| Autor: |
Guzmán-Zapata D; Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología. Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 Mexico City, Mexico. danielguza@gmail.com., Sandoval-Vargas JM; Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología. Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 Mexico City, Mexico. ibt_jose_sandoval@hotmail.com., Macedo-Osorio KS; Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología. Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 Mexico City, Mexico. karla_032214@hotmail.com., Salgado-Manjarrez E; Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología. Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 Mexico City, Mexico. esalgado@ipn.mx., Castrejón-Flores JL; Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología. Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 Mexico City, Mexico. jlcastrejon@ipn.mx., Oliver-Salvador MDC; Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología. Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 Mexico City, Mexico. moliver@ipn.mx., Durán-Figueroa NV; Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología. Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 Mexico City, Mexico. nduranf@ipn.mx., Nogué F; Institut Jean-Pierre Bourgin, INRA, AgroParisTech, CNRS, Université Paris-Saclay, 78000 Versailles, France. fabien.nogue@inra.fr., Badillo-Corona JA; Instituto Politécnico Nacional, Unidad Profesional Interdisciplinaria de Biotecnología. Av. Acueducto S/N., Col. Barrio La Laguna Ticomán, 07340 Mexico City, Mexico. jbadilloc@ipn.mx. |
| Abstrakt: |
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. |
