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. 2024 Oct 9;6:1467449. doi: 10.3389/fgeed.2024.1467449

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Copyright © 2024 Yao, Schank, Zhao, El Gazzar, Wang, Zhang, Hill, Banik, Pyburn and Moorman.

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

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HBV genome and gRNAs design. The genome of HBV is a double-stranded (+/−) circular DNA of about 3.2 kb pairs comprising four overlapping open reading frames (ORFs): surface (S), capsid (C), polymerase (P), and X genes. To select the most specific targets in the HBV genome, we used an online CHOPCHOP program to analyze HBV sequences that have the best-predicted on-target and lowest off-target effects. We compared the genomic DNA sequences of different HBV genotypes originating from distinct geographic regions worldwide and identified 9 target sites in highly conserved regions of the HBV genome (based on the ayw strain, Genbank accession number: NC_003977.2) within the S, C, P, and X genes. The sequence alignments of the gRNA target sites on the HBV genome are shown in our recent publication (Suzuki et al., 2021).