Custom Gene-edited Cell Lines

Your cell lines with your chosen genotype

Overview

With nearly a decade of experience planning and executing nearly 3,000 gene editing experiments, access to a multiple genome editing technologies, and cell line development service options to suit a variety of experimental needs and budgets, Horizon is the global leader in cell line engineering.

Custom Cell Line Generation

Starter
  • Sequence verified biallelic KO* or monoallelic KI via CRISPR-Cas9^#
  • DLD-1, HCT-116, or RKO cell line
  • Predesigned sgRNA (KO) or screening of 2 sgRNAs (KI)

From 10 weeks

Standard
  • Sequence verified biallelic KO* or monoallelic KI via CRISPR-Cas9^#
  • Any standard cancer cell line1**
  • Cell line characterization and optimization (if not used before)***
  • Predesigned sgRNA (KO) or screening of 2 sgRNAs (KI)
  • Mycoplasma Hoechst testing

From 13 weeks

Premium
  • Sequence verified biallelic KO* or monoallelic KI via any/all of our gene editing^ technologies
  • Any cell line1
  • Cell line characterization and optimization
  • Custom sgRNA design (KO) or screening of 5 sgRNAs (KI)
  • Mycoplasma Hoechst and culture isolation testing
  • Extended validation

From 20 weeks

*Knockouts by NHEJ-mediated frame-shift mutations. Defined deletions and gene disruption via targeted insertion are subject to knockin pricing. Some cell lines may be polyploid at some loci. Additional alleles may be modified for an additional fee. **Possessed by Horizon or in ATCC for the commercial use. ***Characterization of more than one cell line is available for an additional fee. 1Cell line must tolerate single cell cloning and display adequate transfection efficiency. ^KI of additional alleles is available for an additional fee. #A plasmid donor carrying a selection cassette will be utilized.

Key Benefits
  • Strong IP position to support your freedom to operate
  • Substantial experience with over 100 cell lines and 200 target genes
  • Emphasis on design to test your hypothesis, not just project execution
  • A collaborative approach including a dedicated project manager to ensure the project meets your needs
  • Development of genomically validated isogenic cell line pairs (all cell lines are provided with a non-edited control)
  • Initial characterization of parental cell line prior to gene-editing (if required)
Case Studies

Case study I: CRISPR-Mediated Homozygous Knockout

Purpose

Disruption of the gene X in the human melanoma A375 cell line (copy number = 3).

Workflow
  • Transfection with plasmids expressing Cas9 nuclease and a validated gRNA
  • Derivation of single cell clones
  • Screening of the clones
  • Genotype analysis by Sanger sequencing
Targeting Strategy

Targeting

  • Sequencing of individual alleles of 7 clones (via molecular cloning) demonstrated that 3 clones had out of frame deletions on all 3 alleles
  • Sequencing of transcripts from individual alleles indicated out of frame deletions were also present at the RNA level for two of the three clones

Clone 1

Case Study II: Heterozygous Knockin in Leukemia Cells

Purpose

To create a heterozygous point mutation K700E (AAA -> GAA) in SF3B1 in chronic myelogenous leukemia (CML) K562 cells using rAAV.

Workflow

  • Design of rAAV targeting vectors
  • rAAV infection
  • Plating of infected clones
  • PCR and Sanger sequencing to identify KI pools
  • Derivation of single cell clones
  • Screening of the clones by PCR
  • Genotype analysis by Sanger sequencing

Vector Design

Targeting

Targeted allele-specific sequencing shows that both clones contain the K700E mutation. Non-allele specific sequencing shows a ratio of 50:50 of the mutant to wild-type peaks, as would be expected for a 2N locus.

Project Plan

Phase I:

Cell Line Characterization

  • Revival, quality control and establishment of the cell line in routine culture
  • Single cell dilution test
  • Transfection/infection tests
  • Gene copy number analysis

Phase II:

Gene-editing Reagent Design

  • Designing, manufacturing and testing CRISPR gRNAs
  • Designing and manufacturing a suitable donor (e.g. AAV, oligonucleotide, plasmid) if needed
  • Designing a suitable screening strategy to identify correctly targeted cells

Phase III:

Cell Line Engineering

  • Introduction of the targeting event
  • Enrichment of the targeted cell population
  • Assessment of targeting in the pools of cells
  • Plating the cells to 96-well plates and growing them for screening
  • Screening followed by in depth genetic assessment to select correctly targeted populations
  • If required, single cell dilution of targeted populations to get targeted clones
  • Expansion of the selected clones and banking

Phase IV:

Cell Line Validation

  • Final sequence validation
  • Recovery viability testing
  • Sterility testing (bacteria and mycoplasma)

Phase V:

Project Delivery

  • Data pack summarizing the process and analysis of generated cell lines; up to 2 clones per cell line

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