Microhomology-Mediated End-Joining Chronicles: Tracing the Evolutionary Footprints of Genome Protection.

The fidelity of genetic information is essential for cellular function and viability. DNA double-strand breaks (DSBs) pose a significant threat to genome integrity, necessitating efficient repair mechanisms. While the predominant repair strategies are usually accurate, paradoxically, error-prone pathways also exist.

Genetic dissection of mutagenic repair and T-DNA capture at CRISPR-induced DNA breaks in .

A practical and powerful approach for genome editing in plants is delivery of CRISPR reagents via transformation. The double-strand break (DSB)-inducing enzyme is expressed from a transferred segment of bacterial DNA, the T-DNA, which upon transformation integrates at random locations into the host genome or is captured at the self-inflicted DSB site. To develop efficient strategies for precise genome editing, it is thus important to define the mechanisms that repair CRISPR-induced DSBs, as well as those that govern random and targeted integration of T-DNA.

Molecular analysis of the role of polymerase theta in gene targeting in Arabidopsis thaliana.

Precise genetic modification can be achieved via a sequence homology-mediated process known as gene targeting (GT). Whilst established for genome engineering purposes, the application of GT in plants still suffers from a low efficiency for which an explanation is currently lacking. Recently reported reduced rates of GT in A.

Dissecting the genetic landscape of GPCR signaling through phenotypic profiling in C. elegans.

G protein-coupled receptors (GPCRs) mediate responses to various extracellular and intracellular cues. However, the large number of GPCR genes and their substantial functional redundancy make it challenging to systematically dissect GPCR functions in vivo. Here, we employ a CRISPR/Cas9-based approach, disrupting 1654 GPCR-encoding genes in 284 strains and mutating 152 neuropeptide-encoding genes in 38 strains in C.

The interplay of DNA repair context with target sequence predictably biases Cas9-generated mutations.

Mutagenic outcomes of CRISPR/Cas9-generated double-stranded breaks depend on both the sequence flanking the cut and cellular DNA damage repair. The interaction of these features has been largely unexplored, limiting our ability to understand and manipulate the outcomes. Here, we measured how the absence of 18 repair genes changed frequencies of 83,680 unique mutational outcomes generated by Cas9 double-stranded breaks at 2,838 synthetic target sequences in mouse embryonic stem cells.

Discrepancies in Embryonic Staging: Towards a Gold Standard.

For over half a century, the Carnegie staging system has been used for the unification of chronology in human embryo development. Despite the system's establishment as a "universal" system, Carnegie staging reference charts display a high level of variation. To establish a clear understanding for embryologists and medical professionals, we aimed to answer the following question: does a gold standard of Carnegie staging exist, and if so, which set of proposed measures/characteristics would it include? We aimed to provide a clear overview of the variations in published Carnegie staging charts to compare and analyze these differences and propose potential explanatory factors.

Chromosomal breaks at the origin of small tandem DNA duplications.

Small tandem DNA duplications in the range of 15 to 300 base-pairs play an important role in the aetiology of human disease and contribute to genome diversity. Here, we discuss different proposed mechanisms for their occurrence and argue that this type of structural variation mainly results from mutagenic repair of chromosomal breaks. This hypothesis is supported by both bioinformatical analysis of insertions occurring in the genome of different species and disease alleles, as well as by CRISPR/Cas9-based experimental data from different model systems.

Sister chromatid exchanges induced by perturbed replication can form independently of BRCA1, BRCA2 and RAD51.

Sister chromatid exchanges (SCEs) are products of joint DNA molecule resolution, and are considered to form through homologous recombination (HR). Indeed, SCE induction upon irradiation requires the canonical HR factors BRCA1, BRCA2 and RAD51. In contrast, replication-blocking agents, including PARP inhibitors, induce SCEs independently of BRCA1, BRCA2 and RAD51.

SIQ: easy quantitative measurement of mutation profiles in sequencing data.

With the emergence of CRISPR-mediated genome editing, there is an increasing desire for easy-to-use tools that can process and overview the spectra of outcomes. Here, we present Sequence Interrogation and Quantification (SIQ), a simple-to-use software tool that enables researchers to retrieve, data-mine and visualize complex sets of targeted sequencing data. SIQ can analyse Sanger sequences but specifically benefit the processing of short- and long-read next-generation sequencing data (e.

THO complex deficiency impairs DNA double-strand break repair via the RNA surveillance kinase SMG-1.

The integrity and proper expression of genomes are safeguarded by DNA and RNA surveillance pathways. While many RNA surveillance factors have additional functions in the nucleus, little is known about the incidence and physiological impact of converging RNA and DNA signals. Here, using genetic screens and genome-wide analyses, we identified unforeseen SMG-1-dependent crosstalk between RNA surveillance and DNA repair in living animals.

Distinct mechanisms for genomic attachment of the 5' and 3' ends of Agrobacterium T-DNA in plants.

Agrobacterium tumefaciens, a pathogenic bacterium capable of transforming plants through horizontal gene transfer, is nowadays the preferred vector for plant genetic engineering. The vehicle for transfer is the T-strand, a single-stranded DNA molecule bound by the bacterial protein VirD2, which guides the T-DNA into the plant's nucleus where it integrates. How VirD2 is removed from T-DNA, and which mechanism acts to attach the liberated end to the plant genome is currently unknown.

Helicase Q promotes homology-driven DNA double-strand break repair and prevents tandem duplications.

DNA double-strand breaks are a major threat to cellular survival and genetic integrity. In addition to high fidelity repair, three intrinsically mutagenic DNA break repair routes have been described, i.e.

Gene targeting in polymerase theta-deficient Arabidopsis thaliana.

Agrobacterium tumefaciens-mediated transformation has been for decades the preferred tool to generate transgenic plants. During this process, a T-DNA carrying transgenes is transferred from the bacterium to plant cells, where it randomly integrates into the genome via polymerase theta (Polθ)-mediated end joining (TMEJ). Targeting of the T-DNA to a specific genomic locus via homologous recombination (HR) is also possible, but such gene targeting (GT) events occur at low frequency and are almost invariably accompanied by random integration events.

Small tandem DNA duplications result from CST-guided Pol α-primase action at DNA break termini.

Small tandem duplications of DNA occur frequently in the human genome and are implicated in the aetiology of certain human cancers. Recent studies have suggested that DNA double-strand breaks are causal to this mutational class, but the underlying mechanism remains elusive. Here, we identify a crucial role for DNA polymerase α (Pol α)-primase in tandem duplication formation at breaks having complementary 3' ssDNA protrusions.

Polθ inhibitors elicit BRCA-gene synthetic lethality and target PARP inhibitor resistance.

To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor.

Preservation of lagging strand integrity at sites of stalled replication by Pol α-primase and 9-1-1 complex.

During genome duplication, the replication fork encounters a plethora of obstacles in the form of damaged bases, DNA-cross-linked proteins, and secondary structures. How cells protect DNA integrity at sites of stalled replication is currently unknown. Here, by engineering "primase deserts" into the genome close to replication-impeding G-quadruplexes, we show that de novo DNA synthesis downstream of the blocked fork suppresses DNA loss.

BRCA1-associated structural variations are a consequence of polymerase theta-mediated end-joining.

Failure to preserve the integrity of the genome is a hallmark of cancer. Recent studies have revealed that loss of the capacity to repair DNA breaks via homologous recombination (HR) results in a mutational profile termed BRCAness. The enzymatic activity that repairs HR substrates in BRCA-deficient conditions to produce this profile is currently unknown.