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Fast Genotyping of Genome-Edited Animals

In the few past years, engineered endonucleases (Meganucleases, ZFN, TALENS) and CRISPR/Cas9 technology have revolutionized genome editing possibilities. Genome editing can now be used in a wide range of species to generate gene inactivation (knockout) or small nucleotide changes (knockin). These endonucleases initiate double-stranded DNA breaks which are repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR). The error-prone NHEJ pathway leads to nucleotide insertion (ins) or deletion (del), termed indels, which disrupt the targeted open reading frame, causing gene inactivation. Single-stranded donor oligonucleotides (ssODN) with single nucleotide or codon changes at the target site are used to make knockin edits via the HDR pathway.

CRISPR/Cas9, the easiest and least costly gene editing system, is widely used in mutation generation in various species. With increasing throughput, an effective mutation detection method is needed. Using on-target gene editing PCR in combination with the LabChip GX Touch instrument reveals both the amplicon of the target site and the heteroduplex (HD) signature, indicating whether NHEJ has occurred, even if it is a single base pair indel. For F0 and F1 generations, PCR sequencing should be performed to confirm the exact nucleotide mutation. For further generation genotyping analysis, the HD pattern alone can be used to determine whether NHEJ has occurred.