Related Experiment Video
Updated: Dec 25, 2025
14:46
Efficient Generation of hiPSC Neural Lineage Specific Knockin Reporters Using the CRISPR/Cas9 and Cas9 Double Nickase System
Published on: May 28, 2015
11.5K
PAM recognition by miniature CRISPR-Cas12f nucleases triggers programmable double-stranded DNA target cleavage
Tautvydas Karvelis1, Greta Bigelyte1, Joshua K Young2
1Institute of Biotechnology, Vilnius University, Vilnius LT-10257, Lithuania.
Nucleic Acids Research
|April 5, 2020
Summary
Researchers discovered exceptionally compact CRISPR-Cas12f nucleases. These miniature genome editing tools recognize specific DNA sequences and show potential for advanced gene editing applications.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- CRISPR-associated (Cas) nucleases, including Cas9 and Cas12, have transformed genome editing.
- The development of more compact Cas nucleases is crucial for simplifying delivery and expanding applications.
- Existing CRISPR systems require specific protospacer adjacent motif (PAM) sequences for DNA cleavage.
Purpose of the Study:
- To identify and characterize exceptionally compact CRISPR-Cas12f nucleases.
- To investigate the PAM specificities and DNA cleavage activities of these novel nucleases.
- To evaluate the potential of these miniature nucleases in bacterial defense systems and genome editing.
Main Methods:
- Bioinformatic analysis to identify novel Cas proteins.
- Biochemical assays to determine DNA cleavage activity and PAM recognition.
- Functional evaluation in *Escherichia coli* to assess anti-dsDNA defense capabilities.
Main Results:
- Discovery of 10 compact CRISPR-Cas12f nucleases (422-603 amino acids).
- Demonstration of PAM-dependent double-stranded DNA (dsDNA) cleavage triggered by 5' T- or C-rich PAM sequences.
- Evidence that some Cas12f nucleases confer resistance against invading dsDNA in *E. coli*.
Conclusions:
- Miniature Cas12f nucleases function similarly to larger class 2 CRISPR effectors in dsDNA targeting.
- These compact nucleases represent a promising new class of programmable tools for genome editing.
- The findings expand the toolkit for CRISPR-based technologies, offering simplified delivery and novel applications.
More Related Videos
11:35Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells
Published on: June 16, 2017
13.1K
10:07A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells
Published on: August 25, 2017
8.3K
Related Concept Videos
01:28CRISPR/Cas9 Genome Editing
1.5K
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...
1.5K
02:53CRISPR and crRNAs
18.5K
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...
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...
18.5K
01:59CRISPR
57.2K
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...
57.2K
01:23The Antiviral System of Bacteria and Archaea: CRISPR
531
CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats is a adaptive immune system found in bacteria and archaea that protects against viral infections. This system enables prokaryotic cells to identify, remember, and neutralize foreign genetic elements, primarily bacteriophages, by storing fragments of the invader’s DNA as a genetic memory.The CRISPR immune response begins during an initial infection. Cas (CRISPR-associated) proteins play a central role in this...
531
02:53Conservative Site-specific Recombination and Phase Variation
6.5K
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...
The recognition sites for Cre recombinase called LoxP...
6.5K
02:31Homologous Recombination
62.0K
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...
62.0K