Multiplex gene editing to promote cell survival using low-pH clustered regularly interspaced short palindromic repeats activation (CRISPRa) gene perturbation

Levis, Hunter; Weston, Jacob; Austin, Brooke; Larsen, Bente; Ginley-Hidinger, Matthew; Gullbrand, Sarah E.; Lawrence, Brandon D.; Bowles, Robby D.

doi:10.1016/j.jcyt.2023.05.001

Cited by 3 publications

(1 citation statement)

References 52 publications

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“…Recently, clustered regularly interspaced short palindromic repeats (CRISPR)-guided gene modulation systems have emerged as powerful tools for profiling complex gene regulatory networks 9 , gene function 10 , improving cell survival 11 , and directing stem cell fate 12 . These systems comprise a single-guide RNA (gRNA) that targets the system to a gene of interest, and a nuclease-null mutant Cas9 (dCas9) that is fused to an effector molecule (VPR 13 or KRAB 14 ) for targeted upregulation (CRISPR-activation) or downregulation (CRISPR-interference).…”

Section: Introductionmentioning

confidence: 99%

ZNF865 Regulates Senescence and Cell Cycle for Applications to Cell Engineering and Gene Therapy

Levis,

Lewis,

Stockham

et al. 2023

Preprint

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Zinc finger (ZNF) proteins represent the largest group of regulatory proteins within eukaryotic genomes. However, despite their broad regulatory function, the majority of ZNF protein function remains unknown. Recently, we discovered ZNF865, which has no in-depth publications and has not been functionally characterized. Utilizing CRISPR-guided gene modulation, we show that ZNF865 regulates key cellular and molecular processes associated with healthy cell function by primarily regulating cellular senescence, cell cycle progression, and protein processing. As a result, regulating this gene acts as a primary titratable regulator of cell activity, and we demonstrate the potential of targeted ZNF865 regulation as a tool to control senescence and protein production in multiple clinically relevant cell types for cell engineering/tissue engineering/gene therapy applications. We demonstrate its ability to rescue human senescent cell populations, boost T-cell activity, and dramatically deposit more cartilaginous tissue in a whole organ tissue-engineered intervertebral disc. Overall, we present novel biology and regulatory mechanisms of senescence and cell cycle that were previously unknown and display the power of CRISPR-cell engineering to enhance cell engineering strategies treating disease.

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Section: Introductionmentioning

confidence: 99%

ZNF865 Regulates Senescence and Cell Cycle for Applications to Cell Engineering and Gene Therapy

Levis,

Lewis,

Stockham

et al. 2023

Preprint

Self Cite

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pH: A major player in degenerative intervertebral disks

Trone,

Stover,

Almarza

et al. 2024

JOR Spine

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Chronic lower back pain is the leading cause of disability worldwide, generating a socioeconomic cost of over $100 billion annually in the United States. Among the prominent causes of low back pain (LBP) is degeneration of the intervertebral disk (IVD), a condition known as degenerative disk disease (DDD). Despite the prevalence of DDD and multiple studies demonstrating its relationship with LBP, the mechanisms by which it contributes to pain remain unknown. Previous studies have identified potential causes for this pain, such as extracellular matrix (ECM) breakdown, changes in biomechanics, and pro‐inflammatory signals. Possible pain treatments targeting these factors have been developed but with limited effects. However, low pH in DDD is a potential pain generator whose role has largely been unexplored and underappreciated. This review highlights hyperacidity's effects on the IVD, such as catabolism of disk cells and ECM, neoinnervation, altered mechanical signaling, and expression of pro‐inflammatory cytokines and ion channels. This review aims to discuss what is known about the contributions of acidity to DDD pain, identify the knowledge gaps on this topic, and propose what research can be conducted to fill these gaps. We must better understand the underlying mechanisms of DDD and the interaction between hyperacidity and nociception to develop better therapeutics for this disease.

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Targeted CRISPR regulation of ZNF865 enhances stem cell cartilage deposition, tissue maturation rates, and mechanical properties in engineered intervertebral discs

Levis,

Lewis,

Fainor

et al. 2025

Acta Biomaterialia

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Multiplex gene editing to promote cell survival using low-pH clustered regularly interspaced short palindromic repeats activation (CRISPRa) gene perturbation

Cited by 3 publications

References 52 publications

ZNF865 Regulates Senescence and Cell Cycle for Applications to Cell Engineering and Gene Therapy

ZNF865 Regulates Senescence and Cell Cycle for Applications to Cell Engineering and Gene Therapy

pH: A major player in degenerative intervertebral disks

Targeted CRISPR regulation of ZNF865 enhances stem cell cartilage deposition, tissue maturation rates, and mechanical properties in engineered intervertebral discs

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