CRISPR base editing of cis-regulatory elements enables the perturbation of neurodegeneration-linked genes

Lim, Colin K.W.; McCallister, Tristan X.; Saporito-Magriñá, Christian; McPheron, Garrett D.; Krishnan, Ramya; C, M. Alejandra Zeballos; Powell, Jackson E.; Clark, Lindsay V.; Pérez-Piñera, Pablo; Gaj, Thomas

doi:10.1016/j.ymthe.2022.08.008

Cited by 14 publications

(8 citation statements)

References 98 publications

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“…To facilitate the isolation of transduced nuclei for a more detailed analysis of RfxCas13d-mediated outcomes, we co-injected C9-BACexp mice with 2 x 10 10 GCs of a second AAV-PHP.eB vector encoding a CBh-driven EGFP variant fused to KASH (Klarsicht/ANC-1/Syne-1 homology; AAV-PHP.eB-EGFP-KASH), a domain that promotes EGFP localization to the outer nuclear membrane 56 , which can enable the isolation of transduced nuclei by fluorescence-activated cell sorting (FACS) 57,58 ( Fig. 2a-b ).…”

Section: Resultsmentioning

confidence: 99%

A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD

McCallister,

Lim,

Terpstra

et al. 2023

Preprint

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An abnormal expansion of a GGGGCC hexanucleotide repeat in the C9ORF72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), two debilitating neurodegenerative disorders driven in part by gain-of-function mechanisms involving transcribed forms of the repeat expansion. By utilizing a Cas13 variant with reduced collateral effects, we developed a high-fidelity RNA-targeting CRISPR-based system for C9ORF72-linked ALS/FTD. When delivered to the brain of a transgenic rodent model, this Cas13-based platform effectively curbed the expression of the GGGGCC repeat-containing RNA without affecting normal C9ORF72 levels, which in turn decreased the formation of RNA foci and reversed transcriptional deficits. This high-fidelity Cas13 variant possessed improved transcriptome-wide specificity compared to its native form and mediated efficient targeting in motor neuron-like cells derived from a patient with ALS. Our results lay the foundation for the implementation of RNA-targeting CRISPR technologies for C9ORF72-linked ALS/FTD.

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Section: Resultsmentioning

confidence: 99%

A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD

McCallister,

Lim,

Terpstra

et al. 2023

Preprint

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“… 55 , 56 While the size of BEs exceeds the packaging capacity of single AAV particles, we and others have previously demonstrated that split-intein BEs can be used to effectively deliver active BEs in vivo via AAV. 26 , 27 , 36 , 57 , 58 …”

Section: Discussionmentioning

confidence: 99%

Targeting Duchenne muscular dystrophy by skipping DMD exon 45 with base editors

Gapinske,

Winter,

Swami

et al. 2023

Molecular Therapy - Nucleic Acids

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“…Studies have highlighted the value of CRISPR screens in general in identifying cis-regulatory elements associated with disease states, [130][131][132][133] including neurodevelopmental disorders and neurodegenerative diseases [134,135] and host-pathogen interactions for viruses such as COVID-19. [136] These screens have also pinpointed specific TFs as potential immunotherapeutic targets in various cancer types [137] and been used to investigate the connection between regulatory elements and drug resistance or sensitivity in cancer cells.…”

Section: Crispr-cas9 In Gene Regulation Researchmentioning

confidence: 99%

Advances in computational and experimental approaches for deciphering transcriptional regulatory networks

Moeckel,

Mouratidis,

Chantzi

et al. 2024

BioEssays

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Understanding the influence of cis‐regulatory elements on gene regulation poses numerous challenges given complexities stemming from variations in transcription factor (TF) binding, chromatin accessibility, structural constraints, and cell‐type differences. This review discusses the role of gene regulatory networks in enhancing understanding of transcriptional regulation and covers construction methods ranging from expression‐based approaches to supervised machine learning. Additionally, key experimental methods, including MPRAs and CRISPR‐Cas9‐based screening, which have significantly contributed to understanding TF binding preferences and cis‐regulatory element functions, are explored. Lastly, the potential of machine learning and artificial intelligence to unravel cis‐regulatory logic is analyzed. These computational advances have far‐reaching implications for precision medicine, therapeutic target discovery, and the study of genetic variations in health and disease.

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CRISPR base editing of cis-regulatory elements enables the perturbation of neurodegeneration-linked genes

Cited by 14 publications

References 98 publications

A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD

A high-fidelity CRISPR-Cas13 system improves abnormalities associated with C9ORF72-linked ALS/FTD

Targeting Duchenne muscular dystrophy by skipping DMD exon 45 with base editors

Advances in computational and experimental approaches for deciphering transcriptional regulatory networks

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