Bioinformatics Biotechnology

CRISPR-Cas12

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Cas12 is a compact and efficient enzyme that produces staggered cuts in dsDNA. Cas12 processes its own guide RNAs, leading to increased multiplexing ability.

Cas12 has also been engineered as a platform for epigenome editing, and it was recently discovered that Cas12a can indiscriminately chop up single-stranded DNA once activated by a target DNA molecule matching its spacer sequence. This property makes Cas12a a powerful tool for detecting tiny amounts of target DNA in a mixture. Cas12 is an RNA-guided protein that binds and cuts any matching DNA sequence. Binding of the Cas12-CRISPR RNA (crRNA) complex to a matching single-stranded DNA (ssDNA) or double-stranded DNA (dsDNA) molecule activates the protein to non-specifically degrade any ssDNA in trans.

Type II CRISPR-Cas9 and type V CRISPR-Cas12a systems naturally evolve to defend against invading foreign DNAs. The defense process includes three phases: spacer acquisition, crRNA biogenesis, and target interference. Interestingly, anti-CRISPR-Cas systems evolved by phages are discovered recently, revealing an evolutionary arms race between CRISPR-Cas systems and foreign DNA invaders. 

Cas12 as important effectors in CRISPR-Cas, have been engineered to generate variants for different purposes. As only bearing the RuvC domain for DNA cut, Cas12 can be changed to a nuclease-deactivated Cas12a, and cannot be engineered to form Cas12a-nickase. Despite functionally irreversible binding, Cas12 distinguishes strongly against mismatches along most of the DNA target sequence. 

CRISPR-Cas12 is well-studied CRISPR nucleases, and thoroughly engineered and optimized in bacteria for broad applications, especially in metabolic engineering and synthetic biology. Bacteria as cell factories can take up simple and cheap feedstock, like renewable biomass and even wastes, for basic cell metabolism and biosynthesis of value-added chemicals. To increase the performance of the cell factory, genetic manipulation on them is often required. Nowadays, CRISPR-Cas12 based technologies have greatly helped the genetic manipulation of model and non-model bacteria for higher editing efficiency and specificity. 

The first Cas12 enzyme, classified as type V-A and known as Cas12a (previously known as Cpf1) was identified in the genomes of Prevotella and Francisella and had a large protein of unknown function. Cas12a is a distinct enzyme unrelated to Cas9. A number of Francisella species have Cas12a in association with putative CRISPR arrays, including F. novicida. In contrast to Cas9, the Cas12a system does not contain a tracrRNA, and its DNA cleavage results in a 5′ overhang instead of a blunt DSB. Also, unlike Cas9, which uses host RNase III to process its CRISPR array, Cas12a itself has RNase activity and processes its own pre-crRNA array into individual crRNAs.

A search for Cas12a orthologs showed two-Cas12a enzymes, from Acidaminococcus and Lachnospiraceae, with strong cleavage activity in human cells, comparable to SpCas9. 

Relative to the Cas9 family of Cas effectors, Cas12 is a much more diverse family. Indeed a number of subtypes of Cas12 systems have recently been reported. The Cas12b effectors (previously known as C2c1) target DNA, but in contrast to Cas12a, they are dual-RNA guided, requiring a tracrRNA. Although initial characterization of Cas12b indicated thermophilic nuclease activity, which prevented application in mammalian cells, subsequent exploration of the Cas12b diversity and protein engineering made possible the development of two-Cas12b systems with robust genome editing activity in human cells. Comparison of Cas12b with SpCas9 showed that Cas12b has substantially reduced off-target activity, indicating it is inherently more specific than wild-type SpCas9 when targeting the human genome. Additional Cas12 effectors have also been identified from bacterial genomic databases, including Cas12c, Cas12d (CasY) and Cas12e (CasX), both of which were found in metagenomic samples and three subtypes of Cas12f (Cas14). 

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