Cas12a: Definition, Applications, & Industry Uses

Cas12a: Definition, Applications, & Industry Uses

Cas12a: Overview

CRISPR-Cas9 system (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein 9) offers adaptive immunity in bacteria and archaea. It uses RNA-guided nucleases to target and cleaves foreign nucleic acids and other mobile genetic elements, such as transposons and plasmids.

The CRISPR-Cas systems are classified into two classes:

  • Class I: It includes CRISPR subtypes I, III, and IV, which involve the use of multiple Cas proteins in effector nucleases and have ∼90% of all identified CRISPR-Cas loci. They are profoundly present in archaea and bacteria.  
  • Class II: It involves the use of single Cas proteins and includes subtypes II, V, and VI CRISPR systems. It consists of a ribonucleoprotein complex made of CRISPR RNA (or crRNA) and a Cas protein and the remaining 10% of CRISPR loci.

The most commonly known family of Cas protein is Cas9 (or spCas9) which cuts double-stranded DNA (dsDNA), complementary to the guide RNA sequence, induced by RuvC and HNH domains. It originated from Streptococcus pyogenes) and has been applied in a range of gene-editing workflows.

One other family of Cas enzyme that cuts dsDNA is Cas12. It’s a compact efficient enzyme that generates sticky ends through the staggered cut of the DNA template. The enzyme has increased multiplexing ability due to having its own guide RNA. It has also been engineered to serve epigenome editing.

One form of Cas12 enzyme is Cas12a, which can cleave ssDNA when activated by a target DNA sequence matching its spacer sequence. As a result, Cas12a is considered an effective tool for detecting tiny amounts of target DNA in a mixture.

Figure: CRISPR-Cas12 system.

What is Cas12a?

Cas12a is a Class-2, type V CRISPR system, identified as CRISPR nucleases in Prevotella and Francisella 1 bacteria. It’s also known as the CRISPR-Cpf1 system. It appears in many other bacterial species but the two forms that show efficient genome editing in human cells are obtained from Acidaminococcus and Lachnospiraceae.

In this article, we will review how do Cas12a enzyme work, its crystal structure, and its applications in life science labs and industries.

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How Does Cas12a Work?

CRISPR-Cpf1 system employs a multistep mechanism to ensure the recognition and cleavage of the target sequence with precision and accuracy.

  • The hybridization of DNA target and PAM recognition is initiated by the WED II-III, REC1, and PAM-interacting domains of the enzyme.
  • After recognition, the helical dsDNA is unwound by the insertion of a dsDNA at a 45° angle. This is carried out by the conserved loop-lysine helix-loop (LKL) region in the PI domain.
  • The unwinding of the DNA target allows the hybridization of crRNA with the PAM-containing strand. And, the uncoupled DNA strand and non-target strands are sent toward the DNase site for degradation.

Figure: Schematic representation of Cas12a crRNA with the target strand DNA association.

Difference Between Cas9 and Cas12a

CRISPR-Cas12a (Cpf1) is a guide RNA-guided endonuclease that cleaves dsDNA using its RuvC domain. It has three homologs, which include LbCas12a (from Lachnospiraceae bacterium), FnCas12a (from Francisella novicida), and AsCas12a (from Acidaminococcus sp.).

There are many ways through which this enzyme differs from the CRISPR-Cas9:

  • Unlike Cas9, Cas12a enzymes catalyze their own guide CRISPR RNA (crRNA) maturation, recognize a T nucleotide–rich protospacer-adjacent motif (PAM), and produce a staggered 5′ and 3′ dsDNA break. These features are the interests of many gene editing applications. To minimize off-target events in lab workflows, scientist Chen and his colleague identified two Cas12a variants (BeCas12a and CeCas12a) having more stringent PAM sites.
  • In contrast to Cas9, Cas12a cleaves the target DNA strand 18-23 nucleotides (nt) distal of the PAM, leaving staggered ends (5 to 8 nt 5′ overhangs).
  • Compared to Cas9, Cas12a enzymes have a lower off target and are similar to spontaneous mutations generated during plant development. But, the enzyme has been shown to cleave random targets in vitro containing about four mismatches.

Figure: Schematic representation of salient differences between CRISPR-Cas9 and Cas12a.

What is Cas12a Used For?

Cas12a has a range of in vitro and in vivo applications. The enzyme is extensively used for its ability to generate targeted, double-stranded DNA breaks for gene editing workflows. It has intrinsic RNase activity that facilitates the processing of its own crRNA array, allowing multigene editing from a single transcript. Further, it’s also used for programmable editing of a target base in genomic DNA without cleaving dsDNA.

However, the Cas12a enzyme also has a limitation in plant genome editing. It required a long TTTV PAM sequence to perform DNA cleavage activity at the target site.

Nucleic Acid Detection

CRISPR-Cpf1 is an effective system to detect nucleic acids of interest. It has been frequently used to detect viral pathogens in contaminated food, clinical samples, soil, and water to reduce the socioeconomic impact of diseases and improve clinical outcomes.

Diagnostic Assay

CRISPR-Cas is used in combination with several analytical assays to identify target DNA, edit specific nucleic acid sequences, or target DNA amplification. It includes:

  • Fluorescence In Situ Hybridization (DNA-FISH) assay
  • CRISPR-Cas triggered isothermal exponential amplification reaction (CAS-EXPAR)
  • CRISPR rolling circular amplification (CRISPR-RCA) assay
  • DNA endonuclease-targeted CRISPR trans-reporter (DETECTR) and one-hour low-cost multipurpose highly efficient system (HOLMES) based CRISPR-Cas12a technique
  • specific high-sensitivity enzymatic reporter unlocking (SHERLOCK) system-based CRISPR-C2c2 technique
  • SHERLOCKv2 assay

What Industries Use Cas12a?

Many research labs and pharmaceutical and medical industries exploit the CRISPR-Cas12a system for a range of applications. Guide RNA (sgRNA) is designed based on the goal of the experiment, which influences the specificity and efficiency of the CRISPR system.

The Cas12a system is a robust, rapid, inexpensive, sensitive, and selective method of detecting viral DNA. It can be done without additional sample purification and amplification. Further, the CRISPR-Cas12a system shows great promise for bioassay research using E-CRISPR.

Additionally, the CRISPR-Cas system offers a better alternative to techniques like chemical mutagenesis as it purges off-target events and has a lower mutational load.


CRISPR-Cas12a system is used in many diagnostic and medical applications. It has been used to treat several genetic illnesses, implement gene drives, cure diseases caused due to mutations, and as cancer treatments.


CRISPR-Cas12a is one the popular genome editing tool. According to a study by Zhang and their team, the system allows reagents to deliver directly into cells without inserting DNA in the genome during genome editing workflows. It creates mutations identical mutations to naturally occurring ones, which might simplify the regulation process for traditional genetically modified crops.

The tool has been successfully used to precisely replace genes in rice by RNA transcript-templated homologous recombination. Further, chemically modified Cpf1-CRISPR RNAs are also used to edit the genome in mammalian cells.

Figure: Application of CRISPR-Cas12a.

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CRISPR-Cas system was discovered as a part of the bacterial immune mechanism. Over time different forms of the system have been found in different bacterial forms. One such system is the CRISPR-Cas12a system. It’s a type V CRISPR protein, which generates double-stranded breaks in DNA.

It’s widely used in genome editing applications and detection and identification of viral DNA combined with other high-throughput workflows, such as next-generation sequencing (NGS), Quantitative real-time PCR (qPCR), and enzyme-linked immunosorbent assays (ELISA).

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