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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.
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...
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CRISPR/Cas9 Editing of the C. elegans rbm-3.2 Gene using the dpy-10 Co-CRISPR Screening Marker and Assembled Ribonucleoprotein Complexes.
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Efficient Single-Nucleotide Microbial Genome Editing Achieved Using CRISPR/Cpf1 with Maximally 3'-End-Truncated

Ho Joung Lee1, Hyun Ju Kim1, Young-Jun Park2

  • 1Department of Systems Biotechnology and Institute of Microbiomics, Chung-Ang University, Anseong 17546, Republic of Korea.

ACS Synthetic Biology
|May 18, 2022
PubMed
Summary

Maximally truncating CRISPR/Cpf1 crRNAs enhances microbial genome editing accuracy. This modified system efficiently introduces single-base substitutions and indels, improving precision for gene editing applications.

Keywords:
FnCpf1crRNAmismatch tolerancesingle-base editing

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR/Cas systems are powerful genome editing tools, but mismatch tolerance causes off-target effects, limiting accuracy.
  • CRISPR/Cpf1 systems offer an alternative for genome editing, with crRNA recognition crucial for target specificity.

Purpose of the Study:

  • To investigate the impact of crRNA 3'-end truncations and mismatches on CRISPR/Cpf1 cleavage activity.
  • To develop a more precise CRISPR/Cpf1-mediated genome editing system for microbial applications.

Main Methods:

  • Systematic comparison of modified crRNAs with varying 3'-end nucleotide truncations and single mismatches using FnCpf1 in *Escherichia coli*.
  • Evaluation of genomic cleavage activity and editing efficiency at specific target genes (*galK*, *xylB*).

Main Results:

  • Maximum cleavage efficiency was achieved with a 5-nucleotide truncation at the crRNA 3'-end.
  • Maximally truncated crRNAs enabled highly efficient single-base substitutions (>80%) and single-nucleotide indel introduction (up to 79% in *galK*, 62% in *xylB*).
  • The truncated crRNA-Cpf1 complex demonstrated discrimination against mismatched targets, enhancing editing specificity.

Conclusions:

  • Maximally 3'-end-truncated crRNAs significantly improve the specificity and efficiency of CRISPR/Cpf1-mediated genome editing.
  • This optimized system provides a simple and effective tool for precise microbial genome editing, including base and indel correction at single-nucleotide resolution.