English (United Kingdom)

https://curated-unify.zendy.io/wp-json/zendy-region/v1/featured_content/oa?rat=en

https://curated-unify.zendy.io/wp-json/zendy-region/v1/highlighted_journal/

Global: Zendy Plus annual

Presents the access of premium content as premium feature

Premium Content

Presents the keyphrase highlighting as premium feature

Keyphrase Highlighting

Presents the summarisation as premium feature

Summarisation

Insights

Presents the pdf analysis as premium feature

PDF Analysis

Presents the zaia usage as premium feature

ZAIA

Global: Zendy Tools annual

Global: Zendy Free Plan

Global: Zendy Tools monthly

Global: Zendy Plus monthly

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.

TIM, a targeted insertional mutagenesis method utilizing CRISPR/Cas9 in Chlamydomonas reinhardtii