AsCas12a

Summary

In 2015, AsCas12a (previously AsCpf1) was characterized as an RNA-guided endonuclease capable of efficient mammalian genome-editing activity ¹ and has subsequently become a staple in the CRISPR toolbox. Recognizing a thymine-rich 5'-TTTV-3' PAM on the 5' side of the protospacer (on the non-target strand), AsCas12a utilizes a single catalytic site to cleave RNA-complementary double-stranded DNA (cis cleavage), as well as indiscriminately cleave single-stranded DNA (trans cleavage), ³ upon RNA-guided DNA binding. This has led to its use in rapid and highly sensitive nucleic acid detection ^{4 3} . AsCas12a and related orthologs LbCas12a and FnCas12a generate staggered cuts within the protospacer but distal to the PAM ¹ . Because AsCas12a does not require tracrRNA and can process the CRISPR array into crRNAs internally, it is a good tool for multiplex editing ⁵ .

Applications

Administering AsCas12a complexed with a guide has biotechnological applications in healthcare, agriculture, and fundamental research. CRISPR-based treatments employing AsCas12a Ultra, an engineered variant of AsCas12a, are currently under investigation in human clinical trials ⁶ . Target-activated trans DNase activity has also been harnessed for detection of specific viral nucleic acid sequences ⁴ in human patient specimens ³ .

Additionally, by leveraging AsCas12a's ability to introduce targeted genetic disruptions, scientists have built successful platforms for genetic screening ⁷ , multiplex editing ⁸ , and model system creation ^{6 9} . AsCas12a, and related enzyme LbCas12a, have also been harnessed for agricultural purposes, improving plant immunity and nutritional content for staple crops like rice ¹⁰ . A more comprehensive listing of all agricultural Cas12a-based engineering can be found in other resources ¹¹ , but several examples may be found in the experimental section of CasPEDIA.

Experimental Considerations

When using AsCas12a and related orthologs, such as LbCas12a, MbCas12a and FnCas12a, several experimental considerations should be taken into account.

Multiple software tools, such as CHOPCHOP ¹² and CRISPOR ¹³ , exist for guide design ¹⁴ to help identify suitable target sites throughout the genomes of several model organisms. The T-rich PAM requirements of AsCas12a allow it to complement the PAM restriction to G-rich genomic sites in Cas9 editing ² . The protospacer-adjacent motif (PAM) sequence (TTTV) of Cas12a lies 5' of the target site on the non-target strand, in contrast to Cas9, whose PAM lies 3' of the target site on the non-target strand. It is important to note that the individual "staggered" cuts reported in the original discovery paper ¹ represent only a subset of the cuts made by AsCas12a in an RNA-complementary DNA target; in reality, AsCas12a forms an ~5-nt gap in the non-target strand and an ~2-nt gap in the target strand ¹⁵ .

AsCas12a's ability to process its own pre-crRNA facilitates multiplex applications, as multiple crRNAs can be synthesized in one transcript off of a single promoter. If the experimentalist's goal is to induce homology-directed repair (HDR), Cas12a may be a particularly efficient choice of CRISPR endonuclease ^{16 17 18} . It is hypothesized that the efficiency with which Cas12a induces HDR stems from its staggered cut and the speed with which it releases the PAM-distal cleavage product ¹⁶ .

Engineered versions of AsCas12a exist that broaden its PAM targeting ¹⁹ , improve its nuclease activity ⁶ , or improve its specificity ²⁰ . These variants may be better suited for certain genome-engineering goals than WT AsCas12a. Because Cas12a homologs cleave both the non-target strand and target strand using a single active site ²¹ , these enzymes cannot be trivially engineered to form DNA nicks in the way that Cas9 can ²² . A variant of AsCas12a originally reported to act as a nickase (AsCas12a R1226A) ²³ was later revealed to have double-stranded DNase activity that was dramatically slowed relative to WT AsCas12a ¹⁵ .

Experimental Details

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Gene Editing 

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Tools and Diagnostics

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Engineered Variants

PFAM Annotation

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Features

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Active Site

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Protein Structure

REFERENCES