Related Experiment Video

Updated: Oct 21, 2025

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage
06:51

Parallel High Throughput Single Molecule Kinetic Assay for Site-Specific DNA Cleavage

Published on: May 6, 2020

4.1K

Cas12a target search and cleavage on force-stretched DNA

Marialucrezia Losito1,2,3, Quentin M Smith1,2, Matthew D Newton1,2

  • 1Department of Infectious Disease, Section of Virology, Faculty of Medicine, Imperial College London, London W12 0NN, UK. david.rueda@imperial.ac.uk.

Physical Chemistry Chemical Physics : PCCP
|September 8, 2021
PubMed

Insights

CRISPR-Cas12a uses 1D hopping to find DNA targets. Low force enables fast binding and cleavage, but mechanical stress slows search and impairs DNA cutting.

Area of Science:

  • Molecular Biology
  • Biophysics
  • Genetics

Background:

  • CRISPR-Cas12a is a versatile gene-editing tool.
  • Understanding its mechanism is crucial for applications.

Purpose of the Study:

  • Investigate CRISPR-Cas12a's DNA search and cleavage mechanism.
  • Determine the role of mechanical forces in Cas12a activity.

Main Methods:

  • Utilized optical tweezers to apply force to λ-DNA.
  • Observed CRISPR-Cas12a interactions with DNA at the single-molecule level.

Main Results:

  • CRISPR-Cas12a employs rapid 1D hopping for target localization.
  • Efficient binding and cleavage occur at low forces (≤5 pN).
  • A 1.8 nm conformational change limits the cleavage rate.

Conclusions:

  • Mechanical forces significantly influence Cas12a dynamics.
  • Low forces optimize Cas12a search and cleavage efficiency.
  • Increased DNA stretching hinders Cas12a function.

More Related Videos

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers08:48

Stretching Short Sequences of DNA with Constant Force Axial Optical Tweezers

Published on: October 13, 2011

13.2K
Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair10:59

Atomic Force Microscopy Investigations of DNA Lesion Recognition in Nucleotide Excision Repair

Published on: May 24, 2017

9.7K

Related Concept Videos

Single-Strand DNA Binding Proteins01:03

Single-Strand DNA Binding Proteins

For successful DNA replication, the unwinding of double-stranded DNA must be accompanied by stabilization and protection of the separated single strands of the DNA. This crucial task is performed by single-strand DNA-binding (SSB) proteins. They bind to the DNA in a sequence-independent manner, which means that the nitrogenous bases of the DNA need not be present in a specific order for binding of SSB proteins to it. The binding of SSB proteins straightens single-stranded DNA (ssDNA) and makes...
15.5K
DNA Helicases00:55

DNA Helicases

DNA unwinding helicase enzymes are a type of motor protein. Motor proteins can translocate along filaments or polymers using energy generated from ATP hydrolysis. Helicases are involved in all the important cellular processes where DNA unwinding is required, such as DNA replication, repair, recombination, and transcription. They are present in all living organisms, but vary in their structure, function, and mechanism of action. For example, in prokaryotes, DnaB helicase binds and translocates...
22.8K
Homologous Recombination02:31

Homologous Recombination

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...
55.9K
Restarting Stalled Replication Forks02:37

Restarting Stalled Replication Forks

DNA replication is initiated at sites containing predefined DNA sequences known as origins of replication. DNA is unwound at these sites by the minichromosome maintenance (MCM) helicase and other factors such as Cdc45 and the associated GINS complex.The unwound single strands are protected by replication protein A (RPA) until DNA polymerase starts synthesizing DNA at the 5’ end of the strand in the same direction as the replication fork. To prevent the replication fork from falling apart,...
6.0K
Maxam-Gilbert Sequencing01:05

Maxam-Gilbert Sequencing

In the same year as the discovery of the Sanger sequencing method, another group of scientists, Allan Maxam and Walter Gilbert, demonstrated their chemical-cleavage method for DNA sequencing. The Maxam-Gilbert method relies on using different chemicals that can cleave the DNA sequence at specific sites, the separation of resulting DNA fragments of variable size using electrophoresis, and deciphering the DNA sequence from the resulting gel bands.
Challenges of the Maxam-Gilbert Method
The...
11.7K
DNA Topoisomerases02:02

DNA Topoisomerases

Topoisomerases are enzymes that relax overwound DNA molecules during various cell processes, including DNA replication and transcription. These enzymes regulate positive and negative DNA supercoiling without changing the nucleotide sequence. DNA overwinding in a clockwise direction results in positively supercoiled DNA, whereas underwinding in a counterclockwise direction produces negatively supercoiled DNA.
Types and Mechanism of action
Topoisomerases are divided into two main types. ...
33.0K