Assaying Cell Cycle Progression via Flow Cytometry in CRISPR/Cas9-Treated Cells.

CRISPR/Cas9 system is a powerful technique for genome editing and engineering but obtaining a sizeable population of edited cells can be challenging for some cell types. CRISPR/Cas9-induced cell cycle arrest is a possible cause of this barrier to efficient editing; thus, it is desirable to know the cell cycle progression profile of any given cell line or type of interest resulting from CRISPR/Cas9 treatment. Here we describe a flow cytometry-based assay that enables the determination of cell cycle progression in the presence of CRISPR/Cas9 treatment, in addition to the transfection and expression efficiencies of Cas9 vectors.

CRISPR/Cas9 treatment causes extended TP53-dependent cell cycle arrest in human cells.

While the mechanism of CRISPR/Cas9 cleavage is understood, the basis for the large variation in mutant recovery for a given target sequence between cell lines is much less clear. We hypothesized that this variation may be due to differences in how the DNA damage response affects cell cycle progression. We used incorporation of EdU as a marker of cell cycle progression to analyze the response of several human cell lines to CRISPR/Cas9 treatment with a single guide directed to a unique locus.

Knock-in Blunt Ligation Utilizing CRISPR/Cas9.

The incorporation of the CRISPR/Cas9 bacterial immune system into the genetic engineering toolbox has led to the development of several new methods for genome manipulation ( Auer , 2014 ; Byrne , 2015 ). We took advantage of the ability of Cas9 to generate blunt-ended double-strand breaks ( Jinek , 2012 ) to introduce exogenous DNA in a highly precise manner through the exploitation of non-homologous end-joining DNA repair machinery ( Geisinger , 2016 ). This protocol has been successfully applied to traditional immortalized cell lines and human induced pluripotent stem cells.

In vivo blunt-end cloning through CRISPR/Cas9-facilitated non-homologous end-joining.

The CRISPR/Cas9 system facilitates precise DNA modifications by generating RNA-guided blunt-ended double-strand breaks. We demonstrate that guide RNA pairs generate deletions that are repaired with a high level of precision by non-homologous end-joining in mammalian cells. We present a method called knock-in blunt ligation for exploiting these breaks to insert exogenous PCR-generated sequences in a homology-independent manner without loss of additional nucleotides.

Using phage integrases in a site-specific dual integrase cassette exchange strategy.

ΦC31 integrase, a site-specific large serine recombinase, is a useful tool for genome engineering in a variety of eukaryotic species and cell types. ΦC31 integrase performs efficient recombination between its attB site and either its own placed attP site or a partially mismatched genomic pseudo attP site. Bxb1 integrase, another large serine recombinase, has a similar level of recombinational activity, but recognizes only its own attB and attP sites.

Recombinase-mediated reprogramming and dystrophin gene addition in mdx mouse induced pluripotent stem cells.

A cell therapy strategy utilizing genetically-corrected induced pluripotent stem cells (iPSC) may be an attractive approach for genetic disorders such as muscular dystrophies. Methods for genetic engineering of iPSC that emphasize precision and minimize random integration would be beneficial. We demonstrate here an approach in the mdx mouse model of Duchenne muscular dystrophy that focuses on the use of site-specific recombinases to achieve genetic engineering.

Site-specific recombinase strategy to create induced pluripotent stem cells efficiently with plasmid DNA.

Induced pluripotent stem cells (iPSCs) have revolutionized the stem cell field. iPSCs are most often produced by using retroviruses. However, the resulting cells may be ill-suited for clinical applications.

Deep congenic analysis identifies many strong, context-dependent QTLs, one of which, Slc35b4, regulates obesity and glucose homeostasis.

Although central to many studies of phenotypic variation and disease susceptibility, characterizing the genetic architecture of complex traits has been unexpectedly difficult. For example, most of the susceptibility genes that contribute to highly heritable conditions such as obesity and type 2 diabetes (T2D) remain to be identified despite intensive study. We took advantage of mouse models of diet-induced metabolic disease in chromosome substitution strains (CSSs) both to characterize the genetic architecture of diet-induced obesity and glucose homeostasis and to test the feasibility of gene discovery.

Modulation of RNA polymerase II subunit composition by ubiquitylation.

Emerging evidence suggests that components of the ubiquitin-proteasome system are involved in the regulation of gene expression. A variety of factors, including transcriptional activators, coactivators, and histones, are controlled by ubiquitylation, but the mechanisms through which this modification can function in transcription are generally unknown. Here, we report that the Saccharomyces cerevisiae protein Asr1 is a RING finger ubiquitin-ligase that binds directly to RNA polymerase II via the carboxyl-terminal domain (CTD) of the largest subunit of the enzyme.