Method for inducing deletion in genomic DNA
A production method of genomic DNA in which a deletion of more than 100 bases of nucleotides is introduced into a target region of the genomic DNA, the method includes a contact step of bringing a type I CRISPR associated complex for anti-viral defense (type I Cascade complex), CRISPR RNA (crRNA), and Cas3 protein into contact with the genomic DNA.
1 . A production method of an induced pluripotent stem (iPS) cell in which genomic DNA is modified, the method comprising:
introducing Cse1 protein, Cse2 protein, Cas7 protein, Cas5 protein, Cas6 protein, and Cas3 protein into the iPS cell in the form of an expression vector or an mRNA; and
contacting a type I CRISPR associated complex for anti-viral defense (type I Cascade complex), CRISPR RNA (crRNA), and the Cas3 protein with the genomic DNA of the iPS cell,
wherein the type I Cascade complex consists of the Cse1 protein, the Cse2 protein, the Cas7 protein, the Cas5 protein, and the Cas6 protein,
wherein the expression vector is for expressing three proteins selected from the group consisting of the Cse1 protein, the Cse2 protein, the Cas7 protein, the Cas5 protein, the Cas6 protein, and the Cas3 protein by one promoter,
wherein the mRNA is for expressing three proteins selected from the group consisting of the Cse1 protein, the Cse2 protein, the Cas7 protein, the Cas5 protein, the Cas6 protein, and the Cas3 protein as individual proteins from a single mRNA.
2 . The production method according to claim 1 , wherein a target region of the crRNA is in a β2-microglobulin (B2M) gene or a regulatory region thereof, or vicinities thereof; a Human Leukocyte Antigen (HLA) gene or a regulatory region thereof, or vicinities thereof; or a dystrophin (DMD) gene or a regulatory region thereof, or vicinities thereof.
3 . The production method according to claim 1 ,
wherein a deletion of more than 100 bases of nucleotides is introduced into a target region of the genomic DNA.