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.

Figure 1. Structure of the human p16INK4a gene in HCT116.

(a) The Gx4 allele is not transcribed because the CpG island (including the promoter region, first exon, and first intron) is CpG-methylated. In the Gx5 allele, a frameshift mutation caused by insertion of a single guanine (G, shown in red) in the coding region of the first exon prevents production of the functional protein. The Gx4 and Gx5 sequences are shown in uppercase. (b) The CpG island of the p16INK4a gene. (Upper) Schematic diagram of the CpG island around the first exon of p16INK4a. Four alternatively spliced mRNAs are transcribed from the CDKN2A locus, one of which is p16INK4a. The CpG island is shown in green. (Lower) DNA sequence of the CpG island in the Gx4 allele. An additional guanine (G) is inserted into the G stretch (shown in uppercase) of the Gx5 allele. The upper image and DNA sequence were generated using the UCSC Genome Browser (https://genome.ucsc.edu/). CpG sites are underlined. (c) Primer positions for bisulfite sequencing. (d) Bisulfite sequencing of genomic DNA extracted from HCT116. The target sites for sgRNA_lef5, sgRNA_mid2, and sgRNA_rig3 are shown in purple, red, and light blue, respectively (b–d).

Figure 2. Effects of CpG methylation of target sites on genome editing in vivo.

(a) DNA sequences targeted by sgRNAs. Seed sequences and PAMs are shown in yellow and green, respectively. The single-guanine insertion in the Gx5 allele is shown in red. CpG sites in the Gx4 allele are underlined. (b) Evaluation of genome editing. Schemes for genome editing and genotyping PCR are shown in Supplementary Fig. S1. Products of genotyping PCR were cloned, and 15 (sgRNA_mid2) or 18 (sgRNA_lef5 and sgRNA_rig3) independent clones were subjected to DNA sequencing analysis to identify the targeted alleles. (c) Evaluation of locus binding, as determined by DNA yields of enChIP. Error bars represent s.e.m. of three enChIP experiments (**t-test P-value < 0.01).

Figure 3. CpG methylation does not directly suppress binding of CRISPR to purified DNA.

Genomic DNA was purified from HCT116 cells and used for in vitro enChIP; DNA yields of enChIP are shown. Error bars represent s.e.m. of three in vitro enChIP experiments. N.D.: not detected.

Figure 4. Evaluation of p14ARF locus binding by CRISPR in vivo.

(a) Structure of the human p14ARF gene in HCT116. One allele of the human p14ARF gene is not transcribed in HCT116 because the CpG island (including the promoter region and first exon) is CpG-methylated. In the other allele, a frameshift mutation caused by deletion of a single guanine in the coding region of the first exon prevents production of the functional protein. (b) The CpG island of the p14ARF gene in HCT116. (Upper) Schematic diagram of the CpG island around the first exon of p14ARF. The CpG islands are shown in green. (Lower) A partial DNA sequence of the CpG island (CpG: 176) in the methylated allele. A guanine (G) is deleted from the G stretch (shown in uppercase) of the non-methylated allele. The upper image and DNA sequence were generated using the UCSC Genome Browser (https://genome.ucsc.edu/). CpG sites are underlined. The target sites for sgRNA_p14ARF_LEF3, sgRNA_p14ARF_MID6, and sgRNA_p14ARF_RIG3 are shown in purple, red, and light blue, respectively. PAMs are shown in yellow. (c) Primer positions for bisulfite sequencing. (d) Bisulfite sequencing of genomic DNA extracted from HCT116. Target sites for sgRNAs are constitutively CpG-methylated in an allele-specific manner. (e) DNA yields of conventional enChIP. enChIP targeting p14ARF was performed similarly to Supplementary Fig. S3a. Error bars represents s.e.m. of three enChIP experiments (*t-test P-value < 0.05).

Figure 5. Allele-specific genome editing using an allele-specific single-nucleotide insertion in vivo.

(a) DNA sequences targeted by sgRNAs. PAMs are shown in green. The inserted single guanine in the Gx5 allele is shown in red. (b) Evaluation of genome editing. Schemes for genome editing and genotyping PCR are shown in Supplementary Fig. S8. Products of genotyping PCR were cloned, and 13 (sgRNA_Gx4#2) or 14 (sgRNA_Gx5#2) independent clones were subjected to DNA sequencing analysis to identify the targeted alleles. (c) Evaluation of locus binding, as determined by DNA yields of conventional in vivo enChIP. The error bar represents s.e.m. of three enChIP experiments (*t-test P-value < 0.05).

Figure 6. Summary of genome editing and locus binding using sgRNA_Gx4#2 or sgRNA_Gx5#2.

Single-nucleotide skipping [“sgRNA jump (DNA bulge)” for sgRNA_Gx4#2 and “sgRNA bulge” for sgRNA_Gx5#2] can occur between the third and seventh positions 5′ of the PAM for each sgRNA. As a representative, single-nucleotide skipping at the seventh nucleotide 5′ of the PAM is shown for each sgRNA. PAMs are shown in green. The single-guanine insertion in the Gx5 allele is shown in red.