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. eCollection 2020.


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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • Chlamydomonas reinhardtii / genetics*
  • DNA / genetics
  • Gene Editing / methods*
  • Mutagenesis, Insertional / methods*
  • RNA, Guide, Kinetoplastida / genetics


  • RNA, Guide
  • DNA