Paula M. Vertino

CRISPR/Cas9 systems are a versatile tool for genome editing due to the highly efficient targeting of DNA sequences complementary to their RNA guide strands. However, it has been shown that RNA-guided Cas9 nuclease cleaves genomic DNA sequences containing mismatches to the guide strand. A better understanding of the CRISPR/Cas9 specificity is needed to minimize off-target cleavage in large mammalian genomes. Here we show that genomic sites could be cleaved by CRISPR/Cas9 systems when DNA sequences contain insertions ('DNA bulge') or deletions ('RNA bulge') compared to the RNA guide strand, and Cas9 nickases used for paired nicking can also tolerate bulges in one of the guide strands. Variants of single-guide RNAs (sgRNAs) for four endogenous loci were used as model systems, and their cleavage activities were quantified at different positions with 1- to 5-bp bulges. We further investigated 114 putative genomic off-target loci of 27 different sgRNAs and confirmed 15 off-target sites, each harboring a single-base bulge and one to three mismatches to the guide strand. Our results strongly indicate the need to perform comprehensive off-target analysis related to DNA and sgRNA bulges in addition to base mismatches, and suggest specific guidelines for reducing potential off-target cleavage.

Cytosine residues in mammalian DNA occur in at least three forms, cytosine (C), 5-methylcytosine (M; 5mC) and 5-hydroxymethylcytosine (H; 5hmC). During semi-conservative DNA replication, hemi-methylated (M/C) and hemi-hydroxymethylated (H/C) CpG dinucleotides are transiently generated, where only the parental strand is modified and the daughter strand contains native cytosine. Here, we explore the role of DNA methyltransferases (DNMT) and ten eleven translocation (Tet) proteins in perpetuating these states after replication, and the molecular basis of their recognition by methyl-CpG-binding domain (MBD) proteins. Using recombinant proteins and modified double-stranded deoxyoligonucleotides, we show that DNMT1 prefers a hemi-methylated (M/C) substrate (by a factor of >60) over hemi-hydroxymethylated (H/C) and unmodified (C/C) sites, whereas both DNMT3A and DNMT3B have approximately equal activity on all three substrates (C/C, M/C and H/C). Binding of MBD proteins to methylated DNA inhibited Tet1 activity, suggesting that MBD binding may also play a role in regulating the levels of 5hmC. All five MBD proteins generally have reduced binding affinity for 5hmC relative to 5mC in the fully modified context (H/M versus M/M), though their relative abilities to distinguish the two varied considerably. We further show that the deamination product of 5hmC could be excised by thymine DNA glycosylase and MBD4 glycosylases regardless of context.

Promoter region CpG island methylation is associated with tumor suppressor gene silencing in neoplasia. GenBank sequence analyses revealed that a number of CpG islands are juxtaposed to multiple Alu repeats, which have been proposed as "de novo methylation centers." These islands also contain multiple Sp1 elements located upstream and downstream of transcription start, which have been shown to protect CpG islands from methylation. We mapped the methylation patterns of the E-cadherin (E-cad) and von Hippel-Lindau (VHL) tumor suppressor gene CpG island regions in normal and neoplastic cells. Although unmethylated in normal tissue, these islands were embedded between densely methylated flanking regions containing multiple Alu repeats. These methylated flanks were segregated from the unmethylated, island CpG sites by Sp1-rich boundary regions. Finally, in human fibroblasts overexpressing DNA methyltransferase, de novo methylation of the E-cad CpG island initially involved sequences at both ends of the island and the adjacent, flanking regions and progressed with time to encompass the entire CpG island region. Together, these data suggest that boundaries exist at both ends of a CpG island to maintain the unmethylated state in normal tissue and that these boundaries may be progressively overridden, eliciting the de novo methylation associated with tumor suppressor gene silencing in neoplasia.