Functional control of CRISPR/Cas9 is essential for precise gene manipulation.Chemical engineering of guide RNA(gRNA)provides diverse approaches for conditional control of CRISPR/Cas9 function with a variety of chemica...Functional control of CRISPR/Cas9 is essential for precise gene manipulation.Chemical engineering of guide RNA(gRNA)provides diverse approaches for conditional control of CRISPR/Cas9 function with a variety of chemical reactive groups.However,previous investigations into chemically engineering gRNA only unidirectionally regulated the CRISPR/Cas9 function via stimuli-induced caging/decaging processes.Herein,we propose a combinatory strategy to engineer the dynamics of gRNA in which photocontrolled strand-displacement reactions coupled with sequence designs of gRNA can achieve lightinduced switching-on/off control of CRISPR/Cas9 function.Biochemical analysis and cellular gene regulation indicate this approach is capable of both activating and deactivating CRISPR/Cas9 activities using light irradiation.Moreover,photocontrolled multiplex modulations of gene expression for opposite regulatory effects have also been achieved simultaneously under the same cellular context.This work establishes an essential principle for construction of stimuli-induced switching-on/off modulations of gRNA that can greatly enrich the versatility of conditional control for a variety of CRISPR/Cas9-based applications.展开更多
基金the National Natural Science Foundation of China(grant nos.21977122 and 22222706)the National Key R&D Program of China(grant no.2020YFA0211200)the Guangdong Basic Research Center of Excellence for Functional Molecular Engineering.
文摘Functional control of CRISPR/Cas9 is essential for precise gene manipulation.Chemical engineering of guide RNA(gRNA)provides diverse approaches for conditional control of CRISPR/Cas9 function with a variety of chemical reactive groups.However,previous investigations into chemically engineering gRNA only unidirectionally regulated the CRISPR/Cas9 function via stimuli-induced caging/decaging processes.Herein,we propose a combinatory strategy to engineer the dynamics of gRNA in which photocontrolled strand-displacement reactions coupled with sequence designs of gRNA can achieve lightinduced switching-on/off control of CRISPR/Cas9 function.Biochemical analysis and cellular gene regulation indicate this approach is capable of both activating and deactivating CRISPR/Cas9 activities using light irradiation.Moreover,photocontrolled multiplex modulations of gene expression for opposite regulatory effects have also been achieved simultaneously under the same cellular context.This work establishes an essential principle for construction of stimuli-induced switching-on/off modulations of gRNA that can greatly enrich the versatility of conditional control for a variety of CRISPR/Cas9-based applications.
