Therapeutic validation of MMR-associated genetic modifiers in a human ex vivo model of Huntington disease DOI Creative Commons
Ross Ferguson,

Robert Goold,

Lucy A. Coupland

et al.

The American Journal of Human Genetics, Journal Year: 2024, Volume and Issue: 111(6), P. 1165 - 1183

Published: May 14, 2024

The pathological huntingtin (HTT) trinucleotide repeat underlying Huntington disease (HD) continues to expand throughout life. Repeat length correlates both with earlier age at onset (AaO) and faster progression, making slowing its expansion an attractive therapeutic approach. Genome-wide association studies have identified candidate variants associated altered AaO many found in DNA mismatch repair (MMR)-associated genes. We examine whether lowering expression of these genes affects the rate human ex vivo models using HD iPSCs iPSC-derived striatal medium spiny neuron-enriched cultures. generated a stable CRISPR interference iPSC line which we can specifically efficiently lower gene from donor carrying over 125 CAG repeats. Lowering each member MMR complexes MutS (MSH2, MSH3, MSH6), MutL (MLH1, PMS1, PMS2, MLH3), LIG1 resulted characteristic deficiencies. Reduced MSH2, MLH1 slowed largest degree, while either or MLH3 it lesser degree. These effects were recapitulated cultures where factor was lowered. CRISPRi-mediated key levels feasibly achievable by current approaches able effectively slow HTT tract. highlight members family as potential targets pathogenic aim delay progression potentially other disorders exhibiting somatic instability.

Language: Английский

Epigenome editing technologies for discovery and medicine DOI

Sean R. McCutcheon,

Dahlia Rohm,

Nahid Iglesias

et al.

Nature Biotechnology, Journal Year: 2024, Volume and Issue: 42(8), P. 1199 - 1217

Published: July 29, 2024

Language: Английский

Citations

18
Systematic reconstruction of molecular pathway signatures using scalable single-cell perturbation screens DOI
Longda Jiang, Carol Dalgarno, Efthymia Papalexi

et al.

Nature Cell Biology, Journal Year: 2025, Volume and Issue: unknown

Published: Feb. 26, 2025

Language: Английский

Citations

2
Maximizing CRISPRi efficacy and accessibility with dual-sgRNA libraries and optimal effectors DOI Creative Commons
Joseph M. Replogle, Jessica L. Bonnar, Angela N. Pogson

et al.

eLife, Journal Year: 2022, Volume and Issue: 11

Published: Dec. 28, 2022

CRISPR interference (CRISPRi) enables programmable, reversible, and titratable repression of gene expression (knockdown) in mammalian cells. Initial CRISPRi-mediated genetic screens have showcased the potential to address basic questions cell biology, genetics, biotechnology, but wider deployment CRISPRi screening has been constrained by large size single guide RNA (sgRNA) libraries challenges generating models with consistent knockdown. Here, we present next-generation sgRNA effector constructs that enable strong knockdown across models. First, combine empirical selection a dual-sgRNA library design generate an ultra-compact (1-3 elements per gene), highly active library. Next, compare effectors show recently published Zim3-dCas9 provides excellent balance between on-target minimal non-specific effects on growth or transcriptome. Finally, engineer suite lines stable robust Our results publicly available reagents establish best practices for screening.

Language: Английский

Citations

66
Targeted epigenomic editing ameliorates adult anxiety and excessive drinking after adolescent alcohol exposure DOI Creative Commons
John Bohnsack,

Huaibo Zhang,

Gabriela M. Wandling

et al.

Science Advances, Journal Year: 2022, Volume and Issue: 8(18)

Published: May 4, 2022

Adolescent binge drinking is a major risk factor for psychiatric disorders later in life including alcohol use disorder. exposure induces epigenetic reprogramming at the enhancer region of activity-regulated cytoskeleton-associated protein (Arc) immediate-early gene, known as synaptic activity response element (SARE), and decreases Arc expression amygdala both rodents humans. The causal role amygdalar epigenomic regulation SARE adult anxiety after adolescent unknown. Here, we show that dCas9-P300 increases histone acetylation normalizes deficits expression, leading to attenuation excessive rat model exposure. Conversely, dCas9-KRAB repressive methylation SARE, produces control rats. These results demonstrate editing can ameliorate psychopathology

Language: Английский

Citations

48
CRISPR/dCas9 Tools: Epigenetic Mechanism and Application in Gene Transcriptional Regulation DOI Open Access

Ruijie Cai,

Runyu Lv, Xin’e Shi

et al.

International Journal of Molecular Sciences, Journal Year: 2023, Volume and Issue: 24(19), P. 14865 - 14865

Published: Oct. 3, 2023

CRISPR/Cas9-mediated cleavage of DNA, which depends on the endonuclease activity Cas9, has been widely used for gene editing due to its excellent programmability and specificity. However, changes DNA sequence that are mediated by CRISPR/Cas9 affect structures stability genome, may accuracy results. Mutations in RuvC HNH regions Cas9 protein lead inactivation into dCas9 with no activity. Despite loss activity, can still bind strand using guide RNA. Recently, proteins active/inhibitory effects have linked end form fusion transcriptional effects, named CRISPRa CRISPRi, respectively. These CRISPR tools mediate transcription protein-coding non-coding genes regulating chromosomal modification states target promoters, enhancers, other functional elements. Here, we highlight epigenetic mechanisms applications common CRISPR/dCas9 tools, hope provide a reference future related regulation, function, high-throughput screening, disease treatment.

Language: Английский

Citations

40
CRISPR/Cas9 Landscape: Current State and Future Perspectives DOI Open Access
Marina A. Tyumentseva, Marina A. Tyumentseva, В. Г. Акимкин

et al.

International Journal of Molecular Sciences, Journal Year: 2023, Volume and Issue: 24(22), P. 16077 - 16077

Published: Nov. 8, 2023

CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 is a unique genome editing tool that can be easily used in wide range of applications, including functional genomics, transcriptomics, epigenetics, biotechnology, plant engineering, livestock breeding, gene therapy, diagnostics, and so on. This review focused on the current CRISPR/Cas9 landscape, e.g., Cas9 variants with improved properties, Cas9-derived fusion proteins, delivery methods, pre-existing immunity against anti-CRISPR their possible roles function improvement. Moreover, this presents detailed outline CRISPR/Cas9-based diagnostics therapeutic approaches. Finally, addresses future expansion editors’ toolbox orthologs other CRISPR/Cas proteins.

Language: Английский

Citations

32
Recent advances and prospects for lipid-based nanoparticles as drug carriers in the treatment of human retinal diseases DOI Creative Commons
Shaoxue Zeng, Yingying Chen, Fanfan Zhou

et al.

Advanced Drug Delivery Reviews, Journal Year: 2023, Volume and Issue: 199, P. 114965 - 114965

Published: June 13, 2023

The delivery of cures for retinal diseases remains problematic. There are four main challenges: passing through multiple barriers the eye, to particular cell types, capability carry different forms therapeutic cargo and long-term efficacy. Lipid-based nanoparticles (LBNPs) potent overcome these challenges due their unique merits: amphiphilic nanoarchitectures pass biological barriers, vary modifications with specific affinity target flexible capacity large mixed types cargos slow-release formulations treatment. We have reviewed latest research on applications LBNPs treating categorized them by payloads. Furthermore, we identified technical discussed possible future development expand potential in diseases.

Language: Английский

Citations

23
Structures of human γδ T cell receptor–CD3 complex DOI Creative Commons
Weizhi Xin, Bangdong Huang, Ximin Chi

et al.

Nature, Journal Year: 2024, Volume and Issue: 630(8015), P. 222 - 229

Published: April 24, 2024

Gamma delta (γδ) T cells, a unique cell subgroup, are crucial in various immune responses and immunopathology

Language: Английский

Citations

15
Systematic reconstruction of molecular pathway signatures using scalable single-cell perturbation screens DOI Open Access
Longda Jiang, Carol Dalgarno, Efthymia Papalexi

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Jan. 30, 2024

ABSTRACT Recent advancements in functional genomics have provided an unprecedented ability to measure diverse molecular modalities, but learning causal regulatory relationships from observational data remains challenging. Here, we leverage pooled genetic screens and single cell sequencing (i.e. Perturb-seq) systematically identify the targets of signaling regulators biological contexts. We demonstrate how Perturb-seq is compatible with recent commercially available advances combinatorial indexing next-generation sequencing, perform more than 1,500 perturbations split across six lines five introduce improved computational framework (Mixscale) address cellular variation perturbation efficiency, alongside optimized statistical methods learn differentially expressed gene lists conserved signatures. Finally, our derived can be used precisely infer changes pathway activation for in-vivo in-situ samples. Our work enhances understanding their targets, lays a towards data-driven inference ‘atlas’

Language: Английский

Citations

12
Engineered CRISPR-Cas12a for higher-order combinatorial chromatin perturbations DOI Creative Commons
Chris C.‐S. Hsiung, Caroline Wilson,

Nicholas A. Sambold

et al.

Nature Biotechnology, Journal Year: 2024, Volume and Issue: unknown

Published: May 17, 2024

Abstract Multiplexed genetic perturbations are critical for testing functional interactions among coding or non-coding elements. Compared to double-stranded DNA cutting, repressive chromatin formation using CRISPR interference (CRISPRi) avoids genotoxicity and is more effective perturbing regulatory elements in pooled assays. However, current CRISPRi screening approaches limited targeting one three genomic sites per cell. We engineer an Acidaminococcus Cas12a (AsCas12a) variant, multiplexed transcriptional AsCas12a (multiAsCas12a), that incorporates R1226A, a mutation stabilizes the ribonucleoprotein–DNA complex via nicking. The multiAsCas12a-KRAB fusion improves activity over DNase-dead AsCas12a-KRAB fusions, often rescuing activities of lentivirally delivered RNAs (crRNA) inactive when used with latter. supports 6-plex crRNA arrays high-throughput screens. Using multiAsCas12a-KRAB, we discover enhancer dissect combinatorial function cis -regulatory human cells. These results instantiate group framework efficiently surveying numerous combinations biological discovery engineering.

Language: Английский

Citations

12