biostudies-literatureUnknownMatsuzaki SHiroshima UniversityJapan Society for the Promotion of ScienceA variety of CRISPR-Cas9-based gene editing technologies have been developed, including gene insertion and gene replacement, and applied to the study and treatment of diseases. While numerous studies have been conducted to improve the efficiency of gene insertion and to expand the system in various ways, there have been relatively few reports on gene replacement technology; therefore, further improvements are still needed in this context. Here, we developed the REMOVER-PITCh system to establish an efficient long-range gene replacement method and demonstrated its utility at two genomic loci in human cultured cells. REMOVER-PITCh depends on microhomology-assisted gene insertion technology called PITCh with highly multiplexed CRISPR-Cas9. First, we achieved gene replacement of about 20-kb GUSIn vitro cellular & developmental biology. Animalhttps://www.ebi.ac.uk/biostudies/studies/S-EPMC11297102biostudies-literatureREMOVER-PITCh: microhomology-assisted long-range gene replacement with highly multiplexed CRISPR-Cas9.PMC1129710217H0140921K06137Matsuzaki SSakuma TYamamoto TfalseREMOVER-PITCh: microhomology-assisted long-range gene replacement with highly multiplexed CRISPR-Cas9.A variety of CRISPR-Cas9-based gene editing technologies have been developed, including gene insertion and gene replacement, and applied to the study and treatment of diseases. While numerous studies have been conducted to improve the efficiency of gene insertion and to expand the system in various ways, there have been relatively few reports on gene replacement technology; therefore, further improvements are still needed in this context. Here, we developed the REMOVER-PITCh system to establish an efficient long-range gene replacement method and demonstrated its utility at two genomic loci in human cultured cells. REMOVER-PITCh depends on microhomology-assisted gene insertion technology called PITCh with highly multiplexed CRISPR-Cas9. First, we achieved gene replacement of about 20-kb GUS2024-01-01T00:00:00Z2024 Feb2025-04-19T20:47:32.635Z2025-02-19T02:27:27.37ZS-EPMC112971023833488010.1007/s11626-024-00850-1