CRISPR-Cas9 for in vivo Gene Therapy: Promise and Hurdles

Dai, Wei-Jing; Zhu, Liyao; Yan, Zhongyi; Xu, Yong; Wang, Qilong; Lu, Xiao‐Jie

doi:10.1038/mtna.2016.58

Cited by 123 publications

(98 citation statements)

References 40 publications

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“…Conversely, all other sgHpd1 and sgHpd2 treated mice achieved indels with frequencies ranging from 35% to 60% (Figure 2E). This level of gene inactivation likely reflects not only the initial editing events but also the competitive expansion of edited cell lineages (after NTBC withdrawal) at the expense of their unedited counterparts (6,46,55). Liver histology revealed that liver damage is substantially less severe in the sgHpd1 and sgHpd2 treated mice compared to Fah mut/mut mice injected with PBS, as indicated by the smaller numbers of multinucleated hepatocytes compared to PBS injected mice (Figure 2F).…”

Section: This Provides Us With a Context In Which To Test The Efficacmentioning

confidence: 95%

All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo

Ibraheim¹,

Song²,

Mir³

et al. 2018

Preprint

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Clustered, regularly interspaced, short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) have recently opened a new avenue for gene therapy. Cas9 nuclease guided by a single-guide RNA (sgRNA) has been extensively used for genome editing. Currently, three Cas9 orthologs have been adapted for in vivo genome engineering applications: SpyCas9, SauCas9 and CjeCas9. However, additional in vivo editing platforms are needed, in part to enable a greater range of sequences to be accessed via viral vectors. Here, we present an additional in vivo editing platform using Neisseria meningitidis Cas9 (NmeCas9). NmeCas9 is compact, edits with high accuracy, and possesses a distinct PAM, making it an excellent candidate for safe gene therapy applications. Here we demonstrate that NmeCas9 can be used to target the Pcsk9 and Hpd genes in mice. Using tail vein hydrodynamic-based delivery of NmeCas9 plasmid to target the Hpd gene, we successfully reprogrammed the tyrosine degradation pathway in Hereditary Tyrosinemia Type I mice. More importantly, we delivered NmeCas9 with its single-guide RNA in a single recombinant adeno-associated vector (rAAV) to target Pcsk9, resulting in lower cholesterol levels in mice. This all-in-one vector yielded >35% gene modification after two weeks of vector administration, with minimal off-target cleavage in vivo. Our findings indicate that NmeCas9 can facilitate future efforts to correct disease-causing mutations by expanding the targeting scope of RNA-guided nucleases.3 SignificanceWe and others have shown that NmeCas9 is an efficient Cas9 nuclease with a high degree of specificity in cultured human cells. NmeCas9 has a unique PAM that expands the targeting range of Cas9-based genome editing. Since NmeCas9 is more compact than SpyCas9, its delivery with its guide-RNA in a single recombinant adeno-associated virus becomes a viable option. Furthermore, NmeCas9 has an unusually low off-target profile. These characteristics make NmeCas9 an ideal candidate for in vivo genome editing, including therapeutic gene knockouts, as we demonstrate in the mouse liver. We anticipate that successful in vivo delivery of this accurate and effective Cas9 can advance therapeutic editing in humans.

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Section: This Provides Us With a Context In Which To Test The Efficacmentioning

confidence: 95%

All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo

Ibraheim¹,

Song²,

Mir³

et al. 2018

Preprint

View full text Add to dashboard Cite

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“…For example, recent reports have shown that CRISPR-Cas9 components are able to in vivo correct a gene mutation in mouse models of Duchenne muscular dystrophy and rescue the disease phenotype (Long et al, 2016;Nelson et al, 2016;Tabebordbar et al, 2016). Although this is a significant step forward on the way to bring CRISPR-Cas9 to the clinic, there are several major difficulties there, such as off target effects, poor fitness of edited cells or immunogenicity of CRISPR-Cas9 components (reviewed in Dai et al, 2016). Application of the system in the area of antisense RNAs might be even more challenging than that, as cis-NATs share genomic regions with their mate genes, making it harder to avoid the off target effect.…”

Section: Bace1-as: Alzheimer's Diseasementioning

confidence: 99%

Natural antisense transcripts in diseases: From modes of action to targeted therapies

et al. 2018

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Antisense transcription is a widespread phenomenon in mammalian genomes, leading to production of RNAs molecules referred to as natural antisense transcripts (NATs). NATs apply diverse transcriptional and post‐transcriptional regulatory mechanisms to carry out a wide variety of biological roles that are important for the normal functioning of living cells, but their dysfunctions can be associated with human diseases. In this review, we attempt to provide a molecular basis for the involvement of NATs in the etiology of human disorders such as cancers and neurodegenerative and cardiovascular diseases. We also discuss the pros and cons of oligonucleotide‐based therapies targeted against NATs, and we comment on state‐of‐the‐art progress in this promising area of clinical research. WIREs RNA 2018, 9:e1461. doi: 10.1002/wrna.1461This article is categorized under: RNA in Disease and Development > RNA in DiseaseRegulatory RNAs/RNAi/Riboswitches > Regulatory RNAsRNA Interactions with Proteins and Other Molecules > Small Molecule–RNA Interactions

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“…CRISPR-Cas nucleases make double-strand breaks (DSBs) at the intended target site but can also introduce unwanted mutations at off-target sites within the genome. To bring CRISPR into the clinic, accurate characterization of on- and off-target nuclease activity in any target cell population is critical ( 1 ).…”

Section: Introductionmentioning

confidence: 99%

Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq

Wienert

Wyman

Richardson

et al. 2018

Preprint

107

172

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21Genome editing using nucleases such as CRISPR-Cas induces programmable DNA damage at a 22 target genomic site but can also affect off-target sites. Here, we develop a powerful, sensitive assay 23 for the unbiased identification of off-target sites that we term DISCOVER-Seq. This approach 24 takes advantage of the recruitment of endogenous DNA repair factors for genome-wide 25 identification of Cas-induced double-strand breaks. One such factor, MRE11, is recruited precisely 26 to double-strand breaks, enabling molecular characterization of nuclease cut sites with single-base 27 resolution. DISCOVER-Seq detects off-targets in cellular models and in vivo upon adenoviral gene 28 editing of mouse livers, paving the way for real-time off-target discovery during therapeutic gene 29 editing. DISCOVER-Seq is furthermore applicable to multiple types of Cas nucleases and provides 30 an unprecedented view of events that precede repair of the affected sites. 31 strengths, they also have certain weaknesses. Naïve prediction algorithms are for the most part 41 based on sequence similarity and currently have limited predictive power with very high false-42 positive rates (10). Assays that induce DSBs in vitro, such as Digenome-Seq (5), CIRCLE-Seq (6) 43 and SITE-Seq (7), have high sensitivity but dramatically under-or overestimate the number of 44 target sites that are actually modified in cellular models or in vivo (11). Nuclease concentration 45 within the cell (7), delivery method (ribonucleoprotein (RNP) vs. plasmid) (7, 12, 13) as well as 46 more complex cellular properties such as chromatin accessibility (14, 15) have been shown to 47

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CRISPR-Cas9 for in vivo Gene Therapy: Promise and Hurdles

Cited by 123 publications

References 40 publications

All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo

All-in-One Adeno-associated Virus Delivery and Genome Editing by Neisseria meningitidis Cas9 in vivo

Natural antisense transcripts in diseases: From modes of action to targeted therapies

Unbiased detection of CRISPR off-targets in vivo using DISCOVER-Seq

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