The engineered single guide RNA structure as a biomarker for gene-editing reagent exposure
Abstract CRISPR arrays and CRISPR-associated (Cas) proteins comprise a prevalent adaptive immune system in bacteria and archaea. These systems defend against exogenous parasitic mobile genetic elements. The adaption of single effector CRISPR-Cas systems has massively facilitated gene-editing due to the reprogrammable guide RNA. The guide RNA affords little priming space for conventional PCR-based nucleic acid tests without foreknowledge of the spacer sequence. Further impeding detection of gene-editor exposure, these systems are derived from human microflora and pathogens ( Staphylococcus pyogenes , Streptococcus aureus , etc.) that contaminate human patient samples. The single guide RNA—formed from the CRISPR RNA (crRNA) and transactivating RNA (tracrRNA)—harbors a variable tetraloop sequence between the two RNA segments, complicating PCR assays. Identical single effector Cas proteins are used for gene-editing and naturally by bacteria. Antibodies raised against these Cas proteins are unable to distinguish CRISPR-Cas gene-editors from bacterial contaminant. To overcome the high potential for false positives, we have developed a DNA displacement assay to specifically detect gene-editors. We leveraged the single guide RNA structure as an engineered moiety for gene-editor exposure that does not cross-react with bacterial CRISPRs. Our assay has been validated for five common CRISPR systems and functions in complex sample matrices.
- Research Organization:
- Sandia National Laboratories (SNL-NM), Albuquerque, NM (United States)
- Sponsoring Organization:
- Defense Threat Reduction Agency (DTRA); USDOE; USDOE Laboratory Directed Research and Development (LDRD) Program; USDOE National Nuclear Security Administration (NNSA)
- Grant/Contract Number:
- NA0003525
- OSTI ID:
- 1988087
- Journal Information:
- Scientific Reports, Journal Name: Scientific Reports Journal Issue: 1 Vol. 13; ISSN 2045-2322
- Publisher:
- Nature Publishing GroupCopyright Statement
- Country of Publication:
- United Kingdom
- Language:
- English
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