Navigating the CRISPR-Cas Family: Distinguishing Cas13a, Cas12a/b, and Cas14

Navigating the CRISPR-Cas Family: A Comprehensive Guide to Cas13a, Cas12a/b, and Cas14

CRISPR-Cas technology has emerged as one of the most transformative tools in gene editing. As the CRISPR landscape continues to expand, an increasing variety of Cas proteins have been characterized, including Cas9, Cas12a, Cas12b, Cas13a, and Cas14. Understanding the unique properties of each can be challenging—this article provides a clear roadmap to help you distinguish these family members and their specialized roles.

Cas12a

Cas12a, also known as Cpf1, recognizes the TTN PAM site guided by crRNA to cleave target dsDNA. Unlike Cas9, Cas12a can self-process pre-crRNA into mature crRNA without the assistance of tracrRNA or other proteins. Therefore, Cas12a achieves target cleavage using only a single crRNA. Furthermore, Cas12a possesses non-specific ssDNA cleavage activity, also termed "trans-cleavage" activity.

Leveraging this property, the DNA-targeted nucleic acid detection system DETECTR was developed. This platform combines the CRISPR/Cas system with RPA isothermal amplification, enabling the highly efficient detection of various pathogenic microorganisms.

While Cas12a offers high sensitivity and practicality for DNA detection—being simple, rapid, and requiring no large-scale instrumentation—it lacks the precision to detect single-base variations, leaving room for improvement compared to other CRISPR detection systems.

Cas12b

Cas12b, also known as C2c1, recognizes the TTN PAM site guided by both crRNA and tracrRNA to cleave target DNA.

Utilizing its trans-cleavage activity, the Cas12b-mediated DNA detection platform "C-Detection" was developed. Compared to earlier Cas12a methods, this platform offers superior sensitivity. By introducing a tertiary guide RNA (tgRNA), the system can distinguish single-base differences, overcoming the limitations of Cas12a and providing a highly efficient and practical platform for DNA diagnostics.

Additionally, the HOLMESv2 platform was developed by combining Cas12b with Loop-mediated Isothermal Amplification (LAMP), enabling one-step nucleic acid detection and DNA methylation quantification.

Cas13a

Cas13a, also known as C2c2, recognizes and cleaves target ssRNA guided by crRNA. Similar to Cas12a, it possesses the collateral ability to non-specifically cleave any ssRNA sequences within the system.

By integrating Cas13a with RPA isothermal amplification, the Cas13a-mediated RNA detection platform SHERLOCK was developed. This method has been successfully used to detect Zika and Dengue viruses and has shown promising results in the detection of oncological diseases.

Cas14

Cas14 is an exceptionally compact nuclease, approximately half the size of other Cas family proteins. Cas14a recognizes and cleaves target ssDNA and similarly possesses collateral cleavage activity, making it ideal for ssDNA detection. Notably, Cas14 enables high-fidelity SNP genotyping.

Because its recognition of DNA molecules does not rely on a PAM site, Cas14 is extremely stringent regarding sequence accuracy; even a single-base mismatch can severely inhibit its cleavage activity.

Summary

• For double-stranded DNA (dsDNA) detection, Cas12a and Cas12b are preferred.
• For RNA detection, Cas13 is the optimal choice.
• For single-stranded DNA (ssDNA) detection, Cas14 is highly recommended.
Detection methods established by combining CRISPR/Cas systems with RPA isothermal amplification offer high resolution, high sensitivity, low cost, and ease of operation. These technologies hold immense potential for applications in clinical diagnostics, environmental assessment, and food safety.

EDITGENE has developed a proprietary protein expression and purification platform, through which our Cas enzyme products demonstrate significantly higher activity compared with other commercial enzymes, as confirmed by rigorous activity testing. This enables the production of enzymes with truly high purity, high activity, and high sensitivity.

In addition, we provide ready-to-use isothermal rapid amplification kits and CRISPR detection kits. These products feature low equipment requirements, short reaction times, and immediate availability—ensuring that customers can achieve fast and reliable results with maximum convenience.