Explore our fast-track multiplex editing workflow
Example Pin-point multiple Gene Knock-out project workflow:
- Reagent delivery conditions are optimised using a highly active control single guide RNA (sgRNA).
- Candidate Pin-point sgRNAs targeting the genes of interest are designed and tested in an arrayed format to identify the gRNAs giving the most efficient gene knock-out.
- Shortlisted sgRNAs are validated individually in larger scale transfections to confirm efficiency.
- Validated sgRNAs are combined in multiplex transfections to generate gene edited pools containing cells with multiple combinations of knock-out edits.
- Clonal cell populations are isolated from the edited pool and DNA sequence analysis is performed to identify single and multiple edited clones.
- Clones with desirable genotypes are expanded and editing is confirmed prior to cryopreservation.
View example multiplex editing data
Identifying active Pin-point sgRNAs for gene knockout via C to T base conversion and protein loss screening.
(A) Transfecting 10 sgRNAs/target in HCT-116 cells with Pin-point nCas9 and Rat APOBEC mRNAs under 3 optimized conditions reveals GRN973 and GRN998 as active for genes A and B knockout, confirmed by DNA sequencing and (B) FACS analysis. (C) Multiplex transfection of GRN973 and GRN998 with a validated gRNA, GRN762, targeting a third gene, shows no loss of viability in single, double, or triple targeted pools. (D) DNA sequence analysis confirms effective C>T conversion across all three genes. (E) FACS analysis indicates 7% of transfected cells lost expression of all three target genes.
What’s included
- Optimization of Pin-point reagent delivery conditions to maximize editing efficiency in your cell line(s) of choice.
- Design and screening of candidate Pin-point guide RNAs to enable highly effective C to T editing of your target(s) of interest.
- Potential for simultaneous editing of multiple gene targets through co-delivery of high activity guide RNAs.
- Isolation and genotyping of clonal cell populations, enabling identification of single and multiple edited clones from a single screen.
- Cryopreserved clonal cell lines and supporting data.
- Regular updates from the Project Management team on project progression.
Additional services
- Discuss with our scientific team to learn more about functional validation and verification services available, as well as additional deliverables such as control clones or pools, to be added onto the project.