Development of compact transcriptional effectors using high-throughput measurements in diverse contexts

Tycko, Josh; Van, Mike V.; Aradhana,; DelRosso, Nicole; Yao, David; Xu, Xiaoshu; Ludwig, Connor; Spees, Kaitlyn; Liu, Katherine; Hess, Gaelen T.; Gu, Mingxia; Mukund, Adi; Suzuki, Peter; Kamber, Roarke A.; Qi, Lei; Bintu, Lacramioara; Beer, M

doi:10.1101/2023.05.12.540558

Cited by 14 publications

(4 citation statements)

References 109 publications

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“…We imagine that this success rate can be improved via a combination of brute force, i.e. testing more gRNAs, improved CRISPRa activation domains 38 , and better CRISPRa-specific gRNA design.…”

Section: Discussionmentioning

confidence: 99%

Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements

Chardon

McDiarmid

Page

et al. 2023

Preprint

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CRISPR-based gene activation (CRISPRa) is a promising therapeutic approach for gene therapy, upregulating gene expression by targeting promoters or enhancers in a tissue/cell-type specific manner. Here, we describe an experimental framework that combines highly multiplexed perturbations with single-cell RNA sequencing (sc-RNA-seq) to identify cell-type-specific, CRISPRa-responsive cis-regulatory elements and the gene(s) they regulate. Random combinations of many gRNAs are introduced to each of many cells, which are then profiled and partitioned into test and control groups to test for effect(s) of CRISPRa perturbations of both enhancers and promoters on the expression of neighboring genes. Applying this method to candidate cis-regulatory elements in both K562 cells and iPSC-derived excitatory neurons, we identify gRNAs capable of specifically and potently upregulating target genes, including autism spectrum disorder (ASD) and neurodevelopmental disorder (NDD) risk genes. A consistent pattern is that the responsiveness of individual enhancers to CRISPRa is restricted by cell type, implying a dependency on either chromatin landscape and/or additional trans-acting factors for successful gene activation. The approach outlined here may facilitate large-scale screens for gRNAs that activate therapeutically relevant genes in a cell type-specific manner.

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Section: Discussionmentioning

confidence: 99%

Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements

Chardon

McDiarmid

Page

et al. 2023

Preprint

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“…To overcome this, three of the components were stably introduced using piggybac (dCas9-SSSavi and BirA, 12 kb) and lentiviral integration (6x sgRNA, 6 kb). Furthermore, testing more compact truncated protein domains ( 24 , 28 , 30 , 32 ) could also increase transfection efficiency. However, we emphasise that the SSSavi system is a valuable tool for probing how different chromatin contexts and epigenetic modifications can be modulated, rather than solely as a transcriptional regulatory tool.…”

Section: Discussionmentioning

confidence: 99%

A modular dCas9-based recruitment platform for combinatorial epigenome editing

Swain,

Pflueger,

Freytag

et al. 2023

Nucleic Acids Research

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Targeted epigenome editing tools allow precise manipulation and investigation of genome modifications, however they often display high context dependency and variable efficacy between target genes and cell types. While systems that simultaneously recruit multiple distinct ‘effector’ chromatin regulators can improve efficacy, they generally lack control over effector composition and spatial organisation. To overcome this we have created a modular combinatorial epigenome editing platform, called SSSavi. This system is an interchangeable and reconfigurable docking platform fused to dCas9 that enables simultaneous recruitment of up to four different effectors, allowing precise control of effector composition and spatial ordering. We demonstrate the activity and specificity of the SSSavi system and, by testing it against existing multi-effector targeting systems, demonstrate its comparable efficacy. Furthermore, we demonstrate the importance of the spatial ordering of the recruited effectors for effective transcriptional regulation. Together, the SSSavi system enables exploration of combinatorial effector co-recruitment to enhance manipulation of chromatin contexts previously resistant to targeted editing.

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“…Human embryonic kidney cells (HEK293T, Takara Bio #632180)) were cultured in DMEM-GlutaMAX media (10566024, Gibco) supplemented with 1x Penicillin-Streptomycin-Glutamine (10378016, Gibco) and 10 % FBS (FB-15, Omega Scientific). The reporter cell line was generated in previous work 55 , by co-transfecting cells with TALEN-L (Addgene #35431), TALEN-R (Addgene #35432) and pJT039 (AAVS1-9xTetO-pEF-IGKleader-Cas9site-hIgG1_FC-Myc-PDGFRb-T2A-Citrine-PolyA) reporter donor (Addgene #161927) 19 , followed by antibiotic selection with 0.25 µg/mL puromycin. To isolate single clones, cells were diluted at 100 cells/20 mL, and then dispensed into a 96-well plate at 200 µL per well (~1 cell/well).…”

Section: Human Cell Culture and Reporter Cell Line Generationmentioning

confidence: 99%

Single-cell chromatin state transitions during epigenetic memory formation

Fujimori,

Rios-Martinez,

Thurm

et al. 2023

Preprint

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Repressive chromatin modifications are thought to compact chromatin to silence transcription. However, it is unclear how chromatin structure changes during silencing and epigenetic memory formation. We measured gene expression and chromatin structure in single cells after recruitment and release of repressors at a reporter gene. Chromatin structure is heterogeneous, with open and compact conformations present in both active and silent states. Recruitment of repressors associated with epigenetic memory produces chromatin compaction across 10-20 kilobases, while reversible silencing does not cause compaction at this scale. Chromatin compaction is inherited, but changes molecularly over time from histone methylation (H3K9me3) to DNA methylation. The level of compaction at the end of silencing quantitatively predicts epigenetic memory weeks later. Similarly, chromatin compaction at the Nanog locus predicts the degree of stem-cell fate commitment. These findings suggest that the chromatin state across tens of kilobases, beyond the gene itself, is important for epigenetic memory formation.

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Development of compact transcriptional effectors using high-throughput measurements in diverse contexts

Cited by 14 publications

References 109 publications

Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements

Multiplex, single-cell CRISPRa screening for cell type specific regulatory elements

A modular dCas9-based recruitment platform for combinatorial epigenome editing

Single-cell chromatin state transitions during epigenetic memory formation

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