Concurrent genome and epigenome editing by CRISPR-mediated sequence replacement

摘要

Experimental design. Overview of the experimental approach showing CRISPR dual cuts for removing and replacing the CpG island with an in vitro methylated DNA sequence through NHEJ-mediated repair. The CpG island was cloned, and synonymous coding SNVs were introduced to create two distinguishable alleles (blue and purple). Cloned CpG island alleles were PCR amplified for linearization and to incorporate PAM mutations. Portions of the resulting amplicons were in vitro methylated (cyan) with M.SssI. For each replicate, the methylated version of one allele amplicon and the unmethylated version of the other allele amplicon, together with plasmids bearing Cas9-2A-GFP and two gRNAs, were co-transfected into Hap1 cells. In one plate of Hap1 cells, allele 1 was methylated and allele 2 was not, and in a parallel experiment, allele 2 was methylated and allele 1 was not. Transfected cells were sorted by FACS and re-plated for genome editing. Edited cells were then either selected with 6-TG, which will select for cells that do not express , or mock selected with DMSO. Cells were harvested before and after selection, DNA was extracted, and the relevant regions PCR amplified and sequenced. The alleles allow tracking of the inserted methylated vs. unmethylated CpG island amplicons without requiring bisulfite conversion. The relative frequencies of the methylated and unmethylated alleles were calculated and compared between the 6-TG-selected, mock-selected, and pre-selection cells. Potential outcomes of genome editing are shown for a hypothetical single cell from a single replicate. After a CRISPR dual cut, the possible outcomes at the DNA level are a deletion of the CpG island, re-insertion of the original wild-type CpG island that was cut out, or insertion of the methylated or unmethylated alleles that were transfected in. Inserted CpG islands can be inserted in an inverted or forward orientation. will be expressed if either the original wild-type or the unmethylated allele is inserted, but will no longer be expressed if a deletion or inversion occurs. Insertion of a forward-oriented, methylated allele should result in methylation-induced silencing. Finally, cells are expected to survive 6-TG selection if they no longer express , which can be a consequence of methylation-induced silencing, deletion of the CpG island, or inversion of the CpG island. Therefore, upon sequencing after 6-TG selection, if the methylated allele is inserted, we predicted that its relative frequency will be increased as compared to the unmethylated allele.