Synthetic macromolecular switches for precision control of therapeutic cell functions

Nature Reviews Bioengineering, Journal Year: 2024, Volume and Issue: unknown

Published: Sept. 17, 2024

Language: Английский

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Integrated compact regulators of protein activity enable control of signaling pathways and genome-editing in vivo

Cell Discovery, Journal Year: 2024, Volume and Issue: 10(1)

Published: Jan. 23, 2024

Abstract Viral proteases and clinically safe inhibitors were employed to build integrated compact regulators of protein activity (iCROP) for post-translational regulation functional proteins by tunable proteolytic activity. In the absence inhibitor, co-localized/fused protease cleaves a target peptide sequence introduced in an exposed loop interest, irreversibly fragmenting structure destroying its functionality. We selected three demonstrated versatility iCROP framework validating it regulate ten different proteins. switches can be delivered either as mRNA or DNA, provide rapid actuation kinetics with large induction ratios, while remaining strongly suppressed off state without inhibitor. iCROPs effectors NF-κB NFAT signaling pathways assembled confirmed enable precise activation/inhibition downstream events response inhibitors. lipopolysaccharide-treated mice, iCROP-sr-IκBα cytokine release (“cytokine storm”) rescuing IκBα, which suppresses signaling. also constructed inducible CRISPR-(d)Cas9 variants showed that iCROP-Cas9-mediated knockout PCSK9 gene liver lowered blood LDL-cholesterol levels mice. iCROP-based will facilitate protein-level basic research translational applications.

Language: Английский

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

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: May 14, 2023

Abstract Human nuclear proteins contain >1000 transcriptional effector domains that can activate or repress transcription of target genes. We lack a systematic understanding which regulate robustly across genomic, cell-type, and DNA-binding domain (DBD) contexts. Here, we developed dCas9-mediated high-throughput recruitment (HT-recruit), pooled screening method for quantifying function at endogenous targets, tested library containing 5092 protein Pfam varied find many effectors depend on DBD contexts, such as HLH act either activators repressors. then confirm these findings further map context dependencies drawn from unannotated regions using larger 114,288 sequences tiling chromatin regulators factors. To enable efficient perturbations, select are potent in diverse engineer (1) improved ZNF705 KRAB CRISPRi tools to silence promoters enhancers, (2) compact human activator combination NFZ better CRISPRa inducible circuit delivery. Together, this effector-by-context functional reveals context-dependence guides selection manipulating transcription.

Language: Английский

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Ultra-high throughput mapping of genetic design space

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2023, Volume and Issue: unknown

Published: March 17, 2023

Massively parallel genetic screens have been used to map sequence-to-function relationships for a variety of elements. However, because these approaches only interrogate short sequences, it remains challenging perform high throughput (HT) assays on constructs containing combinations sequence elements arranged across multi-kb length scales. Overcoming this barrier could accelerate synthetic biology; by screening diverse gene circuit designs, "composition-to-function" mappings be created that reveal part composability rules and enable rapid identification behavior-optimized variants. Here, we introduce CLASSIC, generalizable platform combines long- short-read next-generation sequencing (NGS) modalities quantitatively assess pooled libraries DNA arbitrary length. We show CLASSIC can measure expression profiles >10 5 drug-inducible designs (ranging from 6-9 kb) in single experiment human cells. Using statistical inference machine learning (ML) approaches, demonstrate data obtained with enables predictive modeling an entire design landscape, offering critical insight into underlying principles. Our work shows expanding the understanding gained each design-build-test-learn (DBTL) cycle, dramatically augments pace scale biology establishes experimental basis data-driven complex systems.

Language: Английский

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A programmable protease-based protein secretion platform for therapeutic applications

Nature Chemical Biology, Journal Year: 2023, Volume and Issue: 20(4), P. 432 - 442

Published: Oct. 23, 2023

Language: Английский

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