Targeting transthyretin by one Cas9 variant with superfidelity and broad compatibility

Amyloid transthyretin (ATTR) amyloidosis is a fatal disease caused by the accumulation of misfolded transthyretin proteins. Although knocking down the <i>TTR</i> gene by CRISPR-Cas9 represents a promising strategy for treating ATTR amyloidosis, its efficiency and safety remain to be further investigated. Here, we report a systematic investigation of SpCas9-based <i>TTR</i> editing. Besides the target site, wild-type SpCas9 and the reported variants induced extensive off-target edits. To improve the fidelity, we performed structural analysis and designed a series of SpCas9 variants. Studies demonstrated that SpCas9-Mut5 is an ultrahigh-fidelity variant, which induces extremely low levels of off-target edits and translocations without substantial impairment of on-target editing activity. SpCas9-Mut5 is compatible with the adenine base editor (ABE) system, markedly reducing off-target edits and narrowing the editing window. In conclusion, our study suggests that SpCas9-Mut5 is an excellent candidate for <i>TTR</i> gene editing. Besides ATTR amyloidosis, SpCas9-Mut5 and its derivative ABE could be widely used in the treatment of other diseases.