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Hu, Johnny

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Hu

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Johnny

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Hu, Johnny

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2014 - 20183

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Author's Publications: search.filters.isAuthorOfPublication.ac36c014-b207-4278-a09d-5389e6b499aa

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Now showing 1 - 3 of 3
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    Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents
    (2018) Gao, Xue; Tao, Yong; Lamas, Veronica; Huang, Mingqian; Yeh, Wei-Hsi; Pan, Bifeng; Hu, Yu-Juan; Hu, Johnny; Thompson, David; Shu, Yilai; Li, Yamin; Wang, Hongyang; Yang, Shiming; Xu, Qiaobing; Polley, Daniel; Liberman, M.; Kong, Wei-Jia; Holt, Jeffrey; Chen, Zheng-Yi; Liu, David
    Although genetic factors contribute to almost half of all deafness cases, treatment options for genetic deafness are limited1–5. We developed a genome editing approach to target a dominantly inherited form of genetic deafness. Here we show that cationic lipid-mediated in vivo delivery of Cas9:guide RNA complexes can ameliorate hearing loss in a mouse model of human genetic deafness. We designed and validated in vitro and in primary fibroblasts genome editing agents that preferentially disrupt the dominant deafness-associated allele in the Tmc1 (transmembrane channel-like 1) Beethoven (Bth) mouse model, even though the mutant Bth allele differs from the wild-type allele at only a single base pair. Injection of Cas9:guide RNA:lipid complexes targeting the Bth allele into the cochlea of neonatal Bth/+ mice substantially reduced progressive hearing loss. We observed higher hair cell survival rates and lower auditory brainstem response (ABR) thresholds in injected ears compared with uninjected ears or ears injected with complexes that target an unrelated gene. Enhanced acoustic reflex responses were observed among injected compared to uninjected Bth/+ animals. These findings suggest protein:RNA complex delivery of target gene-disrupting agents in vivo as a potential strategy for the treatment of some autosomal dominant hearing loss diseases.
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    Efficient Delivery of Genome-Editing Proteins In Vitro and In Vivo
    (2014) Zuris, John; Thompson, David; Shu, Yilai; Guilinger, John P.; Bessen, Jeffrey; Hu, Johnny; Maeder, Morgan L.; Joung, J. Keith; Chen, Zheng-Yi; Liu, David
    Efficient intracellular delivery of proteins is needed to fully realize the potential of protein therapeutics. Current methods of protein delivery commonly suffer from low tolerance for serum, poor endosomal escape, and limited in vivo efficacy. Here we report that common cationic lipid nucleic acid transfection reagents can potently deliver proteins that are fused to negatively supercharged proteins, that contain natural anionic domains, or that natively bind to anionic nucleic acids. This approach mediates the potent delivery of nM concentrations of Cre recombinase, TALE- and Cas9-based transcriptional activators, and Cas9:sgRNA nuclease complexes into cultured human cells in media containing 10% serum. Delivery of Cas9:sgRNA complexes resulted in up to 80% genome modification with substantially higher specificity compared to DNA transfection. This approach also mediated efficient delivery of Cre recombinase and Cas9:sgRNA complexes into the mouse inner ear in vivo, achieving 90% Cre-mediated recombination and 20% Cas9-mediated genome modification in hair cells.
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    A programmable Cas9-serine recombinase fusion protein that operates on DNA sequences in mammalian cells
    (Oxford University Press, 2016) Chaikind, Brian; Bessen, Jeffrey; Thompson, David; Hu, Johnny; Liu, David
    We describe the development of ‘recCas9’, an RNA-programmed small serine recombinase that functions in mammalian cells. We fused a catalytically inactive dCas9 to the catalytic domain of Gin recombinase using an optimized fusion architecture. The resulting recCas9 system recombines DNA sites containing a minimal recombinase core site flanked by guide RNA-specified sequences. We show that these recombinases can operate on DNA sites in mammalian cells identical to genomic loci naturally found in the human genome in a manner that is dependent on the guide RNA sequences. DNA sequencing reveals that recCas9 catalyzes guide RNA-dependent recombination in human cells with an efficiency as high as 32% on plasmid substrates. Finally, we demonstrate that recCas9 expressed in human cells can catalyze in situ deletion between two genomic sites. Because recCas9 directly catalyzes recombination, it generates virtually no detectable indels or other stochastic DNA modification products. This work represents a step toward programmable, scarless genome editing in unmodified cells that is independent of endogenous cellular machinery or cell state. Current and future generations of recCas9 may facilitate targeted agricultural breeding, or the study and treatment of human genetic diseases.