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

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.

Fig 1. An overview of the TIM (targeted insertional mutagenesis) method.

(A) Schematic overview of TIM. Following delivery of Cas9/gRNA RNP and double-stranded donor DNA (antibiotic-resistance cassette) into cells by electroporation or the glass-bead method, the RNP causes a double-stranded break (DSB) at the target site (blue box). DSBs are repaired with or without integration of the donor DNA. The left pathway results in insertional mutations, whereas the right pathway may generate insertion/deletion (indel) mutations (small white box within blue box, third row). As with other insertional mutagenesis approaches, donor DNA also can insert randomly in the genome, resulting in antibiotic-resistant cells that may not have a mutation at the target site. Cells are then selected for presence of the donor DNA via growth on antibiotic-specific media. Finally, antibiotic-resistant cells are screened via PCR to identify cell lines with mutations at the target site. L and R, left and right homology arms of the donor DNA, or genomic regions corresponding to left and right homology arms of the donor DNA. Short black arrows (P1, P2) indicate the primers used for screening. In the typical agarose gel shown (in this case, the targeted gene was FAP70), the asterisk and arrowhead on the right side of the gel indicate positive control (in this case FUS1) and wild-type target-specific PCR bands, respectively. Absence of a wild-type target-specific band indicates a mutation at the target site; in most cases here this is accompanied either by the appearance of an ~2-Kb band (arrow on the right side of the gel), suggesting insertion of a single copy of the antibiotic-resistance gene (~1.7 Kb), or by lack of any detectible gene-specific band, suggesting a large insertion/deletion at the target site. Presence of a wild-type target-specific band indicates that the cell line was not mutated at the target site; the antibiotic-resistance gene has inserted into the genome somewhere other than at the target site. Mutants generated by the right-hand pathway (no insertion of donor DNA) would not be selected for unless the donor DNA inserted into another site; in the latter case, mutants with small indels would appear to be wild type in the PCR screen because the PCR product would be at or near wild-type length. Relative to the target site, the exact positions of the primers and of genomic regions corresponding to the homology arms will vary when targeting different genes. (B) Schematic of the antibiotic-resistance cassettes used. Gene-specific homology arms of varying lengths were added to the 5’- and 3’-ends of these cassettes. (C) Representative western blot of whole-cell extracts of g1 (wild type) and an ift81 mutant generated by TIM. Absence of an IFT81 band in the mutant strain confirms disruption of the IFT81 gene. ATP synthase β subunit (βF1 ATPase) was probed as a loading control.