RNA modifications, including m⁶A, are vital in biological processes. However, their specific roles remain elusive due to the limitation in tools to study their context-dependent functions. CRIPSR and nuclease-inactive Cas (dCas) have been employed to achieve site-specific and temporal controlled m⁶A editing. However, the constitutive binding of bulky dCas fusion protein with target RNAs raises the concerns of disrupting endogenous RNA functions. Here, we engineer an inducible split dCas13b-based platform for targeted m⁶A editing. Two partial dCas13b domains can be reconstituted into full dCas13b for conditional m⁶A deposition via chemical control. After achieving m⁶A writing, the assembled dCas13b fusion protein can be quickly released from the target RNA through ABA removal, leaving a wide temporal window to study functions of m⁶A-equipped RNAs without obstruction. This split dCas13b platform offers a minimal disruptive tool for functional studies of RNA modifications or other targeted RNA manipulations in native cellular environments.

<b>The chemically inducible split PspCas13b-based m<sup>6</sup>A writing platform. </b>The N- and C- terminal of dCas13b were fused to PYL and ABI separately, and the METTL3 was fused with C-terminal of dCas13b. The addition of ABA initiates the assembly of functional dCas13b fusion protein and the subsequent m<sup>6</sup>A writing at the sgRNA-targeted RNA locus, which can both be reversed after ABA removal.

The chemically inducible split PspCas13b-based m⁶A writing platform. The N- and C- terminal of dCas13b were fused to PYL and ABI separately, and the METTL3 was fused with C-terminal of dCas13b. The addition of ABA initiates the assembly of functional dCas13b fusion protein and the subsequent m⁶A writing at the sgRNA-targeted RNA locus, which can both be reversed after ABA removal.

Chenwei Zhou

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