Overview

  • Case Study I
    • K-ras locus in human colon cancer cell line, HCT116, was knocked-out by creating indels in exon 4, as shown in the targeting strategy (Fig. 1).
    • Viral encapsulation of Cas9 and gRNA delivery particles were carried out, viral titer was optimized for HCT116 cells.
    • Individual clones were Sanger sequenced to select for homozygous knock-out indels of K-ras at exon 4. Sanger sequencing data (Fig. 2).
    • Absence of K-ras expression in the selected clone was confirmed via Western Blot (Fig. 3).
  • Case Study II
    • A sequence optimized gRNA was designed and synthesized to target a specific region on the GS allele. DG44 cells were transfected with the construct and the cell pool was analyzed by Sanger sequencing.
    • Several single clones were obtained and Sanger sequence analyzed. A single clone containing a frame shift mutation was carried forward (Fig. 1).
    • The GS knockout clone was also analyzed for GS protein expression (Fig. 2).
    • To assess loss of function, GS knockouts were grown in the presence or absence of increasing concentrations of glutamine (Fig. 3). GS knockouts were unable to grow in the absence of glutamine. However growth improved in the presence of increasing concentrations of glutamine, thereby indicating a functional loss in the GS knockout cell line.
    • In conclusion, successful targeting of GS generated a single clone that was fully validated for lack of expression and GS function.
    Deletion on GS allele causes frame shift mutation
    Glutamine synthetase is not detected by an anti-GS andtibody in GS knockout cell lysate
    L-Glutamine Dependence of DG44(GS-/-)
  • Case Study III
    • gRNA pairs are designed to target the flanking locus of a full gene to delete a full gene on genome. gRNAs pairs are optimized with highest cleavage efficiency in the Jurkat cells by GenCRISPR™ technology (Figure A).
    • The paired gRNAs are co-transfected to the Jurkat cells and enriched by FACS sorting. The deletion efficiency is evaluated by PCR. It is 21% in the transfected cell pool as calculated from light intensity of band (Figure B).
    • 200 single clones are screened with PCR in a high-throughput way and a few of full-allele deletion clones are identified and confirmed by Sanger sequencing and RT-PCR (Figure C).
    case-study-f1
    Validation of gRNA pairs with PCR
    Screening of deletion clones

Quotations and Ordering

Click the quote button, provide service information and submit
Our Technical Account Manager will contact you within 24 hours to fnalize quote
You review and approve final order details and price
You provide credit card/PO information and order is placed
Project is initiated immediately
Our Project Manager will be in contact with you to provide updates

Our customer service repsentatives are available 24 hours a day, Monday through Friday, to assist you.

CRISPR Services

REQUEST A QUOTE

Do you like the current new website?

Hate

Dislike

Neutral

Like

Love

*