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

Abstract

Programmable site-specific nucleases, such as the CRISPR/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 double-stranded DNA breaks induced by the ribonucleoproteins 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 (WDTC1) 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.

A novel class of long small RNAs associates with Argonaute1 and is up-regulated by nutrient deprivation in the alga Chlamydomonas

“…To corroborate that the C. reinhardtii long sRNAs associate with native AGO1, we used CRISPR/Cas9 genome editing (Akella et al, 2021) to generate knockout mutants of the AGO1 gene. Cells of the wild type CC-124 strain were electroporated with both a CRISPR/Cas9 ribonucleoprotein (RNP) targeting the AGO1 exon1 and a single-stranded oligodeoxynucleotide (ssODN), with a modified DNA sequence, overlapping the Cas9 cleavage site.…”

“…Following a previously described protocol (Akella et al, 2021), cells of the wild type CC-124 strain were electroporated with an in vitro assembled CRISPR/Cas9 ribonucleoprotein, targeting the AGO1 exon1, and a modified single-stranded oligodeoxynucleotide, to serve as template for homology directed DNA repair. After a 48-h recovery period, electroporated cells were spread on TAP agar plates containing oxyfluorfen (Akella et al, 2021). Surviving colonies were screened by PCR amplification (Cao et al, 2009) of the AGO1 target site, using a primer (AGO1-mut-F1) that anneals exclusively to the edited sequence.…”

Molecular Advancements Establishing Chlamydomonas as a Host for Biotechnological Exploitation

“…Editing frequency was also affected by other factors ( Figure 2 ). A heat shock pretreatment of the cells before or after the electroporation was shown to increase the rate of mutations ( Greiner et al, 2017 ; Dhokane et al, 2020 ; Akella et al, 2021 ), as well as cell synchronization ( Angstenberger et al, 2020 ). A co-targeting strategy was recently designed to allow precise editing.…”

“…A co-targeting strategy was recently designed to allow precise editing. In this case, two CRISPR-RNPs were co-electroporated, both with the classical sgRNA for each target gene and with single-stranded oligodeoxyribonucleotides (ssODNs) to allow precise edits ( Akella et al, 2021 ). One of the target genes was PPX1 encoding protoporphyrinogen IX oxidase.…”

“…The second gene was the FTSY gene mentioned above and the ssODN contained a STOP codon. A few pale green colonies with the desired STOP codon were recovered among the herbicide resistant colonies, which opens the way to create strains with precise mutations in several target genes ( Akella et al, 2021 ). The increasing number of reports based on CRISPR/Cas9 in Chlamydomonas is a proof that this technique is effective ( Ghribi et al, 2020 ) and could represent a method of choice for optimizing fluxes through desired biosynthetic pathways.…”