In this era of rapid technological advancement, CRISPR diagnosis has emerged as a captivating application of biotechnology, drawing widespread attention. At the core of CRISPR diagnosis is the utilization of specific protein families, including Cas12, Cas13, and Cas14, forming complexes with proteins and guide molecules to achieve recognition and cleavage of specific nucleic acid sequences.
Basic Principles of CRISPR Diagnosis
The fundamental principle of CRISPR diagnosis involves a process of non-specific cleavage. This technology comprises two key components: the complex of proteins and guide molecules. These complexes first cleave the specific nucleic acid sequences that users want to detect and then proceed to cleave other non-specific nucleic acids. Modified nucleic acids, also known as reporter genes, generate visual signals upon cleavage, making them easily detectable only after the specific nucleic acids have been cleaved.
Introduction to Cas12, Cas13, and Cas14
Cas12, Cas13, and Cas14 are crucial members of the protein family in CRISPR diagnosis, constituting the protein part of the complex with proteins and guide molecules. These proteins are derived from specific types of bacteria and archaea, yet they share some key features.
- Cas12 proteins directly bind and cleave user-specified DNA sequences, capable of cleaving both single-stranded and double-stranded DNA. After Cas12 proteins cleave their DNA targets, they non-specifically cleave single-stranded DNA. Therefore, diagnostics based on Cas12 can only directly detect DNA and must be combined with proteins that convert RNA to DNA for RNA detection.
- Cas12 proteins are located on the larger side of CRISPR diagnostic proteins, with a length of approximately 1,300 amino acids.
- Users employ 42-44 nt RNA molecules to specify Cas12 DNA targets.
- Cas12 dsDNA targets are restricted as they must be located near short DNA fragments called protospacer adjacent motifs (PAM). For some Cas12 proteins, the PAM sequence is TTTN. Importantly, without PAM, diagnostics based on Cas12 cannot detect DNA sequences.
- Cas12 proteins can easily distinguish very similar dsDNA sequences. This functionality is lost when the target sequence is ssDNA.
- Cas13 proteins directly bind and cleave user-specified RNA sequences, with a length of approximately 1,400 amino acids. Similar to Cas12, Cas13 RNA guide molecules are relatively short, around 64 nt.
- Cas13 proteins lack strong targeting restrictions, but their RNA targets can adopt structures that are challenging to cleave. Cas13 cleaves RNA sequences 1 nt away from specific sequences, requiring careful testing when differentiating highly similar sequences using Cas13-based diagnostics.
- Cas14 proteins bind and cleave specific single-stranded or double-stranded DNA. To detect RNA, diagnostics based on Cas14 must be combined with proteins that can convert RNA to DNA.
- Cas14 proteins have a smaller end, consisting of 400-700 amino acids. However, their guide RNA is located on the longer side, approximately 140 nt.
- Cas14 proteins exhibit no targeting restrictions when cleaving ssDNA, making them versatile. For dsDNA targeting, Cas14 proteins require a PAM sequence rich in T, such as TTTA.
- Cas14 proteins can easily distinguish very similar ssDNA sequences.
Applications and Future Prospects of Protein Combinations
Each protein family has its advantages and disadvantages, but researchers have demonstrated creative ways to combine proteins from multiple families in a single detection. This combination can be used to simultaneously detect multiple targets, opening up new possibilities for the widespread application of CRISPR diagnostics.
Scientists continue to discover and improve new CRISPR systems, and it is believed that more innovative CRISPR diagnostic tools will emerge in the future. These tools will find extensive applications in healthcare, agriculture, food safety, infectious diseases, and other fields, contributing to human health and societal development.
At SBS Genetech, we take pride in offering a comprehensive selection of Cas12, Cas13, and Cas14 proteins, empowering researchers and diagnostic professionals with cutting-edge tools for CRISPR applications. Our diverse range of proteins is designed to cater to various diagnostic needs, ensuring precision and efficiency in nucleic acid sequence recognition and cleavage.