title: Allele-specific locus binding and genome editing by CRISPR at the p16INK4a locus.
abstract: The clustered regularly interspaced short palindromic repeats (CRISPR) system has been adopted for a wide range of biological applications including genome editing. In some cases, dissection of genome functions requires allele-specific genome editing, but the use of CRISPR for this purpose has not been studied in detail. In this study, using the p16INK4a gene in HCT116 as a model locus, we investigated whether chromatin states, such as CpG methylation, or a single-nucleotide gap form in a target site can be exploited for allele-specific locus binding and genome editing by CRISPR in vivo. First, we showed that allele-specific locus binding and genome editing could be achieved by targeting allele-specific CpG-methylated regions, which was successful for one, but not all guide RNAs. In this regard, molecular basis underlying the success remains elusive at this stage. Next, we demonstrated that an allele-specific single-nucleotide gap form could be employed for allele-specific locus binding and genome editing by CRISPR, although it was important to avoid CRISPR tolerance of a single nucleotide mismatch brought about by mismatched base skipping. Our results provide information that might be useful for applications of CRISPR in studies of allele-specific functions in the genomes.
authors.affiliation: Chromatin Biochemistry Research Group, Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, 565-0871 Osaka, Japan.
authors.forename: Toshitsugu
authors.initials: T
authors.lastname: Fujita
authors.affiliation: Chromatin Biochemistry Research Group, Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, 565-0871 Osaka, Japan.
authors.forename: Miyuki
authors.initials: M
authors.lastname: Yuno
authors.affiliation: Chromatin Biochemistry Research Group, Combined Program on Microbiology and Immunology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita, 565-0871 Osaka, Japan.
authors.forename: Hodaka
authors.initials: H
authors.lastname: Fujii
mesh_terms: D000483:Alleles; D001483:Base Sequence; D064113:CRISPR-Cas Systems / Q000235:genetics*; D018899:CpG Islands / Q000235:genetics; D019941:Cyclin-Dependent Kinase Inhibitor p16 / Q000235:genetics*; D004247:DNA / Q000302:isolation & purification; D019175:DNA Methylation / Q000235:genetics; D000072669:Gene Editing; D056426:Genetic Loci; D045325:HCT116 Cells; D006801:Humans; D009711:Nucleotides / Q000235:genetics; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics
chemical_list: D019941:Cyclin-Dependent Kinase Inhibitor p16; D009711:Nucleotides; D000094704:RNA, Guide, CRISPR-Cas Systems; D004247:DNA