foundation

Publisher

Oxford University Press (OUP)

Subject

Plant Science,Genetics,Physiology

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