Establishing a conceptual framework for holistic cell states and state transitions

Rafelski, Susanne M.; Theriot, Julie A.

doi:10.1016/j.cell.2024.04.035

Cited by 7 publications

(3 citation statements)

References 132 publications

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“…These EMT lines allow for real-time tracking of the EMT process, providing insight into the dynamics of this transition. These reporter lines can also enable lineage tracing and fate mapping of cells during early development, which is important for identifying regulatory pathways that control cell differentiation and state transitions (Tosic et al, 2019; Sheppard et al, 2021; Rafelski and Theriot, 2024). The CDH5-mEGFP reporter line enables direct observation of endothelial cells as they differentiate and is critical for studying the role of VE-cadherin in cell-cell adhesion, vascular structure formation, and endothelial layer integrity.…”

Section: Discussionmentioning

confidence: 99%

“…The development of CRISPR/Cas9 and related technologies have revolutionized cell biology because it enables site-specific integration of exogenous sequences at endogenous gene loci, a technology referred to as “endogenous tagging” (Bukhari and Müller, 2019). Endogenous tagging enables live-cell fluorescence imaging studies for understanding complex biological processes by tracking protein abundance, organization, and dynamics (Dambournet et al, 2018; Bukhari and Müller, 2019; Lau et al, 2020; Gerbin et al, 2021; Cho et al, 2022; Rafelski and Theriot, 2024; Viana et al, 2023). Several protocols have emerged to facilitate endogenous gene tagging at varying degrees of throughput and efficiency in mammalian cells.…”

Section: Introductionmentioning

confidence: 99%

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Enhanced endogenous gene tagging in human induced pluripotent stem cells via AAV6-mediated donor delivery

Ehlers,

Klein,

Fuqua

et al. 2024

Preprint

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Systematically tagging endogenous proteins with fluorescent markers in human induced pluripotent stem cells (hiPSCs) allows observation of live cell dynamics in different cell states. However, the precise insertion of fluorescent proteins into live cells via CRISPR/Cas9-induced editing relies on homology-directed repair (HDR). The nonhomologous end-joining (NHEJ) DNA repair pathway often outcompetes HDR, resulting in irreversible insertions and deletions (INDELs) and low knock-in efficiency. Recognizing successful HDR-mediated tagging events is an additional challenge when the target gene is not expressed in stem cells and successful tagging cannot be immediately observed. To address these challenges, we used: 1) adeno-associated virus serotype 6 (AAV6) mediated DNA donors at optimized multiplicity of infection (MOI) to deliver tag payloads at maximal efficiency; 2) titrated, multiplexed Cas9:gRNA ribonucleoprotein (RNP) amounts to assure balanced HDR/INDEL frequency among conditions; 3) long-amplicon droplet digital PCR (ddPCR) to measure the frequency of HDR-generated alleles in edited pools; and 4) simultaneous Inference of CRISPR Edits (ICE) to detect and thereby avoid conditions significantly saturated (>50%) with INDELs. These approaches enabled us to identify efficient and accurate editing conditions and recover tagged cells, including cells tagged at loci not expressed in stem cells. Together these steps allowed us to develop an efficient methodology and workflow to clonally isolate directly from an ideal cell pool with optimal HDR and minimized INDEL frequencies. Using this approach, we achieved both monoallelic and biallelic insertion of fluorescent markers into four genes that are turned on during differentiation but not initially expressed in hiPSCs, where direct selection of tagged cells based on fluorescence was impossible: TBR2, TBXT, CDH2 (pro-differentiation and pro-migratory genes), and CDH5 (endothelial specific gene). Through a systematic evaluation of various gRNA sequences and RNP concentrations, we identified conditions for each gene that achieved high HDR frequencies, peaking at 38.6%, while also avoiding conditions saturated with INDELs, where isolation of clones with a tagged allele in trans with an unedited allele is difficult. Overall, this methodology enhances the efficiency of fluorescent tag knock-in at genes not expressed in hiPSCs, facilitating reliable image-based observation of cellular processes, and enables recovery of accurately edited mono- and biallelically tagged clones. We standardized these approaches to yield an efficient and general workflow for introducing large HDR mediated knock-ins into hiPSCs.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Enhanced endogenous gene tagging in human induced pluripotent stem cells via AAV6-mediated donor delivery

Ehlers,

Klein,

Fuqua

et al. 2024

Preprint

View full text Add to dashboard Cite

show abstract

“…Ongoing cell atlas projects have revealed remarkable cellular diversity within the body, identifying over 500 distinct cell types [1][2][3][4][5] . Subsequent more targeted efforts within specific tissues have revealed that even greater diversity exists at the level of transcriptional state 6,7 , with some tissues like the brain comprising possibly thousands of transcriptionally distinct clusters 8 . Furthermore, many disease processes induce new transcriptional states, making them interesting candidates as drivers of pathology [9][10][11] .…”

Section: Introductionmentioning

confidence: 99%

Comprehensive transcription factor perturbations recapitulate fibroblast transcriptional states

Southard,

Ardy,

Tang

et al. 2024

Preprint

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SummaryCell atlas projects have nominated recurrent transcriptional states as drivers of biological processes and disease, but their origins, regulation, and properties remain unclear. To enable complementary functional studies, we developed a scalable approach for recapitulating cell statesin vitrousing CRISPR activation (CRISPRa) Perturb-seq. Aided by a novel multiplexing method, we activated 1,836 transcription factors in two cell types. Measuring 21,958 perturbations showed that CRISPRa activated targets within physiological ranges, that epigenetic features predicted activatable genes, and that the protospacer seed region drove an off-target effect. Perturbations recapitulatedin vivofibroblast states, including universal and inflammatory states, and identifiedKLF4andKLF5as key regulators of the universal state. Inducing the universal state suppressed disease-associated states, highlighting its therapeutic potential. Our findings cement CRISPRa as a tool for perturbing differentiated cells and indicate thatin vivostates can be elicited via perturbation, enabling studies of clinically relevant statesex vivo.

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Toward a foundation model of causal cell and tissue biology with a Perturbation Cell and Tissue Atlas

Rood,

Hupalowska,

Regev

2024

Cell

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Establishing a conceptual framework for holistic cell states and state transitions

Cited by 7 publications

References 132 publications

Enhanced endogenous gene tagging in human induced pluripotent stem cells via AAV6-mediated donor delivery

Enhanced endogenous gene tagging in human induced pluripotent stem cells via AAV6-mediated donor delivery

Comprehensive transcription factor perturbations recapitulate fibroblast transcriptional states

Toward a foundation model of causal cell and tissue biology with a Perturbation Cell and Tissue Atlas

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