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The engineered single guide RNA structure as a biomarker for gene-editing reagent exposure.
- Source :
-
Scientific reports [Sci Rep] 2023 Jul 04; Vol. 13 (1), pp. 10804. Date of Electronic Publication: 2023 Jul 04. - Publication Year :
- 2023
-
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.<br /> (© 2023. The Author(s).)
Details
- Language :
- English
- ISSN :
- 2045-2322
- Volume :
- 13
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Scientific reports
- Publication Type :
- Academic Journal
- Accession number :
- 37402738
- Full Text :
- https://doi.org/10.1038/s41598-023-37525-y