RT Journal Article
SR Electronic
T1 DNA barcoding reveals that injected transgenes are predominantly processed by homologous recombination in mouse zygote
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 603381
DO 10.1101/603381
A1 Alexander Smirnov
A1 Anastasia Yunusova
A1 Alexey Korablev
A1 Irina Serova
A1 Veniamin Fishman
A1 Nariman Battulin
YR 2019
UL http://biorxiv.org/content/early/2019/04/10/603381.abstract
AB Mechanisms that ensure repair of double-stranded DNA breaks play a key role in the integration of foreign DNA into the genome of transgenic organisms. After pronuclear microinjection, exogenous DNA is usually found in the form of concatemer consisting of multiple co-integrated transgene copies. Here we investigated contribution of various DSB repair pathways to the concatemer formation. We injected a pool of linear DNA molecules carrying unique barcodes at both ends into mouse zygotes and obtained 10 transgenic embryos with transgene copy number ranging from 1 to 300 copies. Sequencing of the barcodes allowed us to assign relative positions to the copies in concatemers and to detect recombination events that happened during integration. Cumulative analysis of approximately 1000 integrated copies revealed that more than 80% of copies underwent recombination when their linear ends were processed by SDSA or DSBR. We also observed evidence of double Holliday junction (dHJ) formation and crossing-over during the formation of concatemers. Additionally, sequencing of indels between copies showed that at least 10% of the DNA molecules introduced into the zygote are ligated by non-homologous end joining (NHEJ). Our barcoding approach documents high activity of homologous recombination after exogenous DNA injection in mouse zygote.