SIRT6 exhibits nucleosome-dependent deacetylase activity.

The SIRT6 deacetylase is a key regulator of mammalian genome stability, metabolism and lifespan. Previous studies indicated that SIRT6 exhibits poor deacetylase activity in vitro. Here, we explored the specific conditions that allow SIRT6 to function as a significant deacetylase. We show that SIRT6 associates with the nucleosome and deacetylates histones H3 and H4 when they are packaged as nucleosomes, but not as free histones. In contrast, SIRT1 shows the opposite characteristics. Thus, our results show that SIRT6 activity is nucleosome dependent, and suggest that its binding to the nucleosome might convert it into an active structure.

gRNA Sequence Heterology Tolerance Catalyzed by CRISPR/Cas in an In Vitro Homology-Directed Repair Reaction.

CRISPR and associated Cas nucleases are genetic engineering tools revolutionizing innovative approaches to cancer and inherited diseases. CRISPR-directed gene editing relies heavily on proper DNA sequence alignment between the guide RNA (gRNA)/CRISPR complex and its genomic target. Accurate hybridization of complementary DNA initiates gene editing in human cells, but inherent gRNA sequence variation that could influence the gene editing reaction has been clearly established among diverse genetic populations. As this technology advances toward clinical implementation, it will be essential to assess what degree of gRNA variation generates unwanted and erroneous CRISPR activity. With the use of a system in which a cell-free extract catalyzes nonhomologous end joining (NHEJ) and homology-directed repair (HDR), it is possible to observe a more representative population of all forms of gene editing outcomes. In this manuscript, we demonstrate CRISPR/Cas complexation at heterologous binding sites that facilitate precise and error-prone HDR. The tolerance of mispairing between the gRNA and target site of the DNA to enable HDR is surprisingly high and greatly influenced by polarity of the donor DNA strand in the reaction. These results suggest that some collateral genomic activity could occur at unintended sites in CRISPR-directed gene editing in human cells.

CRISPR-Directed Gene Editing Catalyzes Precise Gene Segment Replacement In Vitro Enabling a Novel Method for Multiplex Site-Directed Mutagenesis.

Much of our understanding of eukaryotic genes function comes from studies of the activity of their mutated forms or allelic variability. Mutations have helped elucidate how members of an intricate pathway function in relation to each other and how they operate in the context of the regulatory circuitry that surrounds them. A PCR-based site-directed mutagenesis technique is often used to engineer these variants. While these tools are efficient, they are not without significant limitations, most notably off-site mutagenesis, limited scalability, and lack of multiplexing capabilities. To overcome many of these limitations, we now describe a novel method for the introduction of both simple and complex gene mutations in plasmid DNA by using in vitro DNA editing. A specifically designed pair of CRISPR-Cas12a ribonucleoprotein complexes are used to execute site-specific double-strand breaks on plasmid DNA, enabling the excision of a defined DNA fragment. Donor DNA replacement is catalyzed by a mammalian cell-free extract through microhomology annealing of short regions of single-stranded DNA complementarity; we term this method CRISPR-directed DNA mutagenesis (CDM). The products of CDM are plasmids bearing precise donor fragments with specific modifications and CDM could be used for mutagenesis in larger constructs such as Bacterial Artificial Chromosome (BACs) or Yeast Artificial Chromosome (YACs). We further show that this reaction can be multiplexed so that product molecules with multiple site-specific mutations and site-specific deletions can be generated in the same in vitro reaction mixture. Importantly, the CDM method produces fewer unintended mutations in the target gene as compared to the standard site-directed mutagenesis assay; CDM produces no unintended mutations throughout the plasmid backbone. Lastly, this system recapitulates the multitude of reactions that take place during CRISPR-directed gene editing in mammalian cells and affords the opportunity to study the mechanism of action of CRISPR-directed gene editing in mammalian cells by visualizing a multitude of genetic products.

Multiple regulatory layers of SREBP1/2 by SIRT6.

The NAD(+)-dependent protein deacetylase SIRT6 regulates genome stability, cancer, and lifespan. Mice overexpressing SIRT6 (MOSES) have lower low-density lipoprotein cholesterol levels and are protected against the physiological damage of obesity. Here, we examined the role of SIRT6 in cholesterol regulation via the lipogenic transcription factors SREBP1 and SREBP2, and AMP-activated protein kinase (AMPK). We show that SIRT6 represses SREBP1 and SREBP2 by at least three mechanisms. First, SIRT6 represses the transcription levels of SREBP1/SREBP2 and that of their target genes. Second, SIRT6 inhibits the cleavage of SREBP1/SREBP2 into their active forms. Third, SIRT6 activates AMPK by increasing the AMP/ATP ratio, which promotes phosphorylation and inhibition of SREBP1 by AMPK. Reciprocally, the expression of miR33a and miR33b from the introns of SREBP2 and SREBP1, respectively, represses SIRT6 levels. Together, these findings explain the mechanism underlying the improved cholesterol homeostasis in MOSES mice, revealing a relationship between fat metabolism and longevity.