Related Experiment Video

Updated: Jul 19, 2025

Gene Digital Circuits Based on CRISPR-Cas Systems and Anti-CRISPR Proteins
10:46

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

Published on: October 18, 2022

1.8K

Inhibitory mechanism of CRISPR-Cas9 by AcrIIC4

Xuzichao Li1, Fumeng Liao1, Jiaqi Gao1

  • 1State Key Laboratory of Experimental Hematology, Key Laboratory of Immune Microenvironment and Disease (Ministry of Education), The Province and Ministry Co-sponsored Collaborative Innovation Center for Medical Epigenetics, Haihe Laboratory of Cell Ecosystem, Department of Biochemistry and Molecular Biology, School of Basic Medical Sciences, Tianjin Medical University, Tianjin 300070, China.

Nucleic Acids Research
|August 17, 2023
PubMed

Insights

Anti-CRISPR protein AcrIIC4 inhibits Neisseria meningitidis Cas9 (NmeCas9) by locking its REC2 domain, preventing DNA binding and cleavage. This discovery offers insights into CRISPR-Cas regulation and gene editing tool development.

Area of Science:

  • Molecular Biology
  • Microbial Immunity
  • Structural Biology

Background:

  • CRISPR-Cas systems provide adaptive immunity in prokaryotes against foreign genetic elements.
  • Anti-CRISPR (Acr) proteins are viral factors that counteract CRISPR-Cas activity.
  • The inhibition mechanism of AcrIIC4 on Neisseria meningitidis Cas9 (NmeCas9) was previously unclear.

Purpose of the Study:

  • To biochemically and structurally elucidate the anti-CRISPR mechanism of AcrIIC4 against NmeCas9.
  • To understand how AcrIIC4 interacts with NmeCas9 and sgRNA.
  • To provide a basis for developing regulatory tools for Cas9 gene editing.

Main Methods:

  • Biochemical assays to assess inhibitory activity.
  • Structural studies (likely crystallography or cryo-EM) to determine complex structure.
  • Site-directed mutagenesis to identify key interaction residues.

Main Results:

  • AcrIIC4 forms a helix bundle that binds to the REC lobe of NmeCas9 and sgRNA.
  • AcrIIC4 locks the REC2 domain of NmeCas9, disrupting DNA binding and cleavage.
  • Mutations at the AcrIIC4-NmeCas9 or AcrIIC4-sgRNA interfaces significantly reduce inhibition.

Conclusions:

  • AcrIIC4 inhibits NmeCas9 by a novel mechanism involving conformational locking of the REC2 domain.
  • The findings clarify the molecular basis of AcrIIC4-mediated NmeCas9 suppression.
  • This research informs the engineering of controllable Cas9-based gene editing technologies.

More Related Videos

Substrate Generation for Endonucleases of CRISPR/Cas Systems11:53

Substrate Generation for Endonucleases of CRISPR/Cas Systems

Published on: September 8, 2012

27.4K
Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells11:35

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Published on: June 16, 2017

12.6K

Related Concept Videos

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...
52.3K
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
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...
54
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...
56
Experimental RNAi02:15

Experimental RNAi

RNA interference (RNAi) is a cellular mechanism that inhibits gene expression by suppressing its transcription or activating the RNA degradation process. The mechanism was discovered by Andrew Fire and Craig Mello in 1998 in plants. Today, it is observed in almost all eukaryotes, including protozoa, flies, nematodes, insects, parasites, and mammals. This precise cellular mechanism of gene silencing has been developed into a technique that provides an efficient way to identify and determine the...
6.2K
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