ArticlePloS one2020
TIM, a targeted insertional mutagenesis method utilizing CRISPR/Cas9 in Chlamydomonas reinhardtii.
Article in PloS one, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 35 papers.
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Who cites it
35 citing papers in PubMed, 86 citations in OpenAlex.
- Enhancing CRISPR/Cas-Mediated Gene Knockout With Short Non-Homologous Oligonucleotides.Plant biotechnology journal · 2026Article
- Highly efficient genome editing using CRISPR/Cas9 ribonucleoprotein in the marine oleaginous diatom Fistulifera solaris.Scientific reports · 2026Article
- Article
- Tubulin monoglutamylation is sufficient to rescue the ciliary motility defects in aFrontiers in cell and developmental biology · 2026Article
- CRISPR-Cas Technology TurnsMarine drugs · 2025Review
- A quick-to-implement and optimized CRISPR-Cas9 protocol to obtain insertional and small indel mutants inMethodsX · 2025Article
- Activation of the ciliary kinase CDKL5 is mediated by the cyclin-dependent kinase CDK20/LF2 to control flagellar length.PLoS biology · 2025Article
- Transcriptional gene fusions via targeted integration at safe harbors for high transgene expression in Chlamydomonas reinhardtii.The New phytologist · 2025Article
- High-yield zeaxanthin production in Chlamydomonas reinhardtii via advanced metabolic pathway engineering.Biotechnology for biofuels and bioproducts · 2025Article
- Genome editing in the green alga Chlamydomonas: past, present practice and future prospects.The Plant journal : for cell and molecular biology · 2025Review
- Cas9-mediated gene-editing frequency in microalgae is doubled by harnessing the interaction between importin α and phytopathogenic NLSs.Proceedings of the National Academy of Sciences of the United States of America · 2025Article
- Progress and challenges in CRISPR/Cas applications in microalgae.Journal of microbiology (Seoul, Korea) · 2025Review
- ATG8 delipidation is not universally critical for autophagy in plants.Nature communications · 2025Article
- Cilia and transcription: a mini review.Frontiers in cell and developmental biology · 2025Review
- A cyclical marker system enables indefinite series of oligonucleotide-directed gene editing in Chlamydomonas reinhardtii.Plant physiology · 2024Article
- Efficient DNA-free co-targeting of nuclear genes in Chlamydomonas reinhardtii.Biology direct · 2024Article
- Structural basis for an early stage of the photosystem II repair cycle in Chlamydomonas reinhardtii.Nature communications · 2024Article
- Thermoprotection by a cell membrane-localized metacaspase in a green alga.The Plant cell · 2024Article
- Efficient precision editing of endogenousCell reports methods · 2023Article
- A gap-free genome assembly of Chlamydomonas reinhardtii and detection of translocations induced by CRISPR-mediated mutagenesis.Plant communications · 2023Article
Corrections and comments
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Authors and funding
7 authors at 3 institutions in 2 countries.
Funding
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.
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Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.