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Gene editing and CRISPR in the clinic: Current and future perspectives

Bioscience Reports

Hirakawa, Matthew H.; Krishnakumar, Raga K.; Timlin, Jerilyn A.; Carney, James P.; Butler, Kimberly B.

Genome editing technologies, particularly those based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeat DNA sequences)/Cas9 are rapidly progressing into clinical trials. Most clinical use of CRISPR to date has focused on ex vivo gene editing of cells followed by their re-introduction back into the patient. The ex vivo editing approach is highly effective for many disease states, including cancers and sickle cell disease, but ideally genome editing would also be applied to diseases which require cell modification in vivo. However, in vivo use of CRISPR technologies can be confounded by problems such as off-target editing, inefficient or off-target delivery, and stimulation of counterproductive immune responses. Current research addressing these issues may provide new opportunities for use of CRISPR in the clinical space. In this review, we examine the current status and scientific basis of clinical trials featuring ZFNs, TALENs, and CRISPR-based genome editing, the known limitations of CRISPR use in humans, and the rapidly developing CRISPR engineering space that should lay the groundwork for further translation to clinical application.

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TYPE Journal Article YEAR 2020
ScopusOSTIDOI

Use of anti-CRISPR protein AcrIIA4 as a capture ligand for CRISPR/Cas9 detection

Biosensors and Bioelectronics

Johnston, Robert K.; Seamon, Kyle J.; Saada, Edwin A.; Podlevsky, Joshua P.; Branda, Steven B.; Timlin, Jerilyn A.; Harper, Jason C.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) ribonucleoprotein (RNP) complex is an RNA-guided DNA-nuclease that is part of the bacterial adaptive immune system. CRISPR/Cas9 RNP has been adapted for targeted genome editing within cells and whole organisms with new applications vastly outpacing detection and quantification of gene-editing reagents. Detection of the CRISPR/Cas9 RNP within biological samples is critical for assessing gene-editing reagent delivery efficiency, retention, persistence, and distribution within living organisms. Conventional detection methods are effective, yet the expense and lack of scalability for antibody-based affinity reagents limit these techniques for clinical and/or field settings. This necessitates the development of low cost, scalable CRISPR/Cas9 RNP affinity reagents as alternatives or augments to antibodies. Herein, we report the development of the Streptococcus pyogenes anti-CRISPR/Cas9 protein, AcrIIA4, as a novel affinity reagent. An engineered cysteine linker enables covalent immobilization of AcrIIA4 onto glassy carbon electrodes functionalized via aryl diazonium chemistry for detection of CRISPR/Cas9 RNP by electrochemical, fluorescent, and colorimetric methods. Electrochemical measurements achieve a detection of 280 pM RNP in reaction buffer and 8 nM RNP in biologically representative conditions. Our results demonstrate the ability of anti-CRISPR proteins to serve as robust, specific, flexible, and economical recognition elements in biosensing/quantification devices for CRISPR/Cas9 RNP.

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TYPE Journal Article YEAR 2019
ScopusOSTIDOI

Imaging effectiveness calculator for non-design microscope samples

Applied Optics

Anthony, Stephen M.; Miller, Philip R.; Timlin, Jerilyn A.; Polsky, Ronen P.

When attempting to integrate single-molecule fluorescence microscopy with microfabricated devices such as microfluidic channels, fabrication constraints may prevent using traditional coverslips. Instead, the fabricated devices may require imaging through material with a different thickness or index of refraction. Altering either can easily reduce the quality of the image formation (measured by the Strehl ratio) by a factor of 2 or more, reducing the signal-to-noise ratio accordingly. In such cases, successful detection of single-molecule fluorescence may prove difficult or impossible. Here we provide software to calculate the effect of non-design materials upon the Strehl ratio or ensquared energy and explore the impact of common materials used in microfabrication.

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TYPE Journal Article YEAR 2019
ScopusOSTIDOI

Posters for AA/CE Reception

Kuether, Robert J.; Allensworth, Brooke M.; Backer, Adam B.; Chen, Elton Y.; Dingreville, Remi P.; Forrest, Eric C.; Knepper, Robert; Tappan, Alexander S.; Marquez, Michael P.; Vasiliauskas, Jonathan G.; Rupper, Stephen G.; Grant, Michael J.; Atencio, Lauren C.; Hipple, Tyler J.; Maes, Danae M.; Timlin, Jerilyn A.; Ma, Tian J.; Garcia, Rudy J.; Danford, Forest L.; Patrizi, Laura P.; Galasso, Jennifer G.; Draelos, Timothy J.; Gunda, Thushara G.; Venezuela, Otoniel V.; Brooks, Wesley A.; Anthony, Stephen M.; Carson, Bryan C.; Reeves, Michael J.; Roach, Matthew R.; Maines, Erin M.; Lavin, Judith M.; Whetten, Shaun R.; Swiler, Laura P.

Abstract not provided.

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TYPE Presentation YEAR 2018
OSTI
Results 1–25 of 263
Results 1–25 of 263