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

50.4K
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...
50.4K
CRISPR and crRNAs02:53

CRISPR and crRNAs

16.9K
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...
16.9K

You might also read

Related Articles

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

Sort by
Same author

Single-Molecule Visualization of DNase I-Mediated DNA Cleavage by High-Speed Atomic Force Microscopy.

ACS chemical biology·2026
Same author

Phage-encoded factor stimulates DNA degradation by the Hna anti-phage defense system.

Nature communications·2026
Same author

Structure and immunogenicity of an engineered soluble prefusion-stabilized EBV gB antigen.

Nature communications·2026
Same author

Phage-encoded factor stimulates DNA degradation by the Hna anti-phage defense system.

bioRxiv : the preprint server for biology·2025
Same author

One-Pot Isothermal Linear Amplification and Cas12a-Based Nucleic Acid Detection.

ACS synthetic biology·2025
Same author

Correction: Room temperature CRISPR diagnostics for low-resource settings.

Scientific reports·2025

Related Experiment Video

Updated: Jun 21, 2025

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

Substrate Generation for Endonucleases of CRISPR/Cas Systems

Published on: September 8, 2012

27.3K

Kinetic dissection of pre-crRNA binding and processing by CRISPR-Cas12a.

Selma Sinan1, Nathan M Appleby1, Chia-Wei Chou1

  • 1Department of Molecular Biosciences, University of Texas at Austin, Austin, Texas 78712, USA.

RNA (New York, N.Y.)
|July 15, 2024
PubMed
Summary

CRISPR-Cas12a binds pre-crRNA tightly, with binding affinity determining genome editing specificity. Optimized crRNA design is crucial, as secondary structures can hinder DNA targeting.

Keywords:
AsCas12aCRISPR–Cas systemRNA foldingRNA-guided nucleaserate-limiting binding

More Related Videos

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

1.7K
Author Spotlight: Development of Simplified CRISPR-Based Tests for Rapid Detection of Infectious Diseases
10:16

Author Spotlight: Development of Simplified CRISPR-Based Tests for Rapid Detection of Infectious Diseases

Published on: August 16, 2024

1.1K

Related Experiment Videos

Last Updated: Jun 21, 2025

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

Substrate Generation for Endonucleases of CRISPR/Cas Systems

Published on: September 8, 2012

27.3K
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

1.7K
Author Spotlight: Development of Simplified CRISPR-Based Tests for Rapid Detection of Infectious Diseases
10:16

Author Spotlight: Development of Simplified CRISPR-Based Tests for Rapid Detection of Infectious Diseases

Published on: August 16, 2024

1.1K

Area of Science:

  • Molecular Biology
  • Biochemistry

Background:

  • CRISPR-Cas12a is a key enzyme for genome editing.
  • Cas12a processes pre-crRNA for its maturation and function.

Purpose of the Study:

  • To establish a kinetic and thermodynamic framework for Cas12a pre-crRNA processing.
  • To determine the contribution of pre-crRNA regions to binding and processing.
  • To guide crRNA design for improved genome editing applications.

Main Methods:

  • In vitro kinetic and thermodynamic analysis of Cas12a-pre-crRNA interactions.
  • Measurement of binding affinities (Kd) and contributions of pre-crRNA regions.
  • Direct competition assays to assess binding specificity and DNA targeting.

Main Results:

  • Cas12a exhibits extremely tight binding to pre-crRNA (Kd = 0.6 pM), with binding being rate-limiting for processing.
  • The guide sequence significantly contributes to pre-crRNA binding affinity.
  • Mature crRNA remains tightly bound, with increased guide region affinity post-processing.
  • Pre-crRNA binding specificity is largely robust to sequence variations, but secondary structures can inhibit DNA targeting.

Conclusions:

  • Quantitative framework for Cas12a pre-crRNA binding and processing established.
  • Guide sequence and post-processing interactions are critical for Cas12a function.
  • Strategic crRNA design, avoiding stable secondary structures, is essential for efficient genome editing.