Related Experiment Video

Updated: Jul 16, 2025

Substrate Generation for Endonucleases of CRISPR/Cas Systems
11:53

Substrate Generation for Endonucleases of CRISPR/Cas Systems

Published on: September 8, 2012

27.4K

Structural basis for the activation of a compact CRISPR-Cas13 nuclease

Xiangyu Deng1, Emmanuel Osikpa2, Jie Yang2

  • 1Department of BioSciences, Rice University, Houston, TX, 77005, USA.

Nature Communications
|September 20, 2023
PubMed

Insights

Researchers characterized a compact CRISPR-Cas13 variant (Cas13bt3) for adeno-associated virus delivery. This variant shows unique RNA cleavage and activation mechanisms, enabling engineered versions with reduced off-target effects for therapeutic applications.

Area of Science:

  • Molecular Biology
  • Biochemistry
  • Structural Biology

Background:

  • CRISPR-Cas13 systems offer programmable RNA knockdown but face delivery challenges due to large effector size and non-specific cleavage.
  • Adeno-associated virus (AAV) delivery is limited by payload size, hindering the therapeutic application of large Cas13 proteins.

Purpose of the Study:

  • To characterize a compact Cas13 variant (Cas13bt3) suitable for AAV delivery.
  • To elucidate the biochemical and structural basis of Cas13bt3 activation and RNA cleavage.
  • To engineer Cas13bt3 variants with improved specificity for therapeutic RNA targeting.

Main Methods:

  • Biochemical assays to characterize Cas13bt3 activity and RNA cleavage.
  • Cryo-electron microscopy to determine the structure of active Cas13bt3.
  • Structure-guided engineering of Cas13bt3 variants.

Main Results:

  • Identified a compact Cas13bt3 effector suitable for AAV delivery.
  • Cas13bt3 exhibits unique target and non-specific RNA cleavage at internal "UC" sites, activated in a target length-dependent manner.
  • Determined the cryo-electron microscopy structure of active Cas13bt3, revealing its activation mechanism.
  • Engineered Cas13bt3 variants with significantly reduced off-target cleavage while retaining target cleavage activity.

Conclusions:

  • Cas13bt3 possesses a distinct activation mechanism compared to other Cas13 enzymes.
  • Structural insights guide the engineering of highly specific Cas13bt3 variants for RNA-targeting applications.
  • The characterized Cas13bt3 is a promising tool for AAV-mediated RNA modulation in therapeutics.

More Related Videos

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins10:46

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins

Published on: October 18, 2022

1.8K
Genome Editing in Mammalian Cell Lines using CRISPR-Cas07:56

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Published on: April 11, 2019

21.8K

Related Concept Videos

CRISPR and crRNAs02:53

CRISPR and crRNAs

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...
17.1K
CRISPR01:59

CRISPR

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...
52.1K
The Antiviral System of Bacteria and Archaea: CRISPR01:23

The Antiviral System of Bacteria and Archaea: CRISPR

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...
43
CRISPR/Cas9 Genome Editing01:28

CRISPR/Cas9 Genome Editing

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...
39
Caspases01:24

Caspases

Caspase, a family of cysteine proteases, serve as effectors in apoptosis. The ced3 gene in C.elegans was first identified to be involved in apoptosis. This gene encodes the ced-3 caspase that is similar to the interleukin-1-beta converting enzyme or ICE in mammals. In addition to apoptosis, caspases also function in the inflammatory response. Inflammatory caspases are essential in activating pro-inflammatory cytokines that recruit immune cells and block the replication of pathogens inside...
12.6K
Homologous Recombination02:31

Homologous Recombination

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...
50.6K