A multifunctional AAV–CRISPR–Cas9 and its host response

Chew, Wei Leong; Tabebordbar, Mohammadsharif; Cheng, Jason; Mali, Prashant; Wu, Elizabeth Y; Ng, Alex H. M.; Zhu, Kexian; Wagers, Amy J.; Church, George M.

doi:10.1038/nmeth.3993

Cited by 516 publications

(444 citation statements)

References 62 publications

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“…A split Cas9 AAV system, which relies on the trans -splicing machinery, was recently described as a way of circumventing the capacity limitation of AAV vectors (Chew et al, 2016). However, it is unclear whether modest levels of in vivo TGA achievable with this split system are sufficient to induce phenotypic change.…”

Section: Discussionmentioning

confidence: 99%

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In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-epigenetic Modulation

Liao

Hatanaka

Araoka

et al. 2017

Cell

342

261

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SUMMARY Current genome-editing systems generally rely on the creation of DNA double-strand breaks (DSBs). This may limit their utility in clinical therapies, as unwanted mutations caused by DSBs can have deleterious effects. The CRISPR/Cas9 system has recently been repurposed to enable target gene activation, allowing regulation of endogenous gene expression without creating DSBs. However, in vivo implementation of this gain-of-function system has proven difficult. Here we report a robust system for in vivo activation of endogenous target genes through trans-epigenetic remodeling. The system relies on recruitment of Cas9 and transcriptional activation complexes to target loci by modified single guide RNAs. As proof-of-concept, we used this technology to treat several mouse models of human diseases. Results demonstrate that CRISPR/Cas9-mediated target gene activation can be achieved in vivo, leading to observable phenotypic changes, and amelioration of disease symptoms. This establishes new avenues for developing targeted epigenetic therapies against human diseases.

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Section: Discussionmentioning

confidence: 99%

“…In contrast, our in vivo CRISPR/Cas9 TGA system did not generate DNA breaks. Furthermore, it has recently been shown that AAV-mediated delivery of CRISPR-Cas9 does not induce extensive cellular damage in vivo (Chew et al, 2016). Despite these advantages, further studies are needed before this strategy can be applied in the clinic.…”

Section: Discussionmentioning

confidence: 99%

In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-epigenetic Modulation

Liao

Hatanaka

Araoka

et al. 2017

Cell

342

261

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“…However, plasmid DNA delivery is often inefficient, especially in vivo, and can cause integration of small plasmid fragments degraded by endogenous nucleases at on-target and offtarget sites in the genome ). Viral delivery of Cas9 can be highly efficient in vivo (Ran et al 2015;Long et al 2016;Nelson et al 2016;Tabebordbar et al 2016), but may be hampered by antibodies or T cells induced against the protein (Shankar et al 2007;Calcedo et al 2015;Chew et al 2016). We and others have shown that preassembled Cas9 ribonucleoproteins (RNPs) can be delivered to human primary and stem cells and mice to modify target genes Schumann et al 2015;Zuris et al 2015).…”

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confidence: 99%

Genome surgery using Cas9 ribonucleoproteins for the treatment of age-related macular degeneration

et al. 2017

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RNA-guided genome surgery using CRISPR-Cas9 nucleases has shown promise for the treatment of diverse genetic diseases. Yet, the potential of such nucleases for therapeutic applications in nongenetic diseases is largely unexplored. Here, we focus on age-related macular degeneration (AMD), a leading cause of blindness in adults, which is associated with retinal overexpression of, rather than mutations in, the VEGFA gene. Subretinal injection of preassembled, Vegfa gene-specific Cas9 ribonucleoproteins (RNPs) into the adult mouse eye gave rise to mutagenesis at the target site in the retinal pigment epithelium. Furthermore, Cas9 RNPs effectively reduced the area of laser-induced choroidal neovascularization (CNV) in a mouse model of AMD. Genome-wide profiling of Cas9 off-target effects via Digenome-seq showed that off-target mutations were rarely induced in the human genome. Because Cas9 RNPs can function immediately after in vivo delivery and are rapidly degraded by endogenous proteases, their activities are unlikely to be hampered by antibody-and cell-mediated adaptive immune systems. Our results demonstrate that in vivo genome editing with Cas9 RNPs has the potential for the local treatment for nongenetic degenerative diseases, expanding the scope of RNA-guided genome surgery to a new dimension.

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“…Indeed, the development of antibodies against Cas9 has been documented in AAV-treated mice. 62 One potential solution is the inclusion in the vector of an additional guide RNA that targets the Cas9 gene itself, resulting in cleavage of the vector that self-limits the duration of expression. 63 An entirely different solution would be the use of lipid nanoparticles to deliver Cas9 messenger RNA into hepatocytes, relieving the size restriction imposed by AAV, as well as providing Cas9 in a form that is rapidly degraded in cells.…”

Section: Next Stepsmentioning

confidence: 99%

CRISPR-Cas9 Genome Editing for Treatment of Atherogenic Dyslipidemia

Chadwick

Musunuru

2018

ATVB

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Although human genetics has resulted in the identification of novel lipid-related genes that can be targeted for the prevention of atherosclerotic vascular disease, medications targeting these genes or their protein products have short-term effects and require frequent administration during the course of the lifetime for maximal benefit. Genome-editing technologies, such as CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR–associated 9) have the potential to permanently alter genes in the body and produce long-term and even lifelong protection against atherosclerosis. In this review, we discuss recent advances in genome-editing technologies and early proof-of-concept studies of somatic in vivo genome editing in mice that highlight the potential of genome editing to target disease-related genes in patients, which would establish a novel therapeutic paradigm for atherosclerosis.

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A multifunctional AAV–CRISPR–Cas9 and its host response

Cited by 516 publications

References 62 publications

In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-epigenetic Modulation

In Vivo Target Gene Activation via CRISPR/Cas9-Mediated Trans-epigenetic Modulation

Genome surgery using Cas9 ribonucleoproteins for the treatment of age-related macular degeneration

CRISPR-Cas9 Genome Editing for Treatment of Atherogenic Dyslipidemia

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