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

49.1K
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...
49.1K
Homologous Recombination02:31

Homologous Recombination

50.1K
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...
50.1K
CRISPR and crRNAs02:53

CRISPR and crRNAs

16.6K
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...
16.6K
Conservative Site-specific Recombination and Phase Variation02:53

Conservative Site-specific Recombination and Phase Variation

5.9K
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...
5.9K

You might also read

Related Articles

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

Sort by
Same author

Circumventing the Synthesizability Problem in Generative Molecular Design.

Journal of chemical information and modeling·2026
Same author

Characterization of Virulence Factors, Biofilm Production, and Antimicrobial Resistance in Mastitis-Associated <i>Staphylococcus aureus</i> Strains by Phenotypic and Genotypic Methods.

Foodborne pathogens and disease·2026
Same author

Platelet-rich plasma (PRP) versus steroid as an intradiscal injection for lower back aches among degenerative disc disorder patient in India.

Bioinformation·2026
Same author

Antimicrobial Potential of Selected Essential Oils and Essential Oil's Active Components Against Food Spoilage Bacteria and Foodborne Pathogens and Antibiofilm Potential Against Multidrug-Resistant <i>Staphylococcus aureus</i> and <i>Pseudomonas aeruginosa</i>.

Foodborne pathogens and disease·2026
Same author

Antibacterial Efficacy of Recombinant Endolysin S.AgEndo1332 in Combination with Antibiotics against Mastitis-Associated S. aureus in a Mouse Mastitis Model.

Probiotics and antimicrobial proteins·2026
Same author

Sequence-based modeling of low-affinity transcription factor-DNA binding through deep learning.

NAR genomics and bioinformatics·2026

Related Experiment Video

Updated: May 28, 2025

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP
09:26

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

Published on: January 20, 2016

10.4K

PAM-adjacent DNA flexibility tunes CRISPR-Cas12a off-target binding.

Aleique Allen1, Brendon H Cooper2,3, Jaideep Singh1

  • 1Department of Chemistry, University of Southern California, 3430 S Vermont Ave., Los Angeles, CA, 90089, USA.

Scientific Reports
|February 10, 2025
PubMed
Summary
This summary is machine-generated.

DNA flexibility near the protospacer-adjacent motif (PAM) influences Cas12a nuclease off-target binding. Unpairing at PAM+1, +2, and +3 positions increases Cas12a off-target activity, impacting genome editing applications.

More Related Videos

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
11:37

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

Published on: February 26, 2019

9.6K
Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
11:35

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Published on: June 16, 2017

12.4K

Related Experiment Videos

Last Updated: May 28, 2025

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP
09:26

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

Published on: January 20, 2016

10.4K
Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution
11:37

Using Sniper-Cas9 to Minimize Off-target Effects of CRISPR-Cas9 Without the Loss of On-target Activity Via Directed Evolution

Published on: February 26, 2019

9.6K
Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
11:35

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Published on: June 16, 2017

12.4K

Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • Cas12a is a CRISPR-associated nuclease used for genome manipulation.
  • DNA flexibility adjacent to the protospacer-adjacent motif (PAM) is implicated in Cas12a target recognition.

Purpose of the Study:

  • To investigate the relationship between PAM-adjacent DNA flexibility and Cas12a off-target binding.
  • To understand how DNA sequence variations near the PAM affect Cas12a specificity.

Main Methods:

  • Adapted a SELEX-seq approach to analyze DNA-DNA mismatches at PAM+1 to +6 positions.
  • In vitro binding assays with FnCas12a were performed using a DNA library with mismatches.
  • Sequencing of bound and unbound DNA populations determined off-target binding propensity.

Main Results:

  • Cas12a off-target binding is dependent on PAM-adjacent DNA flexibility.
  • Unpairing of the protospacer at PAM+1 is required for off-target binding.
  • Off-target binding increases with unpairing at PAM+2 and +3 positions.

Conclusions:

  • PAM-adjacent DNA flexibility can modulate Cas12a off-target binding.
  • Physical properties of DNA influence Cas12a target discrimination.
  • Findings have implications for optimizing Cas12a-based genome editing tools.