Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

CRISPR01:59

CRISPR

53.6K
Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
53.6K
CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

709
The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
709
CRISPR and crRNAs02:53

CRISPR and crRNAs

17.9K
Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
17.9K
Homologous Recombination02:31

Homologous Recombination

56.4K
The basic reaction of homologous recombination (HR) involves two chromatids that contain DNA sequences sharing a significant stretch of identity. One of these sequences uses a strand from another as a template to synthesize DNA in an enzyme-catalyzed reaction. The final product is a novel amalgamation of the two substrates. To ensure an accurate recombination of sequences, HR is restricted to the S and G2 phases of the cell cycle. At these stages, the DNA has been replicated already and the...
56.4K
Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

6.3K
Because the DNA segments are cut and reorganized in a direction-specific manner, site-specific recombination has emerged as an efficient genetic engineering technique. Flippase and Cyclization recombinases or Flp and Cre, respectively, are two members of the tyrosine recombinase family derived from bacteriophages, that are used to mediate site-specific DNA insertions, deletions, and targeted expression of proteins in mammalian cell lines.
The recognition sites for Cre recombinase called LoxP...
6.3K

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Bacterial spores as a modular platform for the production of amyloids for materials.

Trends in biotechnology·2026
Same author

iFLinkC-EZ: A scalable and automatable method for the assembly of complex fusion proteins and multi-gene expression constructs based on the iFLinkC framework.

Synthetic and systems biotechnology·2026
Same author

Identification of Phage RNA Polymerases That Minimize Double-Stranded RNA By-Product Formation and Their Characterization via In Vitro Transcription.

Microorganisms·2026
Same author

Iterative design of a NAND hybrid riboswitch by deep batch Bayesian optimization.

Nucleic acids research·2026
Same author

Spore immobilized enzymes for the multi-step synthesis of cellobiose.

Microbial cell factories·2026
Same author

The Coli Toolkit (CTK): An Extension of the Modular Yeast Toolkit for Use in <i>E. coli</i>.

ACS synthetic biology·2026

Related Experiment Video

Updated: Oct 24, 2025

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins
10:46

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Published on: October 18, 2022

2.0K

Functionalizing Cell-Free Systems with CRISPR-Associated Proteins: Application to RNA-Based Circuit Engineering.

François-Xavier Lehr1,2, Alina Kuzembayeva1,2, Megan E Bailey1,2

  • 1Centre for Synthetic Biology, Technische Universität Darmstadt, Darmstadt 64287, Germany.

ACS Synthetic Biology
|August 12, 2021
PubMed
Summary

This study integrates CRISPR-associated proteins (Csy4 and dCas9) into cell-free expression systems for enhanced RNA-based synthetic circuit prototyping. Pre-expression improves dynamic range and enables new logic gates.

Keywords:
CRISPR-associated systemsRNA-based circuitscell-free extractcell-free synthetic biologymalachite green aptamerprototyping

More Related Videos

Substrate Generation for Endonucleases of CRISPR/Cas Systems
11:53

Substrate Generation for Endonucleases of CRISPR/Cas Systems

Published on: September 8, 2012

27.6K
A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization
08:20

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

Published on: September 2, 2021

4.3K

Related Experiment Videos

Last Updated: Oct 24, 2025

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins
10:46

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Published on: October 18, 2022

2.0K
Substrate Generation for Endonucleases of CRISPR/Cas Systems
11:53

Substrate Generation for Endonucleases of CRISPR/Cas Systems

Published on: September 8, 2012

27.6K
A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization
08:20

A New Toolkit for Evaluating Gene Functions using Conditional Cas9 Stabilization

Published on: September 2, 2021

4.3K

Area of Science:

  • Synthetic Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Cell-free systems are valuable for synthetic circuit prototyping.
  • Producing cell-free extracts and functionalized proteins are often separate processes.
  • CRISPR-associated (Cas) proteins offer precise molecular manipulation capabilities.

Purpose of the Study:

  • To develop a cell-free expression system with pre-expressed CRISPR-associated proteins (Csy4 and dCas9).
  • To demonstrate the utility of these enhanced extracts for RNA-based synthetic circuit design.
  • To expand the repertoire of Boolean logic gates and reporting mechanisms in cell-free systems.

Main Methods:

  • Utilized an *E. coli*-based cell-free expression system.
  • Incorporated pre-expression of Csy4 and dCas9 proteins.
  • Employed RNA circuitry including single guide RNAs, small transcriptional activator RNAs, and toehold switches.
  • Assessed performance using AND-logic gates, NAND-logic gates, and simultaneous transcriptional/translational reporters.

Main Results:

  • Pre-expression of Csy4 and dCas9 did not compromise extract performance.
  • Csy4 processing enhanced the dynamic range of an AND-logic gate by 4-fold.
  • CRISPR-enhanced extracts facilitated reduced leakage and enabled NAND-logic gates.
  • Simultaneous monitoring of mRNA and protein levels for AND-gate circuits was achieved.

Conclusions:

  • Pre-expressing CRISPR-associated proteins in cell-free extracts is feasible and beneficial for synthetic circuit prototyping.
  • This approach expands the capabilities of RNA-based circuits, offering greater control and functionality.
  • The developed system facilitates advanced RNA circuit design and analysis in a cell-free environment.