TIM, a targeted insertional mutagenesis method utilizing CRISPR/Cas9 in Chlamydomonas reinhardtii
Authors
Picariello, TylerHou, Yuqing
Kubo, Tomohiro
McNeill, Nathan A.
Yanagisawa, Haru-Aki
Oda, Toshiyuki
Witman, George B.
Document Type
Journal ArticlePublication Date
2020-05-13Keywords
Guide RNAElectroporation
Chlamydomonas reinhardtii
Insertion mutation
Sequence motif analysis
Mutagenesis
DNA
Polymerase chain reaction
Algae
Amino Acids, Peptides, and Proteins
Biochemistry
Cell Biology
Genetics and Genomics
Nucleic Acids, Nucleotides, and Nucleosides
Metadata
Show full item recordAbstract
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.Source
Picariello T, Hou Y, Kubo T, McNeill NA, Yanagisawa HA, Oda T, Witman GB. TIM, a targeted insertional mutagenesis method utilizing CRISPR/Cas9 in Chlamydomonas reinhardtii. PLoS One. 2020 May 13;15(5):e0232594. doi: 10.1371/journal.pone.0232594. PMID: 32401787; PMCID: PMC7219734. Link to article on publisher's site
DOI
10.1371/journal.pone.0232594Permanent Link to this Item
http://hdl.handle.net/20.500.14038/48441PubMed ID
32401787Related Resources
Rights
Copyright: © 2020 Picariello et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.Distribution License
http://creativecommons.org/licenses/by/4.0/ae974a485f413a2113503eed53cd6c53
10.1371/journal.pone.0232594
Scopus Count
Except where otherwise noted, this item's license is described as Copyright: © 2020 Picariello et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.