Purpose
CRISPR/Cas9 technology allows users to direct double stranded breaks (DSB) nearly anywhere in the genome with the only constraint being it must be near a PAM (NGG) sequence. DSB are then repaired through the cells inherit DNA repair pathways via non-homology end joining (NHEJ) or Microhomology-Mediated end joining (MMEJ), with the latter leaving insertions and deletions (indels). Choosing where you want to make indels can lead to gene knockout (i.e. by disrupting the ATG site or causing frameshifts).
Screening cell lines with CRISPR/Cas9 edits can be challenging due to the variation of possible indels and diploid cells can give rise to complex genotypes, whereby even Sanger sequencing won't be sufficient to resolve the genotype. In order to reliably screen these edited cell lines, we offer a cost-effective NGS genotyping service to determine the exact genotype on both alleles. Furthermore, it can also it can also be used to identify HDR events and aid in isolating the cleanest clone.
Guidelines
In order to genotype your clones, please provide the GTF with 25ul of crude DNA lysate and a SNAP Gene file highlighting the sgRNA used. Up to 96 DNA samples can be analysed in one run and genotyping DNA will be returned within 3 weeks.
Please contact Asif Nakhuda (asif.nakhuda@babraham.ac.uk) in the first instance.