Optimizing a CRISPR-Cas13d Gene Circuit for Tunable Target RNA Downregulation with Minimal Collateral RNA Cutting DOI Creative Commons
Yiming Wan, Christopher Helenek,

Damiano Coraci

et al.

ACS Synthetic Biology, Journal Year: 2024, Volume and Issue: 13(10), P. 3212 - 3230

Published: Oct. 8, 2024

The invention of RNA-guided DNA cutting systems has revolutionized biotechnology. More recently, RNA by Cas13d entered the scene as a highly promising alternative to interference engineer cellular transcriptomes for biotechnological and therapeutic purposes. Unfortunately, "collateral damage" indiscriminate off-target tampered enthusiasm these systems. Yet, how collateral activity, or even target reduction depends on guide abundance remained unclear due lack expression-tuning studies address this question. Here we use precise gene circuits show that both nonspecific specific, on-target depend levels, from trans cleavage might contribute reduction. Using RNA-level control techniques, develop new Multi-Level Optimized Negative-Autoregulated crRNA Hybrid (MONARCH) achieve high dynamic range with low basal while minimizing activity in human kidney cells green monkey most frequently used virology. MONARCH should bring forefront, easily applicable, programmable tools transcriptome engineering medical applications.

Language: Английский

CRISPR-Cas tools for simultaneous transcription & translation control in bacteria DOI Creative Commons
Ryan Cardiff, Ian Faulkner,

Juliana G Beall

et al.

Nucleic Acids Research, Journal Year: 2024, Volume and Issue: 52(9), P. 5406 - 5419

Published: April 13, 2024

Abstract Robust control over gene translation at arbitrary mRNA targets is an outstanding challenge in microbial synthetic biology. The development of tools that can regulate will greatly expand our ability to precisely genes across the genome. In Escherichia coli, most are contained multi-gene operons, which subject polar effects where targeting one for repression leads silencing other same operon. These pose a independently regulating individual operons. Here, we use CRISPR-dCas13 address this challenge. We find dCas13-mediated exhibits up 6-fold lower compared dCas9. then show selectively activate single operon by coupling dCas9 transcriptional activation with dCas13 translational within also and be multiplexed improved biosynthesis medically-relevant human milk oligosaccharide. Taken together, findings suggest combining access difficult achieve either mode independently. combined regulation abilities engineer bacteria biotechnology perform systematic genetic screens.

Language: Английский

Citations

6
Biocomputing at the crossroad between emulating artificial intelligence and cellular supremacy DOI Creative Commons
Xinyuan Qiu,

Lingyun Zhu,

Hui Wang

et al.

Current Opinion in Biotechnology, Journal Year: 2025, Volume and Issue: 92, P. 103264 - 103264

Published: Jan. 20, 2025

Language: Английский

Citations

0
Enhanced RNA-targeting CRISPR-Cas technology in zebrafish DOI Creative Commons
Ismael Moreno-Sánchez,

Luis Hernández-Huertas,

Daniel Nahón-Cano

et al.

Nature Communications, Journal Year: 2025, Volume and Issue: 16(1)

Published: March 16, 2025

CRISPR-Cas13 RNA-targeting systems are widely used in basic and applied sciences. However, its application has recently generated controversy due to collateral activity mammalian cells mouse models. Moreover, competence could be improved vivo. Here, we optimized transient formulations as ribonucleoprotein complexes or mRNA-gRNA combinations enhance the CRISPR-RfxCas13d system zebrafish. We i) use chemically modified gRNAs allow more penetrant loss-of-function phenotypes, ii) improve nuclear RNA targeting, iii) compare different computational models determine most accurate predict gRNA Furthermore, demonstrate that can effectively deplete endogenous mRNAs zebrafish embryos without inducing effects, except when targeting extremely abundant ectopic RNAs. Finally, implement alternative CRISPR-Cas such CRISPR-Cas7-11 CRISPR-DjCas13d. Altogether, these findings contribute technology optimization for through approaches assist progression of vivo applications. comprise an invaluable set tools fields Moreno-Sánchez, Hernández-Huertas, Nahón-Cano et al. enhanced targeted depletion mRNAs.

Language: Английский

Citations

0
Dynamic Multiplexed Control and Modeling of Optogenetic Systems Using the High-Throughput Optogenetic Platform, Lustro DOI Creative Commons
Zachary P. Harmer, Jaron Thompson,

David Cole

et al.

ACS Synthetic Biology, Journal Year: 2024, Volume and Issue: 13(5), P. 1424 - 1433

Published: April 29, 2024

The ability to control cellular processes using optogenetics is inducer-limited, with most optogenetic systems responding blue light. To address this limitation, we leverage an integrated framework combining Lustro, a powerful high-throughput platform, and machine learning tools enable multiplexed over light-sensitive systems. Specifically, identify light induction conditions for sequential activation as well preferential switching between pairs of split transcription factors in the budding yeast, Saccharomyces cerevisiae. We use data generated from Lustro build Bayesian optimization that incorporates data-driven learning, uncertainty quantification, experimental design prediction system behavior identification optimal control. This work lays foundation designing more advanced synthetic biological circuits incorporating optogenetics, where multiple circuit components can be controlled designer programs, broad implications biotechnology bioengineering.

Language: Английский

Citations

3
Enhanced RNA-targeting CRISPR-Cas technology in zebrafish DOI Creative Commons
Ismael Moreno-Sánchez,

Luis Hernández-Huertas,

Daniel Nahón-Cano

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: Oct. 10, 2024

CRISPR-Cas13 systems are widely used in basic and applied sciences. However, its application has recently generated controversy due to collateral activity mammalian cells mouse models. Moreover, efficiency could be improved

Language: Английский

Citations

3
Engineering conditional protein-protein interactions for dynamic cellular control DOI
Anthony M Stohr,

Derron Ma,

Wilfred Chen

et al.

Biotechnology Advances, Journal Year: 2024, Volume and Issue: 77, P. 108457 - 108457

Published: Sept. 27, 2024

Language: Английский

Citations

2
Inducible RNA targeting and N6-methyladenosine editing by a split-Cas13 architecture DOI Creative Commons
Yang Li, Qiang Sun, Zhi Yang

et al.

Journal of Molecular Cell Biology, Journal Year: 2024, Volume and Issue: 16(1)

Published: Jan. 1, 2024

Language: Английский

Citations

1
CRISPR-Cas systems in DNA functional circuits: Strategies, challenges, prospects DOI
Xiaolong Li, Changjiang Li, Canghong Shi

et al.

Chinese Chemical Letters, Journal Year: 2024, Volume and Issue: unknown, P. 110507 - 110507

Published: Sept. 1, 2024

Language: Английский

Citations

1
Optimizing a CRISPR-Cas13d gene circuit for tunable target RNA downregulation with minimal collateral RNA cutting DOI Creative Commons
Yiming Wan, Christopher Helenek,

Damiano Coraci

et al.

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: May 11, 2024

SUMMARY The invention of RNA-guided DNA cutting systems has revolutionized biotechnology. More recently, RNA by Cas13d entered the scene as a highly promising alternative to interference engineer cellular transcriptomes for biotechnological and therapeutic purposes. Unfortunately, “collateral damage” indiscriminate off-target tampered enthusiasm these systems. Yet, how collateral activity, or even target reduction depends on guide abundance remained unclear due lack expression-tuning studies address this question. Here we use precise gene circuits show that both nonspecific specific, on-target depend levels, from trans cleavage might contribute reduction. Using RNA-level control techniques, develop new Multi-Level Optimized Negative-Autoregulated crRNA Hybrid (MONARCH) achieve high dynamic range with low basal while minimizing activity in human kidney cells green monkey most frequently used virology. MONARCH should bring forefront, easily applicable, programmable tools transcriptome engineering medical applications.

Language: Английский

Citations

0
Optimizing a CRISPR-Cas13d Gene Circuit for Tunable Target RNA Downregulation with Minimal Collateral RNA Cutting DOI Creative Commons
Yiming Wan, Christopher Helenek,

Damiano Coraci

et al.

ACS Synthetic Biology, Journal Year: 2024, Volume and Issue: 13(10), P. 3212 - 3230

Published: Oct. 8, 2024

The invention of RNA-guided DNA cutting systems has revolutionized biotechnology. More recently, RNA by Cas13d entered the scene as a highly promising alternative to interference engineer cellular transcriptomes for biotechnological and therapeutic purposes. Unfortunately, "collateral damage" indiscriminate off-target tampered enthusiasm these systems. Yet, how collateral activity, or even target reduction depends on guide abundance remained unclear due lack expression-tuning studies address this question. Here we use precise gene circuits show that both nonspecific specific, on-target depend levels, from trans cleavage might contribute reduction. Using RNA-level control techniques, develop new Multi-Level Optimized Negative-Autoregulated crRNA Hybrid (MONARCH) achieve high dynamic range with low basal while minimizing activity in human kidney cells green monkey most frequently used virology. MONARCH should bring forefront, easily applicable, programmable tools transcriptome engineering medical applications.

Language: Английский

Citations

0