Studying concatenation of the Cas9-cleaved transgenes using barcodes

Bibliographic Details
Title: Studying concatenation of the Cas9-cleaved transgenes using barcodes
Authors: A. V. Smirnov, A. N. Korablev, I. A. Serova, A. M. Yunusova, A. A. Muravyova, E. S. Valeev, N. R. Battulin
Source: Вавиловский журнал генетики и селекции, Vol 29, Iss 1, Pp 26-34 (2025)
Publisher Information: Siberian Branch of the Russian Academy of Sciences, Federal Research Center Institute of Cytology and Genetics, The Vavilov Society of Geneticists and Breeders, 2025.
Publication Year: 2025
Collection: LCC:Genetics
Subject Terms: crispr/cas9, pronuclear microinjection, dna barcoding, transgenic animals, dsb repair, concatemer, homologous recombination (hr), non-homologous end-joining, nhej, mouse embryos, Genetics, QH426-470
More Details: In pronuclear microinjection, the Cas9 endonuclease is employed to introduce in vivo DNA double-strand breaks at the genomic target locus or within the donor vector, thereby enhancing transgene integration. The manner by which Cas9 interacts with DNA repair factors during transgene end processing and integration is a topic of considerable interest and debate. In a previous study, we developed a barcode-based genetic system for the analysis of transgene recombination following pronuclear microinjection in mice. In this approach, the plasmid library is linearized with a restriction enzyme or a Cas9 RNP complex at the site between a pair of barcodes. A pool of barcoded molecules is injected into the pronucleus, resulting in the generation of multicopy concatemers. In the present report, we compared the effects of in vivo Cas9 cleavage (RNP+ experiment) and in vitro production of Cas9-linearized transgenes (RNP– experiment) on concatenation. In the RNP+ experiment, two transgenic single-copy embryos were identified. In the RNP– experiment, six positive embryos were identified, four of which exhibited low-copy concatemers. Next-generation sequencing (NGS) analysis of the barcodes revealed that 53 % of the barcoded ends had switched their initial library pairs, indicating the involvement of the homologous recombination pathway. Out of the 20 transgene-transgene junctions examined, 11 exhibited no mutations and were presumably generated through re-ligation of Cas9-induced blunt ends. The majority of mutated junctions harbored asymmetrical deletions of 2–4 nucleotides, which were attributed to Cas9 end trimming. These findings suggest that Cas9-bound DNA may present obstacles to concatenation. Conversely, clean DNA ends were observed to be joined in a manner similar to restriction-digested ends, albeit with distinctive asymmetry. Future experiments utilizing in vivo CRISPR/Cas cleavage will facilitate a deeper understanding of how CRISPR-endonucleases influence DNA repair processes.
Document Type: article
File Description: electronic resource
Language: English
Russian
ISSN: 2500-3259
Relation: https://vavilov.elpub.ru/jour/article/view/4473; https://doaj.org/toc/2500-3259
DOI: 10.18699/vjgb-25-04
Access URL: https://doaj.org/article/423196588de040ae873243aa16f0b0e1
Accession Number: edsdoj.423196588de040ae873243aa16f0b0e1
Database: Directory of Open Access Journals
More Details
ISSN:25003259
DOI:10.18699/vjgb-25-04
Published in:Вавиловский журнал генетики и селекции
Language:English
Russian