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

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

Selective reduction of hazardous volatiles from baijiu using spent grain-derived activated carbon: Flavor preservation and DFT-elucidated adsorption mechanisms.

Food chemistry·2026
Same author

Investigating the flavor contributions of sulfur compounds in soy sauce flavor Baijiu using flavoromics and machine learning.

NPJ science of food·2026
Same author

Event-Triggered Predefined-Time-Synchronized Model Predictive Selective Impedance Control.

IEEE transactions on cybernetics·2026
Same author

PythiaStudio: a one-stop protein engineering platform powered by Pythia model suite.

Nucleic acids research·2026
Same author

Sequential inoculation with indigenous non-Saccharomyces yeasts drives multi-dimensional quality improvement of cabernet Gernischt wine: Mechanisms for greenness masking, color stabilization, and phenolic modulation.

Food research international (Ottawa, Ont.)·2026
Same author

Inhibitory effect of non-alcoholic compounds from spontaneously fermented beverage on <i>Helicobacter pylori</i>.

Frontiers in cellular and infection microbiology·2026

Related Experiment Video

Updated: May 22, 2025

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

Small RNA Toxin-Assisted Evolution of GC-Preferred ErCas12a for Enhanced Genome Targeting Range.

Zehua Chen1, Junyuan Xue2, Ziying Wang3

  • 1AIM center, College of Life Sciences and Technology, Beijing University of Chemical Technology, Institute of Microbiology, Chinese Academy of Sciences, Beijing, 100101, China.

Advanced Science (Weinheim, Baden-Wurttemberg, Germany)
|May 20, 2025
PubMed
Summary

Researchers developed enhanced Cas12a (enErCas12a) to overcome thymine-rich PAM limitations. This new gene editing tool efficiently targets guanine-cytosine-rich PAMs in bacterial and mammalian cells, expanding genomic accessibility.

Keywords:
CRISPR/Cas12aRNA toxin‐assisted evolutionexpanded PAM profilesgene editing

More Related Videos

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
09:51

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Published on: May 25, 2018

33.6K
A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins
08:37

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Published on: April 30, 2018

7.5K

Related Experiment Videos

Last Updated: May 22, 2025

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
Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms
09:51

Enhanced Genome Editing with Cas9 Ribonucleoprotein in Diverse Cells and Organisms

Published on: May 25, 2018

33.6K
A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins
08:37

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins

Published on: April 30, 2018

7.5K

Area of Science:

  • Molecular Biology
  • Gene Editing Technologies
  • Biotechnology

Background:

  • CRISPR/Cas12a systems are powerful gene editing tools but are limited by their requirement for thymine-rich protospacer adjacent motifs (PAMs).
  • Existing Cas12a variants struggle to access GC-rich genomic loci, hindering broader applications.
  • The need for Cas12a variants with expanded PAM specificity is critical for versatile gene editing.

Purpose of the Study:

  • To evolve the ErCas12a enzyme to target guanine-cytosine (GC)-rich PAMs.
  • To develop a novel small RNA toxin-aided strategy for enzyme evolution.
  • To create an enhanced Cas12a (enErCas12a) variant with an expanded PAM profile.

Main Methods:

  • Employed a small RNA toxin-aided evolution strategy to modify ErCas12a.
  • Tested enErCas12a's ability to recognize and target GC-rich PAMs (e.g., GCCC, CGCC, GGCC).
  • Assessed gene editing efficiency and specificity in both bacterial and mammalian cell systems.

Main Results:

  • Created enhanced ErCas12a (enErCas12a) capable of recognizing GC-rich PAMs.
  • enErCas12a targets five times more PAM sequences compared to wild-type ErCas12a.
  • Achieved efficient gene editing at GC-rich PAMs inaccessible to previous Cas12a variants in mammalian cells (e.g., >75% GC content).
  • Demonstrated high specificity for both canonical and non-canonical PAM targets.

Conclusions:

  • enErCas12a significantly expands the targeting range of Cas12a systems by enabling GC-rich PAM recognition.
  • This engineered enzyme offers a valuable new tool for gene editing in prokaryotes and eukaryotes.
  • The developed evolution strategy provides a framework for creating Cas enzymes with tailored PAM specificities.