PT  - JOURNAL ARTICLE
AU  - Christopher V. McCabe
AU  - Gemma F. Codner
AU  - Alasdair J. Allan
AU  - Adam Caulder
AU  - Skevoulla Christou
AU  - Jorik Loeffler
AU  - Matthew Mackenzie
AU  - Elke Malzer
AU  - Joffrey Mianné
AU  - Fran J. Pike
AU  - Marie Hutchison
AU  - Michelle E. Stewart
AU  - Hilary Gates
AU  - Sara Wells
AU  - Nicholas D. Sanderson
AU  - Lydia Teboul
TI  - Application of long-read sequencing for robust identification of correct alleles in genome edited animals
AID  - 10.1101/838193
DP  - 2019 Jan 01
TA  - bioRxiv
PG  - 838193
4099  - http://biorxiv.org/content/early/2019/11/14/838193.short
4100  - http://biorxiv.org/content/early/2019/11/14/838193.full
AB  - Recent developments in CRISPR/Cas9 genome editing tools have facilitated the introduction of more complex alleles, often spanning genetic intervals of several kilobases, directly into the embryo. These techniques often produce mosaic founder animals and the introduction of donor templates, via homologous directed repair, can be erroneous or incomplete. Newly generated alleles must be verified at the sequence level across the targeted locus. Screening for the presence of the desired mutant allele using traditional sequencing methods can be challenging due to the size of the desired edit(s) together with founder mosaicism. In order to help disentangle the genetic complexity of these animals, we tested the application of Oxford Nanopore long read sequencing of the targeted locus. Taking advantage of sequencing the entire length of the segment in each single read, we were able to determine whether the entire intended mutant sequence was present in both mosaic founders and their offspring.bpbase-pairCas9CRISPR associated protein 9CRISPRclustered regularly interspaced short palindromic repeatddPCRdroplet digital PCRDNAdeoxyribonucleic acidHAhomology armkbkilobasesKIknock-inlssDNAlong single-stranded DNAONTOxford Nanopore TechnologiessgRNAsingle guide RNASNPsingle nucleotide polymorphismssODNsingle-stranded oligo-deoxynucleotideWTwild type.