Standardization of Recombinant CRISPR/Cas13a-nuclease Preparations by Using RNase A of Known Activity

Abstract

The approach to characterize preparations of recombinant Cas13a-nuclease in terms of specific collateral activity has been proposed for standardization of enzyme preparations. The standardization of Cas13a preparations by the specific activity may benefit both the development of assays employing Cas13a collateral ribonuclease activity and the optimization of ribonuclease expression, purification, and storage. The approach is based on measurement of the initial rate of a cleavage of specially designed commercially available RNA molecules (�reporters� labelled with a fluorophore and a quencher) by a preparation of recombinant Cas13a-nuclease and commercial RNase A of known activity. This requires the optimization of a molar ratio for the formation of Cas13a complexes with guide RNA as well as the optimization of amount of the RNA target. The use of a synthetic RNA target appears preferable compared with total RNA preparations.