1. Efficient Editing of the Nuclear APT Reporter Gene in Chlamydomonas reinhardtii via Expression of a CRISPR-Cas9 Module.
- Author
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Guzmán-Zapata D, Sandoval-Vargas JM, Macedo-Osorio KS, Salgado-Manjarrez E, Castrejón-Flores JL, Oliver-Salvador MDC, Durán-Figueroa NV, Nogué F, and Badillo-Corona JA
- Subjects
- Clustered Regularly Interspaced Short Palindromic Repeats genetics, Electroporation methods, Gene Editing methods, Plasmids genetics, RNA, Guide, CRISPR-Cas Systems genetics, Ribonucleoproteins genetics, Adenine Phosphoribosyltransferase genetics, CRISPR-Associated Protein 9 genetics, CRISPR-Cas Systems genetics, Chlamydomonas reinhardtii genetics, Genes, Reporter genetics
- Abstract
The clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9 (CRISPR/Cas9) technology is a versatile and useful tool to perform genome editing in different organisms ranging from bacteria and yeast to plants and mammalian cells. For a couple of years, it was believed that the system was inefficient and toxic in the alga Chlamydomonas reinhardtii . However, recently the system has been successfully implemented in this model organism, albeit relying mostly on the electroporation of ribonucleoproteins (RNPs) into cell wall deficient strains. This requires a constant source of RNPs and limits the application of the technology to strains that are not necessarily the most relevant from a biotechnological point of view. Here, we show that transient expression of the Streptococcus pyogenes Cas9 gene and sgRNAs, targeted to the single-copy nuclear apt9 gene, encoding an adenine phosphoribosyl transferase ( APT ), results in efficient disruption at the expected locus. Introduction of indels to the apt9 locus results in cell insensitivity to the otherwise toxic compound 2-fluoroadenine (2-FA). We have used agitation with glass beads and particle bombardment to introduce the plasmids carrying the coding sequences for Cas9 and the sgRNAs in a cell-walled strain of C. reinhardtii (CC-125). Using sgRNAs targeting exons 1 and 3 of apt9 , we obtained disruption efficiencies of 3 and 30% on preselected 2-FA resistant colonies, respectively. Our results show that transient expression of Cas9 and a sgRNA can be used for editing of the nuclear genome inexpensively and at high efficiency. Targeting of the APT gene could potentially be used as a pre-selection marker for multiplexed editing or disruption of genes of interest.
- Published
- 2019
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