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Related Concept Videos

CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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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...
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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...
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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.
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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.
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Related Experiment Video

Updated: Sep 12, 2025

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins
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CRISPR/Cas system-guided plasmid mutagenesis without sequence restriction.

Fengjiao Zhao1, Feng Chen1, Huahang Yu1

  • 1Institute of Analytical Chemistry and Instrument for Life Science, The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi'an Jiaotong University, Xi'an 710049, China.

Fundamental Research
|August 8, 2025
PubMed
Summary
This summary is machine-generated.

This study introduces CRISPR/Cas system-guided plasmid mutagenesis for engineering protein variants. The new method efficiently creates user-defined mutation libraries on plasmids, improving protein engineering and genome editing tools.

Keywords:
CRISPR/Cas systemDNA assemblyPlasmid mutationProtein engineeringSequence programmability

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Protein Engineering

Background:

  • Plasmid mutagenesis is crucial for engineering proteins with novel functions.
  • Current methods like PCR-based techniques and subcloning have limitations, particularly in generating single-stranded circular plasmids required for certain mutagenesis strategies.
  • Directly generating mutations on plasmids offers advantages but faces sequence restrictions.

Purpose of the Study:

  • To develop a novel CRISPR/Cas system-guided method for plasmid mutagenesis.
  • To enable direct generation of user-defined mutation libraries on plasmids, overcoming existing sequence limitations.
  • To engineer genome-editing protein FnCpf1 variants with improved properties.

Main Methods:

  • Utilizing a CRISPR/Cas system with a guide RNA (gRNA) and Cas9 nickase to generate single-stranded circular plasmids.
  • Employing these single-stranded plasmids as templates for direct mutagenesis.
  • Combining the mutagenesis method with rational design principles for protein engineering.

Main Results:

  • The CRISPR/Cas system-guided mutagenesis method successfully generated user-defined mutation libraries on plasmids.
  • The approach demonstrated broad sequence programmability and compatibility with methylated plasmids.
  • Engineered FnCpf1 variants showed expanded PAM range and reduced off-target effects, enhancing genome editing specificity.

Conclusions:

  • CRISPR/Cas system-guided plasmid mutagenesis is an effective tool for creating mutation libraries and engineering proteins.
  • This method overcomes sequence restrictions associated with previous plasmid mutagenesis techniques.
  • The engineered FnCpf1 variants offer improved genome editing capabilities, loosening PAM constraints and increasing target specificity.