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PAM-flexible adenine base editing rescues hearing loss in a humanized MPZL2 mouse model harboring an East Asian founder mutation

ABSTRACT: PAM-flexible adenine base editing rescues hearing loss in a humanized MPZL2 mouse model harboring an East Asian founder mutation

PROVIDER: PRJNA1272495 | ENA |

REPOSITORIES: ENA

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PAM-flexible adenine base editing rescues hearing loss in a humanized MPZL2 mouse model harboring an East Asian founder mutation

Project description:Hearing loss is one of the most prevalent sensory disorders, but no commercial biological treatments are currently available. Here, we identify an East Asia-specific founder mutation, the homozygous c.220C>T mutation in MPZL2, that contributes to a significant proportion of hereditary deafness cases in our cohort study. We find that the disease-causing mutation can be targetable by adenine base editors (ABEs) that enable A·T-to-G·C base corrections without DNA double-strand breaks. To demonstrate this, we develop a humanized mouse model (hMPZL2Q74X/Q74X) that recapitulates human MPZL2 deafness and leads to progressive hearing loss. A PAM-flexible ABE variant with reduced bystander and off-target effects (ABE8eWQ-SpRY:sgRNA3) is packaged in dual adeno-associated viruses (AAVs) and injected into the inner ear of hMPZL2Q74X/Q74X mice and effectively corrects the mutation. This treatment significantly restores hearing function, improves inner ear structural integrity, and reverses altered gene expression. Base editing may hold therapeutic potential for hereditary deafness, including most cases of MPZL2 deafness.

2025-07-01 | GSE299064 | GEO

High-throughput screens of PAM-flexible Cas9 variants for gene knock-out and transcriptional modulation

Project description:A key limitation of the commonly-used CRISPR enzyme S. pyogenes Cas9 is the strict requirement of an NGG protospacer-adjacent motif (PAM) at the target site, which reduces the number of accessible genomic loci. This constraint can be limiting for genome editing applications that require precise Cas9 positioning. Recently, two Cas9 variants with a relaxed PAM requirement (NG) have been developed (xCas9 and Cas9-NG) but their activity has been measured at only a small number of endogenous sites. Here we devised a high-throughput Cas9 pooled competition screen to compare the performance of both PAM-flexible Cas9 variants and wild-type Cas9 at thousands of genomic loci and across 3 modalities (gene knock-out, transcriptional activation and suppression). We show that PAM flexibility comes at a substantial cost of decreased DNA targeting and cutting. Of the PAM-flexible variants, we found that Cas9-NG outperforms xCas9 regardless of genome engineering modality or PAM. Finally, we combined xCas9 mutations with those of Cas9-NG, creating a stronger transcriptional modulator than existing PAM-flexible Cas9 variants.

2020-01-26 | GSE143892 | GEO

Expanding the Scope of Bacterial CRISPR Activation with PAM-flexible dCas9 Variants

Project description:CRISPR-Cas transcriptional tools have been widely applied for programmable regulation of complex biological networks. In comparison to eukaryotic systems, bacterial CRISPR activation (CRISPRa) has stringent target site requirements for effective gene activation. While genes may not always have an NGG protospacer adjacent motif (PAM) at the appropriate position, PAM-flexible dCas9 variants can expand the range of targetable sites. Here we systematically evaluate a panel of PAM-flexible dCas9 variants for their ability to activate bacterial genes. We observe that dxCas9-NG provides a high dynamic range of gene activation for sites with NGN PAMs while dSpRY permits modest activity across almost any PAM. Similar trends were observed for heterologous and endogenous promoters. For all variants tested, improved PAM-flexibility comes with the tradeoff that CRISPRi-mediated gene repression becomes less effective. Weaker CRISPR interference (CRISPRi) gene repression can be partially rescued by expressing multiple sgRNAs to target many sites in the gene of interest. Our work provides a framework to choose the most effective dCas9 variant for a given set of gene targets, which will further expand the utility of CRISPRa/i gene regulation in bacterial systems.

2022-11-15 | GSE217559 | GEO

Expanding the Scope of Bacterial CRISPR Activation with PAM-flexible dCas9 Variants

Project description:Expanding the Scope of Bacterial CRISPR Activation with PAM-flexible dCas9 Variants

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High-throughput screens of PAM-flexible Cas9 variants for gene knock-out and transcriptional modulation

Project description:High-throughput screens of PAM-flexible Cas9 variants for gene knock-out and transcriptional modulation

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Randomized PAM depletion of AsCas12a

Project description:We report the PAMs of AsCas12a using a cell-free TXTL-based cleavage assay. By adding randomized PAM library and AsCas12a-gRNA in vitro, functional PAM sequences were cleaved, while non-functional PAMs remained. By amplifying the non-cleaved DNA, we use next-generation sequencing to analyze the depletion of functional PAMs of AsCas12a.

2019-01-01 | GSE123443 | GEO

Randomized PAM depeletion of NmeCas9

Project description:We report the PAMs of NmeCas9 using a cell-free TXTL-based cleavage assay. By adding randomized PAM library and NmeCas9-gRNA in vitro, functional PAM sequences were cleaved, while non-functional PAMs remained. By amplifying the non-cleaved DNA, we use next-generation sequencing to analyze the depletion of functional PAMs of NmeCas9.

2017-12-21 | GSE108357 | GEO

seRNA PAM-1 interacts with Ddx5 to regulate skeletal muscle satellite cell activation and aging through trans regulation of Timp2 expression [PAM-1 ChIRP-seq]

Project description:There is an absolute requirement of Pax7 for the normal function of MuSCs during regenerative myogenesis in skeletal muscle at any stage of life. Here using RNA-seq, H3K27ac and Pax7 ChIP-seq, we discover PAM-1 (Pax7 Associated Muscle lncRNA) that is enriched in activated skeletal muscle satellite cells (ASCs) 24 and 48 hours after activation. Knockdown of PAM-1 reduces proliferating Pax7+Myod+ ASCs number, while overexpression of PAM-1 increases ASCs number. Mechanistically, PAM-1 is located on ASCs and myoblast specific super-enhancer (SE), and we categorize it as seRNA. Through a series of multiomics analysis of PAM-1 interactome in myoblast including PAM-1-DNA interaction by ChIRP-seq, PAM-1 SE-DNA interaction by 4C-seq, PAM-1-protein interaction by mass spectrometry and ChIP-seq, we identify a novel class of transcriptional regulation that seRNA PAM-1 interacts with RNA binding protein Ddx5 and tethers PAM-1 SE to regulate inter-chromosomal targets Timp2. Altogether, our findings identify PAM-1 is driven by Pax7 in ASC and myoblast to regulate myogenic activation through binding with Ddx5 and targeting Timp2.

2022-07-12 | GSE180071 | GEO

seRNA PAM-1 interacts with Ddx5 to regulate skeletal muscle satellite cell activation and aging through trans regulation of Timp2 expression [PAM-1 KO ChIP-seq]

2022-07-12 | GSE180069 | GEO

Randomized PAM depletion of phylogenetically-diverse Cas12a nucleases

Project description:We report the PAMs of phylogenetically-diverse Cas12a nucleases using a cell-free TXTL-based PAM screen. By adding a 5N randomized PAM library and Cas12a-gRNA in vitro, recognized PAM sequences were cleaved, while non-recognized PAMs remained. By amplifying the non-cleaved DNA, we used next-generation sequencing to analyze the depletion of functional PAMs of these Cas12a nucleases.

2020-03-02 | GSE130377 | GEO

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