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CRISPR-Cas12a

CRISPR-Cas12a

CRISPR-Cas12a is is a genome editing tool similar to CRISPR-Cas9. CRISPR-Cas12a is a set of RNA guided DNA targeting proteins capable of producing targeted double-stranded DNA (dsDNA) breaks, targeted single-stranded DNA breaks, and indiscriminate ssDNA degradation in trans.

CRISPR-Cas12a is a genome editing tool similar to CRISPR-Cas9. Both systems use guide RNA, which are designed by the researcher, to target the protein complex to DNA and both produce targeted, site-specific double stranded DNA (dsDNA) breaks. CRISPR-Cas12a is more precise and less tolerant of mismatches between the guide RNA and target sequence than CRISPR-Cas9 .

In addition to this activity, Jennifer Doudna’s lab at University of California, Berkeley and Jin Wang’s lab at Chinese Academy of Sciences, Shanghai reported that CRISPR-Cas12a indiscriminately degrades single-stranded DNA (ssDNA) in trans to ssDNA not complimentary to the guide RNA . Wang’s lab also described guide RNA directed ssDNA cleavage for CRISPR-Cas12a.

Since ssDNA degradation in trans is activated by CRISPR-Cas12a recognition of target DNA, Doudna’s group developed the DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) system to detect specific DNA sequences like dsDNA viruses . The system includes a ssDNA fluorophore quencher that fluoresces upon ssDNA degradation to report detection of the HPV DNA sequence by CRISPR-Cas12a. DETECTR detected human papillomavirus (HPV) and distinguished between two types of HPV in DNA extracted from human anal swabs.

Wang’s group developed a similar system called HOur Low-cost Multipurpose highly Efficient System (HOMES) to detect DNA sequences using CRISPR-Cas12a with a quenched ssDNA reporter . In extracted DNA from human cell lines or PCR amplified saliva samples HOMES distinguished sequence variations between samples at single nucleotide polymorphism (SNP) sites that are related to human health and personal characteristics. HOMES also detected viruses and distinguished between different viral strains. To detect RNA viruses, RNA samples need to be first reverse transcribed into cDNA.

The CRISPR-Cas12a non-specific ssDNA degradation is similar to the non-specific cleavage of RNA observed by CRISPR-Cas13a. Similarly, non-specific cleavage of RNA is used as a readout for when CRISPR-Cas13a detects viral RNA sequences in the SHERLOCK system.

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Characterized CRISPR-Cas12 activity, developed DETECTR

Jin Wang

Characterized CRISPR-Cas12 activity developed HOMES

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