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    Zhigang Li, Haojun Liang. Sequence-Independent CRISPR-Based Transcription Regulators Responding Endogenous and Exogenous Molecules[J]. Chinese Journal of Chemical Physics . DOI: 10.1063/1674-0068/cjcp2312129
    Citation: Zhigang Li, Haojun Liang. Sequence-Independent CRISPR-Based Transcription Regulators Responding Endogenous and Exogenous Molecules[J]. Chinese Journal of Chemical Physics . DOI: 10.1063/1674-0068/cjcp2312129

    Sequence-Independent CRISPR-Based Transcription Regulators Responding Endogenous and Exogenous Molecules

    • CRISPR-based transcription regulators (CRISPR-TRs) have revolutionized the field of synthetic biology by enabling targeted activation or repression of any desired gene. However, the majority of existing inducible CRISPR-TRs are limited by their dependence on specific sequences, which restricts their flexibility and controllability in genetic engineering applications. In this study, we proposed a novel strategy to construct sequence-independent inducible CRISPR-TRs, which is achieved by design of stem loop 2 in the single guide RNA (sgRNA). Under this strategy, By utilizing toehold-mediated strand displacement (TMSD) reactions between small endogenous molecules (miR-20a and TK1 mRNA) and bridge RNA (bRNA) to link bRNA with sgRNA, we achieved synergistic transcriptional activation of VP64 and p65-HSF1 in response to endogenous molecules. To enable response to exogenous molecules, we added response sequences and bRNA sequences to the 5' end of sgRNA to block sgRNA activity, and achieved activation of sgRNA by shearing the response sequence, called sequential unlimited interlocking (SUI). Compared with conventinal sequence-restricted interlocking (spacer-blocking hairpin (SBH)), the transcriptional activation ratio between response and non-response to the Cas6A protein using our approach was increased by 2.28-fold. Our work presents a modular and versatile framework for endogenous and exogenous molecule-responsive CRISPR-TRs in mammalian cells, without limitations imposed by sequence dependence.
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