TIM, a targeted insertional mutagenesis method utilizing CRISPR/Cas9 in Chlamydomonas reinhardtii
McNeill, Nathan A.
Witman, George B.
UMass Chan AffiliationsWitman Lab
Division of Cell Biology and Imaging, Department of Radiology
Sequence motif analysis
Polymerase chain reaction
Amino Acids, Peptides, and Proteins
Genetics and Genomics
Nucleic Acids, Nucleotides, and Nucleosides
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AbstractGeneration 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.
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