(AGENPARL) – LONDON (UNITED KINGDOM), mer 09 settembre 2020
Cas12a ribonucleoprotein (RNP) is an RNA-guided CRISPR-associated nuclease used widely for genome editing and molecular diagnostics. Accurate quantification of Cas12a RNPs in diverse conditions, especially within biological samples, is critical for assessing RNP delivery efficiency and genome-editing efficacy. Conventional detection methods rely on adopting antibody-based reagents that are expensive and lack of scalability, and moreover, only detect Cas12 enzymes rather than Cas12 RNPs which are the true effectors. Here we describe a method for rapid and cost-effective detection of the effective Cas12a RNPs by combined use of anti-CRISPR protein AcrVA1 and RT-qPCR. The principle is that a 19 nt ssRNA (termed dRNA) was generated from the cleavage of the Cas12a-bound guide RNA when an AcrVA1 bound one Cas12a RNP. We harnessed the stem-loop RT/Real-time qPCR to measure the dRNA, achieving a limit of detection of 1 fM in reaction buffer and 0.1 pM in biologically representative conditions. Moreover, our method indicated that on ~80% Cas12a RNPs were effective by in vitro assembly of Cas12a proteins and crRNAs. Comparingly, the self-assembling Cas12a RNPs produced from E.coli showed a much higher assembly efficiency of close to 100%. In addition, this method enabled detecting multiple Cas12a RNPs with different crRNAs simultaneously, as well as estimating the effective time of Cas12a RNPs within cells. Together, we develop a method for quantitative detection of the effective Cas12a RNPs, showing great potential for facilitating the CRISPR/Cas12a-based genome engineering, clinical treatment, and molecular diagnostics.
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