Saeko Ishida, Yusuke Sato, Keisuke Chosa, Eri Ezawa, Yuko Yamauchi, Masaaki Oyama, Hiroko Kozuka-Hata, Rina Ito, Rikako Sato, Masatoshi Maeki, Tomo-o Ishikawa, Kenichi Yamamura, Kohei Takeshita, Kensuke Yamaguchi, Yuta Kochi, Fumitaka Hashiya, Yiwei Liu, Naoko Abe, Hiroshi Abe, Yoshiki Sekijima, Kazuto Yoshimi, Tomoji Mashimo
CRISPR-Cas3 represents a mechanistically distinct genome-editing system compared to Cas9 that generates long-range deletions rather than small indels, thereby reducing the risk of residual protein function from in-frame mutations. Here we evaluated CRISPR-Cas3 to correct mutations in the TTR gene causing transthyretin amyloidosis, a systemic proteinopathy where loss of mutant TTR in the liver offers therapeutic benefit. Through CRISPR RNA optimization we achieved 58.9% ± 0.5% editing at the TTR locus in vitro, inducing large deletions that abolished TTR expression. Cas3 generated mostly directional deletions up to 75 kb without reproducible off-target mutations, in contrast to Cas9, which induced indels at several off-target sites. In vivo, a single lipid-nanoparticle-based treatment achieved 48.7% ± 1.1% hepatic editing and reduced serum TTR levels by 80.1% ± 4.6%. Deletion size was limited to 21 kb. In TTR exon-humanized mice, Cas3 editing reduced serum TTR without in-frame mutations and attenuated macrophage-associated TTR deposition. These findings highlight Cas3 as an efficient and distinct sytem for in vivo genome editing.