SERVICES

Custom Knockout Cell Lines

CRISPR-Cas9 knockouts in standard cell lines with functional validation, Sanger sequencing at target locus, and Western blot confirmation using matched TPB antibodies.

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Overview

Most CRISPR services treat all knockout targets equivalently. For proteinase and proteinase-inhibitor targets specifically, reagent quality at the validation step matters: a Western blot run with a poorly characterized third-party antibody provides ambiguous evidence of protein loss. Because TPB has produced and characterized antibodies against proteinase-family targets since 1994, in-house matched antibodies are available for the validation Western blot on many common targets. Where a matched TPB antibody exists, it is used for the knockout-confirmation blot before the clone is released — the same antibody lot and conditions used in our standard QC panel.

Single-gene knockouts are the standard service. Multiplex (multi-gene) knockout is offered, with each additional gene adding approximately four weeks to the timeline for independent guide RNA design, selection, and sequencing confirmation at each locus. Knock-in and targeted point mutation projects are reviewed case-by-case; submission of a project inquiry form is the appropriate first step.

All cell lines are handled under BSL-1/BSL-2 conditions. Work is research-use-only; no GMP documentation, regulatory dossiers, or clinical-grade release testing is performed or implied.

Workflow

Guide RNA design and off-target analysis — Target locus analysis, guide RNA selection, and computational off-target scoring. ~2 weeks
Transfection and selection — Delivery of Cas9/gRNA complex into the chosen cell line background; selection of edited cells. ~3 weeks
Single-cell cloning — Limiting dilution or fluorescence-assisted sorting to isolate individual clones. ~3 weeks
Sequencing and expansion — Sanger sequencing at the target locus to identify bi-allelic edited clones; expansion of confirmed candidates. ~2 weeks
Validation — RT-qPCR for transcript loss; Western blot for protein loss (TPB matched antibody used where available); optional functional activity assay confirming protein-function loss. ~2 weeks
Delivery — Cryovial(s) of validated clone shipped with full characterization report and mycoplasma-negative certification.

Typical total timeline: 12–14 weeks for a single-gene knockout. Each additional gene in a multiplex project adds approximately 4 weeks.

Deliverables

Cryovial(s) of validated knockout clone (defined passage number)
Characterization report including:
- Sanger chromatogram(s) at the target locus confirming bi-allelic editing
- RT-qPCR data showing transcript loss relative to parental line
- Western blot image showing protein loss (matched TPB antibody used where available; antibody identity and lot noted in report)
- Functional activity assay data (optional, project-specific)
Passage history from transfection to delivery
Mycoplasma-negative certification (test date and method recorded)
Guide RNA sequences and genomic editing coordinates

Options

Single-gene knockout — Standard service; CRISPR-Cas9 bi-allelic disruption, fully validated clone.
Multiplex knockout — Two or more genes knocked out in the same cell line; add ~4 weeks per additional gene.
Knock-in / point mutation — Available on request; reviewed case-by-case (submit project inquiry).
Cell line backgrounds (standard) — HEK293, HeLa, MCF-7, A549, primary macrophages.
Custom cell line backgrounds — Reviewed case-by-case; not all backgrounds are supported.
Optional functional activity assay — Confirmatory assay for protein-function loss, where an appropriate assay exists for the target; discussed at project scoping.
Matched TPB antibody for validation WB — Included where a TPB antibody exists for the target; antibody details provided in the characterization report.

Timing and pricing

Typical duration: 12–14 weeks for a single-gene knockout from guide RNA design to delivery of validated clone. Multiplex knockouts requiring additional gene targets add approximately 4 weeks per gene. Knock-in and point mutation timelines are determined during project scoping.

Pricing: Project-based quote. Pricing depends on target gene, cell line background, number of loci, and inclusion of optional assays. Submit a project inquiry with your target gene(s), preferred cell line, and any specific validation requirements to receive a formal quote.

Scope

The following are outside the scope of this service:

iPSC generation or editing — Not offered.
Viral vector generation — Not offered.
In vivo delivery or animal work — Not offered.
GMP-grade or clinical-grade cell lines — All output is research-use-only (RUO); no regulatory documentation is generated.
BSL-3 or BSL-4 pathogens — Work is conducted under BSL-1/BSL-2 conditions only.
Off-target whole-genome sequencing — Not included as standard; computational off-target analysis is performed at the guide RNA design stage.

Frequently asked questions

What QC data is included with every knockout clone, and do I receive the raw files?

Every clone ships with a characterization report containing the Sanger chromatogram at the target locus, RT-qPCR knockdown data relative to the parental line, and a Western blot image confirming protein loss. Where a matched TPB antibody is available for the target, that antibody is used for the blot and its identity and lot number are recorded in the report. Raw Sanger trace files are included. Additional raw data files (e.g., qPCR amplification curves) are provided on request.

How do you confirm bi-allelic knockout rather than heterozygous editing?

Single-cell cloning is performed to isolate individual clones. Sanger sequencing is then carried out at the target locus for each candidate clone. Chromatogram analysis and, where appropriate, TIDE or ICE deconvolution are used to confirm that both alleles carry loss-of-function edits before a clone is advanced to the expansion and validation steps.

What is the typical turnaround time, and what can extend it?

A single-gene knockout in a standard cell line (HEK293, HeLa, MCF-7, A549) typically takes 12–14 weeks from project start to delivery of a validated clone. Factors that can extend this include low transfection efficiency in a particular cell background, a low rate of bi-allelic editing at the chosen locus requiring screening of more clones, or the addition of an optional functional activity assay. Multiplex projects add approximately 4 weeks per additional gene.

Can I request a specific cell line not listed — for example, a cancer line or primary cells other than macrophages?

Custom cell line backgrounds are reviewed case-by-case. Submit a project inquiry describing the cell line, its growth characteristics, and your target gene. We will assess feasibility and advise on likely timeline and cost implications before committing to a project.

Do I receive the guide RNA sequences, and can I use them independently?

Yes. The guide RNA sequences and the genomic coordinates of the editing event are provided in the characterization report as part of the standard deliverables. There are no restrictions on your use of that sequence information for your research purposes.

How is pricing structured, and what is included in the base quote?

Pricing is project-based. A formal quote is issued after review of the project inquiry and covers guide RNA design, transfection, single-cell cloning, Sanger sequencing, RT-qPCR, Western blot validation, mycoplasma testing, and delivery of one validated cryovial with the characterization report. Optional items — such as a functional activity assay, additional cryovials, or a custom cell background — are quoted as line items. Submit a project inquiry with your target gene(s), preferred cell line, and any specific requirements to receive a quote.

What happens if no validated bi-allelic clone is identified within the project scope?

This is addressed during project scoping. The inquiry review process includes a feasibility assessment of the target locus and cell line combination. If screening of the agreed number of clones does not yield a confirmed bi-allelic knockout, we will discuss options — which may include screening additional clones or redesigning the guide RNA — and agree on next steps and any associated costs before proceeding.

Are these cell lines suitable for use in animal studies or clinical applications?

No. All cell lines generated by Triple Point Biologics are research-use-only (RUO) reagents. They are not manufactured under GMP conditions, carry no regulatory documentation, and are not intended for therapeutic, diagnostic, or clinical use.