UMMS Affiliation

Division of Cell Biology and Imaging, Department of Radiology; Witman Lab

Publication Date

2020-05-13

Document Type

Article

Disciplines

Algae | Amino Acids, Peptides, and Proteins | Biochemistry | Cell Biology | Genetics and Genomics | Nucleic Acids, Nucleotides, and Nucleosides

Abstract

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.

Keywords

Guide RNA, Electroporation, Chlamydomonas reinhardtii, Insertion mutation, Sequence motif analysis, Mutagenesis, DNA, Polymerase chain reaction

Rights and Permissions

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.

DOI of Published Version

10.1371/journal.pone.0232594

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

Journal/Book/Conference Title

PloS one

Related Resources

Link to Article in PubMed

PubMed ID

32401787

Creative Commons License

Creative Commons Attribution 4.0 License
This work is licensed under a Creative Commons Attribution 4.0 License.

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