Cytosolic CRISPR RNA for RNA-targeting CRISPR-Cas systems

Abstract

RNA-targeting Clustered, Regularly Interspaced, Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) technologies have been evolving rapidly over the past decade with the continuous discovery of new subtypes and development of novel applications. In particular, the type VI Cas13 proteins are promising single-effector systems for modulation or editing of target mRNAs. Nevertheless, the Cas13 systems are still restrained by suboptimal efficiencies and limited clinical applications. In this study, we showed that replacing the conventional U6 promoter with U1 promoter for the expression of cytosolic crRNA would be beneficial to enhance the efficiencies of existing RNA-targeting Cas13 technologies. In addition to the effect of effector-crRNA co-localization, our data interestingly suggested an unknown mechanistic factor for U1-driven crRNA to perform better than the conventional U6-driven crRNA. With the new engineering of U1 3′ regulatory sequences, we used Rapid amplification of cDNA ends (RACE) analysis to define the 5′ and 3′ end sequences of our new U1-driven CRISPR-RNA (crRNA). To fully utilize the characteristic cytosolic localization of our U1-driven crRNA, we validated the translational repression of reporter genes using catalytically inactive PspCas13b and U1-driven crRNA targeting start codons. This proof-of-principle finding underpinned the potential of our new cytosolic RNA-targeting system to efficiently mediate the correction of disease-associated abnormalities on either mRNA or protein level.