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Cas12a ribonucleoprotein (RNP) is an RNA-guided CRISPR-associated nuclease used widely for genome editing and molecular diagnostics. Conventional detection methods rely on adopting antibody-based reagents that are expensive and lack scalability, and, moreover, only detect Cas12 enzyme rather than RNP, which is the true effector. Here, we describe a method for the rapid and quantitative detection of the effective Cas12a RNPs by the combined use of anti-CRISPR protein AcrVA1 and stem-loop RT-qPCR, achieving a limit of detection (LOD) of 1 fM in reaction buffer and 0.1 pM under biologically representative conditions. [ABSTRACT FROM AUTHOR]

Though plenty of viral and non-viral methods have been rapidly developed for the delivery of the CRISPR/Cas9 system, the direct delivery of Cas9 ribonucleoproteins (RNPs) into the nucleus for genome editing via the non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathway, especially the latter one, remains a challenge. Here we report a delivery vehicle achieved by encapsulating red fluorescent protein (RFP) within chitosan (CS), which can simultaneously deliver engineered Cas9 RNPs with a poly-glutamate peptide tag (E-tag) and DNA donors into a range of cell types with high genome editing efficacy and non-cytotoxicity. [ABSTRACT FROM AUTHOR]