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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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CRISPR01:59

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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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CRISPR and crRNAs02:53

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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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Updated: Jan 11, 2026

A Rapid and Facile Pipeline for Generating Genomic Point Mutants in C. elegans Using CRISPR/Cas9 Ribonucleoproteins
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A Practical CRISPR-Based Method for Rapid Genome Editing in Caulobacter crescentus.

Xuezhou Yuan1,2,3, Xin Yu1,2, Wei Zhao1,2

  • 1State Key Laboratory of Quantitative Synthetic Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen, China.

Bio-Protocol
|November 12, 2025
PubMed
Summary

This study introduces a CRISPR/SpCas9M-reporting system for efficient genome editing in recalcitrant bacteria like Caulobacter crescentus. The novel system enables rapid, marker-less gene deletion and other modifications within a week.

Keywords:
CRISPRCaulobacter crescentusGenome editingReporting systemSpCas9M

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Area of Science:

  • Microbiology
  • Molecular Biology
  • Biotechnology

Background:

  • CRISPR-Cas systems offer precise genome editing but face challenges in certain bacteria.
  • Caulobacter crescentus and related species are important industrially and biomedically, yet difficult to genetically manipulate.
  • Existing CRISPR/Cas9 methods struggle with SpCas9 expression and CRISPR escape in these organisms.

Purpose of the Study:

  • To develop an efficient genome editing system for CRISPR/Cas-recalcitrant bacteria.
  • To overcome limitations of SpCas9 expression and CRISPR escape in Caulobacter and relatives.
  • To establish a rapid, marker-less gene deletion protocol for C. crescentus.

Main Methods:

  • Developed a CRISPR/SpCas9M-reporting system by fusing a reporter gene to SpCas9M.
  • Optimized SpCas9M expression for enhanced editing efficiency.
  • Applied the system for marker-less gene deletion in Caulobacter crescentus.

Main Results:

  • The CRISPR/SpCas9M-reporting system achieved high apparent editing efficiency by overcoming CRISPR escape.
  • The method enabled in-frame, marker-less chromosomal modifications.
  • A complete genome editing cycle was achieved within one week.
  • The system demonstrated applicability for C. crescentus, A. fabrum, and S. meliloti.

Conclusions:

  • The CRISPR/SpCas9M-reporting system provides an efficient solution for genome editing in previously recalcitrant bacteria.
  • This technology facilitates rapid, marker-less genetic modifications, accelerating research and applications.
  • The protocol is adaptable for diverse genome editing applications in industrially relevant bacteria.