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