Schematic overview of conventional insertional mutagenesis, TIM, and TIM-tagging in <i>C</i>. <i>reinhardtii</i>.

(A) Conventional insertional mutagenesis to create mutants randomly. Donor DNA encoding a selectable marker is introduced into the cell; it then inserts randomly into the nuclear genome, causing a large insertion, deletion, and/or reorganization at the site of integration. (B) Targeted insertional mutagenesis (TIM) to disrupt a specific gene of interest. Based on insertional mutagenesis, but a CRISPR/Cas9 RNP is included in the transformation step and short gene-specific homology arms are added on both ends of the donor DNA. The RNP creates a DSB at the target site, which facilitates the incorporation of donor DNA at this site. The homology arms further increase the probability that the donor DNA will insert at this site, but they are not critical for success. Transformants expressing the selectable marker are screened by gene-specific PCR analysis to identify those in which the target gene has been disrupted. (C) TIM-tagging to specifically tag a gene of interest, as illustrated here for C-terminal tagging. Based on TIM, but the RNP target site is placed close to and upstream of the intended tagging site. The donor DNA starts with an essential homology arm to promote precise integration via homology-directed repair (HDR) at this end of the donor DNA, and then continues with the rest of the gene, including the tag sequence, followed by a selectable marker. A second homology arm at the 3’ end of the donor DNA is optional but might be helpful. In the final result, the allele contains the tag sequence, and is followed by the selectable marker. Following selection of transformants, cells that have integrated the tag sequence are identified by gene-specific PCR analysis. Green star: start codon; red star: stop codon; black and white boxes: homology arms on donor DNA or sequence on gene matching homology arms.