Related Experiment Video

Updated: May 13, 2025

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

Substrate Generation for Endonucleases of CRISPR/Cas Systems

Published on: September 8, 2012

27.3K

Trans-cleavage activity of Cas12a effectors can be unleashed by both double-stranded DNA and single-stranded RNA

Guohui Xiao1, Hongyu Shi2, Meixia Liu1

  • 1National Clinical Research Center for Infectious Diseases, Guangdong Provincial Clinical Research Center for Tuberculosis, Shenzhen Third People's Hospital, Southern University of Science and Technology, Shenzhen 518112, China.

International Journal of Biological Macromolecules
|April 13, 2025
PubMed

Insights

CRISPR-Cas12a can detect nucleic acids without needing a protospacer adjacent motif (PAM). This discovery expands the use of CRISPR-Cas12a technology for sensitive detection of diverse DNA and RNA targets.

Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetics

Background:

  • CRISPR-Cas12a is a key tool for nucleic acid detection.
  • Its trans-cleavage activity and dependence on protospacer adjacent motif (PAM) sequences are not fully understood.

Purpose of the Study:

  • To investigate the relationship between CRISPR-Cas12a trans-cleavage activity and PAM sequences.
  • To explore novel applications of CRISPR-Cas12a in nucleic acid detection.

Main Methods:

  • Synthesized diverse PAM-sequence substrates.
  • Conducted systematic cis-cleavage and trans-cleavage experiments with three Cas12a orthologs.
  • Utilized AlphaFold 3 for structural prediction.

Main Results:

  • Double-stranded DNA (dsDNA) activates Cas12a trans-cleavage independently of PAM and cis-cleavage.
  • Single-stranded RNA (ssRNA) can directly initiate Cas12a trans-cleavage.
  • Validated CRISPR-Cas12a for detecting PAM-lacking dsDNA, including clinical mutations.

Conclusions:

  • CRISPR-Cas12a exhibits PAM-independent trans-cleavage activity activated by dsDNA and ssRNA.
  • This expands the utility of CRISPR-Cas12a for detecting nucleic acids beyond PAM-dependent targets.
  • Paves the way for developing versatile and sensitive diagnostic platforms.
Keywords:
CRISPR-Cas12aCis-cleavageProtospacer adjacent motifTrans-cleavage

More Related Videos

Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System07:59

Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System

Published on: April 25, 2025

56
Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection08:16

Identifying Caspases and their Motifs that Cleave Proteins During Influenza A Virus Infection

Published on: July 21, 2022

1.4K

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...
16.3K
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...
11.5K
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...
48.6K
Single-Strand DNA Binding Proteins01:03

Single-Strand DNA Binding Proteins

For successful DNA replication, the unwinding of double-stranded DNA must be accompanied by stabilization and protection of the separated single strands of the DNA. This crucial task is performed by single-strand DNA-binding (SSB) proteins. They bind to the DNA in a sequence-independent manner, which means that the nitrogenous bases of the DNA need not be present in a specific order for binding of SSB proteins to it. The binding of SSB proteins straightens single-stranded DNA (ssDNA) and makes...
13.8K
DNA Helicases00:55

DNA Helicases

DNA unwinding helicase enzymes are a type of motor protein. Motor proteins can translocate along filaments or polymers using energy generated from ATP hydrolysis. Helicases are involved in all the important cellular processes where DNA unwinding is required, such as DNA replication, repair, recombination, and transcription. They are present in all living organisms, but vary in their structure, function, and mechanism of action. For example, in prokaryotes, DnaB helicase binds and translocates...
21.0K
Translesion DNA Polymerases02:10

Translesion DNA Polymerases

Translesion (TLS) polymerases rescue stalled DNA polymerases at sites of damaged bases by replacing the replicative polymerase and installing a nucleotide across the damaged site. Doing so, TLS allows additional time for the cell to repair the damage before resuming regular DNA replication.
TLS polymerases are found in all three domains of life - archaea, bacteria, and eukaryotes. Of the different classes of TLS polymerases, members of the Y family are fitted with specialized structures that...
9.7K