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New Method Streamlines sgRNA Production for Cas9 Research

3/27/2026

Summary

RNA Transcription Method

The article details an in vitro transcription method for single-guide RNA (sgRNA) DNA templates, beginning with annealing.
RNA polymerase transcribes the annealed dsDNA overnight at 37 °C using template, dNTPs, and MgCl2.
Purification uses denaturing PAGE and ethanol precipitation to isolate the transcription product.
The article describes protein expression and labeling of wild-type Cas9 (WT Cas9) and dead Cas9 (dCas9).
The product is quantified and stored at -80 °C.

Summary

Minicircle Cleavage Kinetics

Minicircles were assembled by heat-annealing with T4 DNA ligase and T4 DNA polymerase; supercoiled by E. coli gyrase.
For bulk cleavage assays, CRISPR-associated protein 9 (Cas9) was complexed with sgRNA and incubated with minicircles.
Timepoints were used to analyze cleavage kinetics by native PAGE.
DNA minicircles were adsorbed on mica; AFM captured force-distance curves.
Cleavage fractions were calculated with a specific equation, and kinetics were fitted to a single exponential model.

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RNA Transcription Method

The article details an in vitro transcription method for single-guide RNA (sgRNA) DNA templates, beginning with annealing.
RNA polymerase transcribes the annealed dsDNA overnight at 37 °C using template, dNTPs, and MgCl2.
Purification uses denaturing PAGE and ethanol precipitation to isolate the transcription product.
The article describes protein expression and labeling of wild-type Cas9 (WT Cas9) and dead Cas9 (dCas9).
The product is quantified and stored at -80 °C.

1 / 2