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. 2024 Feb 17;25:189. doi: 10.1186/s12864-024-10050-6

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 6 — Precise evaluation of NHEJ and HDR efficiency through NP-Seq analysis of long Alb-F8 insert junction PCR products. (A) Diagram showing forward and reverse integrations of the BDDF8 donor carrying homology arms. Four primer pairs were strategically designed to amplify the junctions: Left F8-Forward (F1-R1), Right F8-Forward (F2-R2), Left F8-Reverse (F3-R3), and Right F8-Reverse (F4-R4). The sgAlb target site is indicated by red scissors. (B) Demonstration of successful amplification of junctions using the designated primers. Shown here is a representative result of long-range PCR products obtained from the liver genomic DNA of an edited mouse. The identities of these PCR products were confirmed by Nanopore sequencing. (C) Visualization of 15 randomly selected reads depicting forward insertion of F8 at the left junction (FL) and forward insertion at the right junction (FR) of the donor with HA190-130. Labels indicate HDR and NHEJ alleles, plasmid backbone inserts, and their respective percentages. (D) Effect of homology arm length on HDR efficiency. Various homology lengths, ranging from 85 to 600 bp, were assessed. Error bars represent mean ± SEM, based on data from 3 mice. Unpaired two-sided Student’s t-tests were utilized for statistical analysis