Light-inducible CRISPR/Cas9 system for genome editing
AssigneeThe Johns Hopkins University
InventorsYang Liu, Roger Zou, Bin Wu, Taekjip Ha
Life of this patent
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Abstract
A very fast and efficient CRISPR/Cas9 system is provided. Compositions include light-sensitive caged nucleotides at the PAM distal region of guide RNAs (gRNAcaged) to create artificial mismatches as a “roadblock”. Upon light stimulation, the caging group (“roadblock”) is removed and the gRNA fully hybridizes with the target DNA. Thus, the pre-bound inactive Cas9/gRNAcaged is rapidly converted to active Cas9.
Claims17
A guide RNA (gRNA) comprising a sequence having one or more caged nucleotides or analogs thereof in a guiding sequence of the gRNA, wherein the caged nucleotides or analogs thereof are in the PAM distal-half of the guiding sequence.
A crRNA comprising one or more photocleavable caged nucleotides or analogs thereof in the PAM-distal half of the guiding sequence of the crRNA.
A composition comprising a clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease and at least one guide RNA, wherein the at least one guide RNA comprises at least one photocleavable caged nucleotide or analogs thereof in a crRNA region of the guide RNA, wherein the at least one photocleavable caged nucleotide or analogs thereof is in the PAM-distal half of the guide sequence of the crRNA region.
A method of modulating activity of a gene editing complex in a host cell, comprising:
- contacting the host cell with a composition comprising:
- a clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease and at least one guide RNA (gRNA), wherein the at least one gRNA comprises at least one photocleavable caged nucleotide or analogs thereof in a guide sequence of the at least one gRNA, wherein the at least one photocleavable caged nucleotide or analogs thereof is in the PAM-distal half of the guide sequence;
- subjecting the host cell to an electromagnetic radiation, thereby cleaving the at least one photocleavable caged nucleotide or analogs thereof;
- and,
- modulating activity of the gene-editing complex.
A method of sequential activation of a gene-editing complex in a host cell comprising;
- contacting the host cell with a composition comprising a clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease and at least two guide RNAs (gRNAs), wherein each of the at least two gRNAs comprises at least one photocleavable caged nucleotide or analogs thereof in a guide sequence, wherein the at least one photocleavable caged nucleotide or analogs thereof is in the PAM-distal half of the guide sequence of each of the at least two gRNAs;
- subjecting the host cell to varying wavelengths of electromagnetic radiation over intervals of time, thereby cleaving the at least one photocleavable caged nucleotide or analogs thereof in each of the at least two gRNAs;
- and,
- modulating activity of the gene-editing complex.
Dependencies are inferred from the claim text.