Fluorescent CRISPR Reporters: SRIRACCHA and GEmCherry2

By Alyssa Cecchetelli

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Cartoon depictions of SHERLOCK and DETECTR nucleic acid detection methods.  SHERLOCK can be used to detect dsDNA or RNA. The material is amplified using RPA (for dsDNA, producing more dsDNA) or RT-RPA (for RNA, producing cDNA). Either way, T7 transcription then produces RNA from the amplified material. A target RNA sequence is specifically recognized by Cas13, which then promiscuously cleaves nearby RNA reporters, freeing a fluorescent marker from a linked quencher.  DETECTR is used to detect DNA. The material is amplified using RPA, and a target DNA sequence is specifically recognized by Cas12a, which then promiscuously cleaves nearby ssDNA reporters, freeing a fluorescent marker from a linked quencher.
Workflow of phage-assisted evolution of base-editing activity. Fresh host cells contain Cas, mutated gIII, and mutagenesis plasmids. Phage infection delivers the deaminase plasmid. After mutagenesis, if the base editor is inactive, no pIII is produced, and the progeny phage are not infectious. If the base editor is active, however, pIII is produced and the progeny phage are infectious, allowing them to continue propagating.

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