In vivo genome editing via CRISPR/Cas9 mediated homology-independent targeted integration

By Keiichiro Suzuki, Yuji Tsunekawa, R. Hernández-Benítez, Jun Wu, Jie Zhu, Euiseok J. Kim, Fumiyuki Hatanaka, Mako Yamamoto, T. Araoka, Zhe Li and 25 more. Nature, 2016

1,069 citations recorded by the source corpus.

Targeted genome editing via engineered nucleases is an exciting area of biomedical research and holds potential for clinical applications. Despite rapid advances in the field, in vivo targeted transgene integration is still infeasible because current tools are inefficient, especially for non-dividing cells, which compose most adult tissues. This poses a barrier for uncovering fundamental biological principles and developing treatments for a broad range of genetic disorders. Based on clustered regularly interspaced short palindromic repeat/Cas9 (CRISPR/Cas9) technology, here we devise a homology-independent targeted integration (HITI) strategy, which allows for robust DNA knock-in in both dividing and non-dividing cells in vitro and, more importantly, in vivo (for example, in neurons of postnatal mammals). As a proof of concept of its therapeutic potential, we demonstrate the efficacy of HITI in improving visual function using a rat model of the retinal degeneration condition retinitis pigmentosa. The HITI method presented here establishes new avenues for basic research and targeted gene therapies.

Subfields: Cellular and Molecular Neuroscience, Molecular Biology

View Paper: https://www.semanticscholar.org/paper/a533dd1f9a21efc753a95ba6f7fe2a27036ea639

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Citations in the corpus

CRISPR-Cas12a: Functional overview and applications (2020, background): “[65] Suzuki K, Tsunekawa Y, Hernandez-Benitez R, Wu J, Zhu J, Kim EJ, et al.”
Engineered AAVs for efficient noninvasive gene delivery to the central and peripheral nervous systems (2017, background): “Although both systems are viable, we focused on the tTA-TRE system because (1) the short (417 bp) TRE promoter is compatible with longer transgenes, and (2) the positive-feedback loop in the AAV-tTA vector provides high levels of expression (Supplementary Fig.”
Identification of Pre-Existing Adaptive Immunity to Cas9 Proteins in Humans (2018, positive): “Much of the effort to treat disease in vivo has used Cas9 delivery systems that express the Cas9 protein for prolonged periods of time, such as through a non-integrating AAV vector or even through nanoparticle delivery of mRNA to non-dividing hepatocytes (8, 10-12).”
RNA-guided DNA insertion with CRISPR-associated transposases (2019, background): “Although it is possible to achieve precise integration of new DNA following Cas9 cleavage either through homologous recombination (10) or nonhomologous end-joining (11, 12), these processes are inefficient and vary greatly depending on cell type.”