Our work has yielded a number of protocols and reagents that will be made available upon request (firstname.lastname@example.org or email@example.com).
1. Oligonucleotide-mediated gene modification (oligo targeting)
A method to substitute a single base-pair in the genome of mammalian cells using only short single-stranded oligonucleotides. Includes information on oligonucleotide design, chemical modification and delivery to recipient cells as well as methods to detect and purify successful gene modification events.
2. Oligonucleotide-directed mutation screening (ODMS)
A cellular assay based on oligo targeting to test variants of uncertain significance (VUS) of DNA mismatch repair genes in order to help identification of Lynch syndrome cases who can be offered appropriate counseling.
3. Introduction of break-distal mutations by CRISPR/Cas9 technology
The protocol describes which precautions need to be taken in template design and choice of cell type to introduce mutations located distally from a CRISPR/Cas9-induced DNA double-strand break.
4. Improved recovery of single base-pair substitutions by CRISPR/Cas9 technology
The method presents “hideRNA” as a simple way for preventing re-cutting of the target site after templated repair of a CRISPR/Cas9-induced double-strand break. Re-cutting often frustrates the recovery of desired base substitution events. Although not always effective, the method can easily be implemented in current protocols, does not do any harm and may particularly be suitable to enhance recovery of gene editing events in difficult-to-analyze cell systems like primary cells and zygotes. Importantly, the method obviates the need for additional target-site-disrupting mutations.
5. Improved recovery of single base-pair substitutions by co-CRISPR/Cas9
The method proposes simultaneous introduction of two guideRNAs, one conferring a selectable or detectable phenotype, the other targeting a gene of interest. Co-CRISPR confers enrichment for the non-selectable event among selected cells, in particular when the non-selectable event can only be achieved with a poorly-performing guideRNA.
6. LEGO (Low-complexity by Enrichment for genes shut Off) to facilitate shRNA screening
The method describes the enzymatic production of low-complexity shRNA libraries from subtracted transcriptomes to identify genes that suppress a certain phenotypic transition. E.g., by PCR-based Suppression Subtractive Hybridization (SSH) of transcriptomes from transformed and non-transformed counterparts tumor suppressor transcripts can be effectively enriched and processed into a custom-made LEGO shRNA library that can be screened for transforming activity. The method greatly outperforms whole-genome screening in terms of labor, costs and false-positive and off-target results.
1. Cell lines to perform ODMS of MMR genes
We generated mouse embryonic stem cell (ESC) lines and human diploid cell (DIP1) lines in which one allele of a MMR gene was completely deleted and the wild-type allele was labeled with an adjacent puromycin-resistance marker. These cell lines can be used for ODMS, or the generation of cells expressing a variant MMR allele by oligo targeting or CRISPR/Cas9 followed by phenotypic analysis.
2. A human diploid cell line to perform co-selection for CRISPR/Cas9-mediated base substitution
The cell line stably expresses a Cas9-Geminin fusion protein to direct double-strand break induction to cell cycle phases optimal for homology-directed repair. Furthermore the cell line contains an active BFP and a non-functional GFP sequence that can effectively serve as a template for HDR of a DSB induced by a guideRNA specifically targeting the BFP chromophore. Preselection for green-fluorescent cells enriches for gene modification events induced by a simultaneously introduced second guideRNA.