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

作者：Soujanya Akella, Xinrong Ma, Romana Bačová, Zachary P. Harmer, Martina Koláčková, Xiaoxue Wen, David A. Wright, Martin H. Spalding, Donald P. Weeks, Heriberto Cerutti · 发表于：PLANT PHYSIOLOGY · 年份：2021 · DOI：10.1093/plphys/kiab418 · 被引用次数：44 · 研究领域：CRISPR and Genetic Engineering、Innovation and Socioeconomic Development、Evolution and Genetic Dynamics

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 synthe...