Biology subjects

Layer, J. V.

Publications and source records attributed to Layer, J. V..

3 recordsLinked to original sources

bioRxiv2020

Site-specific targeting of a light activated dCas9-KIllerRed fusion protein generates transient, localized regions of oxidative DNA damage.

DNA repair requires reorganization of the local chromatin structure to facilitate access to and repair of the DNA. Studying DNA double-strand break (DSB) repair in specific chromatin domains has been aided by the use of sequence-specific endonucleases to generate targeted breaks. Here, we describe a new approach that combines KillerRed, a photosensitizer that generates reactive oxygen species (ROS) when exposed to light, and the genome-targeting properties of the CRISPR/Cas9 system. Fusing KillerRed to catalytically inactive Cas9 (dCas9) generates dCas9-KR, which can then be targeted to any desired genomic region with an appropriate guide RNA. Activation of dCas9-KR with green light generates a local increase in reactive oxygen species, resulting in "clustered" oxidative damage, including both DNA breaks and base damage. Activation of dCas9-KR rapidly (within minutes) increases both {gamma}H2AX and recruitment of the KU70/80 complex. Importantly, this damage is repaired within 10 minutes of termination of light exposure, indicating that the DNA damage generated by dCas9-KR is both rapid and transient. Further, repair is carried out exclusively through NHEJ, with no detectable contribution from HR-based mechanisms. Surprisingly, sequencing of repaired DNA damage regions did not reveal any increase in either mutations or INDELs in the targeted region, implying that NHEJ has high fidelity under the conditions of low level, limited damage. The dCas9-KR approach for creating targeted damage has significant advantages over the use of endonucleases, since the duration and intensity of DNA damage can be controlled in "real time" by controlling light exposure. In addition, unlike endonucleases that carry out multiple cut-repair cycles, dCas9-KR produces a single burst of damage, more closely resembling the type of damage experienced during acute exposure to reactive oxygen species or environmental toxins. dCas9-KR is a promising system to induce DNA damage and measure site-specific repair kinetics at clustered DNA lesions.

cancer biology↗

bioRxiv2019

Extensive Mammalian Germline Genome Engineering

Xenotransplantation, specifically the use of porcine organs for human transplantation, has long been sought after as an alternative for patients suffering from organ failure. However, clinical application of this approach has been impeded by two main hurdles: 1) risk of transmission of porcine endogenous retroviruses (PERVs) and 2) molecular incompatibilities between donor pigs and humans which culminate in rejection of the graft. We previously demonstrated that all 25 copies of the PERV elements in the pig genome could be inactivated and live pigs successfully generated. In this study, we improved the scale of porcine germline editing from targeting a single repetitive locus with CRISPR to engineering 13 different genes using multiple genome engineering methods. we engineered the pig genome at 42 alleles using CRISPR-Cas9 and transposon and produced PERVKO{middle dot}3KO{middle dot}9TG pigs which carry PERV inactivation, xeno-antigen KO and 9 effective human transgenes. The engineered pigs exhibit normal physiology, fertility, and germline transmission of the edited alleles. In vitro assays demonstrated that these pigs gain significant resistance to human humoral and cell mediated damage, and coagulation dysregulations, similar to that of allotransplantation. Successful creation of PERVKO{middle dot}3KO{middle dot}9TG pigs represents a significant step forward towards safe and effective porcine xenotransplantation, which also represents a synthetic biology accomplishment of engineering novel functions in a living organism. One Sentence SummaryExtensive genome engineering is applied to modify pigs for safe and immune compatible organs for human transplantation

synthetic biology↗

bioRxiv2018

Parp3 promotes long-range end-joining in murine cells

Chromosomal rearrangements, including translocations, are early and essential events in the formation of many tumors. Previous studies that defined the genetic requirements for rearrangement formation have identified differences between murine and human cells, most notably in the role of classical- and alternative-nonhomologous end joining factors (NHEJ). We reported that poly(ADP)ribose polymerase 3 (PARP3) promotes chromosomal rearrangements induced by endonucleases in multiple human cell types. In contrast to c-NHEJ factors, we show here that Parp3 also promotes rearrangements in murine cells, including translocations in murine embryonic stem cells (mESCs), class switch recombination in primary B cells and inversions in tail fibroblasts that generate Eml4-Alk fusions. In mESCs, Parp3-deficient cells had shorter deletion lengths at translocation junctions. This was corroborated using next-generation sequencing of Eml4-Alk junctions in tail fibroblasts and is consistent with a role for Parp3 in promoting the processing of DNA double-strand breaks. We confirmed a previous report that Parp1 also promotes rearrangement formation. In contrast with Parp3, rearrangement junctions in the absence of Parp1 had longer deletion lengths, suggesting Parp1 may suppress DSB processing. Together, these data indicate that Parp3 and Parp1 promote rearrangements with distinct phenotypes.

cancer biology↗