TY - JOUR

T1 - Studying concatenation of the Cas9-cleaved transgenes using barcodes

AU - Smirnov, A. V.

AU - Korablev, A. N.

AU - Serova, I. A.

AU - Yunusova, A. M.

AU - Muravyova, A. A.

AU - Valeev, E. S.

AU - Battulin, N. R.

N1 - This work was supported by the Russian Science Foundation grant No. 24-74-10013. The experiments involving the visualization of fluorescent embryos were conducted with support from State Project FWNR-2022-0019 at the Institute of Cytology and Genetics SB RAS. The calculations were conducted using the computational resources of the Computational Center of the Novosibirsk State University.

PY - 2025

Y1 - 2025

N2 - 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.

AB - 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.

KW - CRISPR/Cas9

KW - DNA barcoding

KW - DSB repair

KW - concatemer

KW - homologous recombination (HR)

KW - mouse embryos

KW - non-homologous end-joining, NHEJ

KW - pronuclear microinjection

KW - transgenic animals

UR - https://www.scopus.com/pages/publications/86000558411

UR - https://elibrary.ru/item.asp?id=80489903

UR - https://www.mendeley.com/catalogue/1211e109-d353-3135-bfc5-55e759a8bc30/

U2 - 10.18699/vjgb-25-04

DO - 10.18699/vjgb-25-04

M3 - Article

C2 - 40144376

VL - 29

SP - 26

EP - 34

JO - Вавиловский журнал генетики и селекции

JF - Вавиловский журнал генетики и селекции

SN - 2500-0462

IS - 1

M1 - 3

ER -