CRISPR Knockout Cell Line Service

SKU :
ASC-6012-1
Catalog # :
ASC-6012-1
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Overview

Gene knockout cell lines offer a powerful tool to understand and elucidate phenotype-genotype relationships, functional role of genes, antibody validation and more. ASC provides comprehensive CRISPR knockout cell line service using its successful CRISPR/Cas9 cell line generation platform. As one of the earliest CRISPR service providers, we have engineered >1300 unique cell line models to-date and in >200 distinct mammalian cell lines. We can generate a CRISPR knockout cell line model to your specifications, in as little as 2-3 months.

We offer different modalities for generating knockout for your gene of interest using our CRISPR Knockout Cell Line Service:

Frameshift mutation

This is the most common strategy to generate knockout cell lines. Using a validated CRISPR guide RNA (sgRNA) and Cas9, small nucleotide insertions or deletions (indels) are introduced into the selected exon leveraging the non-homologous end joining (NHEJ) repair pathway. This results in a frameshift of the open reading frame (ORF) of the targeted gene, thereby altering stop codon(s) which could lead to abnormally short or long protein after translation or change in amino acid sequence resulting in an abnormal protein.

 

Fragment excision

Using two sgRNAs, large targeted region of the gene of interest can be deleted resulting in complete loss of function. Fragment excision/ deletion is useful for both protein coding and non-coding parts of the genome such as promoter and enhancer regions, regulatory elements, and more. 

 

Stop cassette insertion

Knock-in or transcription terminator signal using homology-directed repair (HDR) into an early exon to generate constitutive knockout cell lines. This is another design option available to knockout your gene of interest: when quality gRNA(s) with low off-targets are not available for exon regions but available for intronic regions.

 

Double gene knockout

ASC can also efficiently knockout two or more genes for multiplexed genetic modification and cell line model generation in even hard-to-transfect cell lines. We customize our design strategy, transfection and cell culture protocol for the characteristics cell lines to minimize toxicity and attain high efficiency genetic modifications.

  

Gene fusion with interstitial deletion

Generating cell line models with gene fusion (such as chromosomal translocation, deletion, inversion) are very clinically representative research models. But, generating these models with high efficiency and without footprints is very difficult. ASC has optimized the strategy to generate footprint-free gene fusion cell lines with deletion/ translocation/ inversion using CRISPR/Cas9 to get you physiologically relevant cell line models for better translation of your results.

 

We use the most up-to-date CRISPR designing strategies, optimized and high efficiency protocols to deliver projects with a very high (>98%) success rate and customizable deliverables. With our dedicated project management, we will work with you every step of the way to ensure that your CRISPR knockout cell line meets your requirements:

  • Homozygous/ heterozygous knockout clones
  • Single cell clones or knockout cell pools
  • Variety of cell lines: cancer, hard-to-transfect, most mammalian species


Workflow for our CRISPR knockout cell line service includes: 

ASC-6012-1-CRISPR-Knockout-Cell-Line-Service-workflow

Some milestones will be done in parallel.

Deliverables:

  • One clone with homozygous (or heterozygous, if specified) for the targeted null allele; two vials cells at 1 x 10^6 cells/vial. (Optional! additional clones may be available purchase)
  • Milestone update/report; final report with detailed description of each procedure, including targeting design, construction and validation, transfection condition, genotyping strategy, and results.

Timeline: 3-5 months

Applications:

  • Antibody validation
  • Disease modeling for immuno-oncology, pharmacogenomic studies
  • Drug discovery and drug efficacy and toxicity screening; drug combination studies
  • Deriving diagnostic reference standards and materials


Case Study for CRISPR Knockout Cell Line Service

ASC-6012-1-crispr-knockout-cell-line-service-case-1.        B. ASC-6012-1-crispr-knockout-cell-line-service-case-2

 

Figure. (A) Schematic representation of CRISPR/Cas9 targeting strategy for fragment deletion in a gene of interest in T2 lymphoblastic cell line (left). (B) PCR gel electrophoresis of single cell clones indicate clones 3, 7, and 12 contained deletion of the desired fragment as compared to wild type (WT). Lane M: GeneRuler 1kb plus DNA ladder.