Edited Cell Determination for CRISPR-Cas9 Using Next-Generation Amplicon Sequencing (NGS)
CRISPR-Cas9, an RNA-guided DNA endonuclease, is used as a tool for genome editing techniques of directed mutations in the eukaryotic genome. CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. This technology was developed to screen ‘loss of function’ experiments in cultured cells that produce insertion or deletion mutants.
Screening for mutated clones is time-consuming and laborious. Avance Biosciences, Inc. has adopted an accurate and sensitive method for edited cell percentage determination using NGS amplicon sequencing of CRISPR-Cas9 transfected clones. This method can identify homozygous or heterozygous mutants and/or mixed clones*. All of our services are available under GMP/GLP regulatory compliance or as research grade projects.
Services provided for CRISPR-Cas9 Edited Cell Screening
- Screening primers are designed to create an amplicon over the targeted region of pooled DNA
- Typically the amplicon length of 200 bps maximizes detection of a variety of mutations ensuring the majority of the reads reach the forward and reverse barcodes
- Unique barcoded amplicons are produced with up to 96 clones/plate using 12 barcoded forward primers and 8 barcoded reverse primers
- The amplicons are PCR amplified and clones pooled
- Adapters are ligated to the clone pools
- A library is created of the fragment pools
- The library is sequenced with NGS
- Gapped mapping is employed to identify indels and their percentage to wild-type determined
- A report is prepared and delivered
Our services include:
- Methods validated per ICH GLP/GMP
- Research grade testing also available
- Projects can be tailored to suit your needs
- Professional project reports that include raw and analyzed data
- Competitive pricing
- Fast turn-around time
- Weekly progress reports
*This method is also applicable to TALEN (transcription activator-like effector nuclease system) or Zinc Finger Nucleases
Need more info?
We are dedicated to timely, high-quality service from the start of your project to the finish. Send us a quick message with your questions to find out what Avance can do for you.
Ready to get started?
Give us a little background on your needs and the goals of your project, and we’ll send a preliminary quote including: tasks, deliverables and a no-obligation cost estimate.