I hope this message finds you well. My name is Ana Gonzalez. I have been attempting to assemble ribonucleoprotein (RNP) complexes with sgRNA and Cas9 for an in vitro cleavage assay, but I haven't had successful results so far. Despite following different ratios and protocols, the expected shift indicating RNP complex formation is not appearing on the agarose gel. Below are some of the details of my experimental setup:

sgRNA purification: Using the GENEART PRECISION gRNA synthesis kit (ThermoFisher). The A280/260 ratio of my sgRNA is 2.1, and the A260/230 ratio is 2.2.

Cas9: Using CAS9GFPPRO from Sigma-Aldrich (5 mg/ml), incubated with sgRNA at room temperature for 15 minutes.

Buffer: NEB 3.1 (1X) with BSA. I’ve tried various sgRNA

ratios (e.g., 4:1, 3:1, 2:1, 1:1, 1:2, etc.), and run the samples on a 2% agarose gel in 1X TAE buffer at 80 volts.

Issue: I consistently observe the sgRNA band between 100 and 200 bp, but I am not seeing the smeared, higher molecular weight band (~500 bp) that would indicate the formation of the sgRNA-Cas9 complex (attached figure).

I have tried adjusting the concentration of Cas9, using different incubation times and conditions, but the complex does not seem to form properly.

I would greatly appreciate any insights, suggestions, or detailed protocols that have worked for similar CRISPR/Cas9 in vitro cleavage assays.

Thank you so much for your time and help!