CRISPR-Cas9, an RNA-guided DNA endonuclease, is used as a tool for genome editing techniques of directed mutations in the eukaryotic genome. 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.
NGS Amplicon sequencing is a highly targeted approach that enables researchers to analyze genetic variation in specific genomic regions. The sequencing of PCR products targeting the genome regions modified by the CRISPR technology allows accurate identification of gene editing profiles.
Avance Biosciences has developed and validated (in compliance with GLP and per ICH guidance) custom NGS amplicon sequencing assays to analyze the percentage of edited cells and the ratio between various indels and wild-type genes, in animal tissues or human blood collected for preclinical and clinical pharmacokinetics studies.
Digital PCR can accurately determine the ratio between edited genes and wild-type genes, especially when the size of the deletions introduced by CRISPR is too large to be accurately quantified using qPCR or amplicon sequencing.
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