CRISPR-based self-cleaving mechanism for controllable gene delivery in human cells

Moore, Richard; Spinhirne, Alec; Lai, Michael J.; Preisser, Samantha; Li, Yang; Kang, Taek Jin; Bleris, Leonidas

doi:10.1093/nar/gku1326

Cited by 49 publications

(39 citation statements)

References 29 publications

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“…Using the proposed methodology, individual nodes of a network can be perturbed from their steady-state using transcriptional or posttranscriptional inhibitors [e.g., TALEs/CRISPR (37,38) or siRNAs]. The pre-and postperturbation steady states can be measured at the mRNA or protein levels, and fed into MRA to predict divergent LRC and accordingly the network structure.…”

Section: Discussionmentioning

confidence: 99%

Discriminating direct and indirect connectivities in biological networks

Kang

Moore

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Reverse engineering of biological pathways involves an iterative process between experiments, data processing, and theoretical analysis. Despite concurrent advances in quality and quantity of data as well as computing resources and algorithms, difficulties in deciphering direct and indirect network connections are prevalent. Here, we adopt the notions of abstraction, emulation, benchmarking, and validation in the context of discovering features specific to this family of connectivities. After subjecting benchmark synthetic circuits to perturbations, we inferred the network connections using a combination of nonparametric single-cell data resampling and modular response analysis. Intriguingly, we discovered that recovered weights of specific network edges undergo divergent shifts under differential perturbations, and that the particular behavior is markedly different between topologies. Our results point to a conceptual advance for reverse engineering beyond weight inference. Investigating topological changes under differential perturbations may address the longstanding problem of discriminating direct and indirect connectivities in biological networks. A focal point of systems biology is the reverse engineering of gene regulatory networks (1-5). The methods have shifted from intuitive inference of local connectivities to comprehensive analysis of large networks, involving heterogeneous data sets from high-throughput experiments and complex theoretical tools (6-10). Despite significant advances, a fundamental reverse engineering bottleneck is the ability to discriminate between direct and indirect connections. In a simple case, assuming three nodes in a cascade formulation, where an input node is activating an intermediary node which in turn is activating an output node, a reverse engineering algorithm may infer an activating edge from the input node to the output, even though there is no direct biological interaction.Unfortunately, the limitation in correctly distinguishing the effects stemming from indirect connectivities is pervasive (11-13) and justifies the urgent need for new and reliable methods to eliminate spurious edges. Importantly, remedies to address this problem should not further muddle the interpretation by removing true network edges (14). A number of theoretical approaches have been proposed to overcome this hurdle (4, 15-18), but the ability to experimentally verify the conclusions drawn by reverse engineering tools remains paramount.The majority of efforts to address the verification issue adopt in silico benchmark suites that are based on biological pathway approximations (19). Although these models do include a number of commonly observed topologies and have provided significant insights, they do not fully capture the complexity of the biological realm and the associated heterogeneity and intrinsic variability. On the other hand, engineered synthetic gene circuits are orthogonal to the endogenous pathways yet operate within the natural cellular context using the available resources. Thus, sy...

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Section: Discussionmentioning

confidence: 99%

Discriminating direct and indirect connectivities in biological networks

Kang

Moore

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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“…So far, AAV-mediated nuclease expression has been demonstrated to be successful in several tissue types, including liver and brain 48,127 . In the case of viral mediated Cas9 delivery, which may result in constitutive expression of nuclease proteins and cause genome instability and toxicity, self-cleaving mechanisms may be used to inactivate the nuclease transgene on the delivery vector 128 .…”

Section: Challenges To Clinical Translationmentioning

confidence: 99%

Therapeutic genome editing: prospects and challenges

Cox

Platt

Zhang

2015

Nat Med

1,140

875

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Recent advances in the development of genome editing technologies based on programmable nucleases have significantly improved our ability to make precise changes in the genomes of eukaryotic cells. Genome editing is already broadening our ability to elucidate the contribution of genetics to disease by facilitating the creation of more accurate cellular and animal models of pathological processes. A particularly tantalizing application of programmable nucleases is the potential to directly correct genetic mutations in affected tissues and cells to treat diseases that are refractory to traditional therapies. Here we discuss current progress towards developing programmable nuclease-based therapies as well as future prospects and challenges.

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“…Alternatively, these size constraints can be sidestepped by creating a shorter Cas9 ortholog. In addition to size incompatibility, viral vectors have the drawback of possible constitutive nuclease expression, resulting in cell toxicity and genomic instability [145]. …”

Section: Part 3 Future Perspectivesmentioning

confidence: 99%

CRISPR-Cas9: a promising tool for gene editing on induced pluripotent stem cells

Kim

Kang

2017

Korean J Intern Med

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Recent advances in genome editing with programmable nucleases have opened up new avenues for multiple applications, from basic research to clinical therapy. The ease of use of the technology—and particularly clustered regularly interspaced short palindromic repeats (CRISPR)—will allow us to improve our understanding of genomic variation in disease processes via cellular and animal models. Here, we highlight the progress made in correcting gene mutations in monogenic hereditary disorders and discuss various CRISPR-associated applications, such as cancer research, synthetic biology, and gene therapy using induced pluripotent stem cells. The challenges, ethical issues, and future prospects of CRISPR-based systems for human research are also discussed.

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CRISPR-based self-cleaving mechanism for controllable gene delivery in human cells

Cited by 49 publications

References 29 publications

Discriminating direct and indirect connectivities in biological networks

Discriminating direct and indirect connectivities in biological networks

Therapeutic genome editing: prospects and challenges

CRISPR-Cas9: a promising tool for gene editing on induced pluripotent stem cells

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