| Autor: |
Tyler Picariello, Yuqing Hou, Tomohiro Kubo, Nathan A McNeill, Haru-Aki Yanagisawa, Toshiyuki Oda, George B Witman |
| Jazyk: |
angličtina |
| Rok vydání: |
2020 |
| Předmět: |
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| Zdroj: |
PLoS ONE, Vol 15, Iss 5, p e0232594 (2020) |
| Druh dokumentu: |
article |
| ISSN: |
1932-6203 |
| DOI: |
10.1371/journal.pone.0232594 |
| Popis: |
Generation and subsequent analysis of mutants is critical to understanding the functions of genes and proteins. Here we describe TIM, an efficient, cost-effective, CRISPR-based targeted insertional mutagenesis method for the model organism Chlamydomonas reinhardtii. TIM utilizes delivery into the cell of a Cas9-guide RNA (gRNA) ribonucleoprotein (RNP) together with exogenous double-stranded (donor) DNA. The donor DNA contains gene-specific homology arms and an integral antibiotic-resistance gene that inserts at the double-stranded break generated by Cas9. After optimizing multiple parameters of this method, we were able to generate mutants for six out of six different genes in two different cell-walled strains with mutation efficiencies ranging from 40% to 95%. Furthermore, these high efficiencies allowed simultaneous targeting of two separate genes in a single experiment. TIM is flexible with regard to many parameters and can be carried out using either electroporation or the glass-bead method for delivery of the RNP and donor DNA. TIM achieves a far higher mutation rate than any previously reported for CRISPR-based methods in C. reinhardtii and promises to be effective for many, if not all, non-essential nuclear genes. |
| Databáze: |
Directory of Open Access Journals |
| Externí odkaz: |
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