A practical and powerful approach for genome editing in plants is delivery of CRISPR reagents via Agrobacterium tumefaciens transformation. The double-strand break (DSB)-inducing enzyme is... Show moreA practical and powerful approach for genome editing in plants is delivery of CRISPR reagents via Agrobacterium tumefaciens 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. In this study, we present a detailed and comprehensive genetic analysis of Cas9-induced DSB repair and T-DNA capture in the model plant Arabidopsis thaliana. We found that classical nonhomologous end joining (cNHEJ) and polymerase theta-mediated end joining (TMEJ) are both, and in part redundantly, acting on CRISPR-induced DSBs to produce very different mutational outcomes. We used newly developed CISGUIDE technology to establish that 8% of mutant alleles have captured T-DNA at the induced break site. In addition, we find T-DNA shards within genomic DSB repair sites indicative of frequent temporary interactions during TMEJ. Analysis of thousands of plant genome-T-DNA junctions, followed up by genetic dissection, further reveals that TMEJ is responsible for attaching the 3' end of T-DNA to a CRISPR-induced DSB, while the 5' end can be attached via TMEJ as well as cNHEJ. By identifying the mechanisms that act to connect recombinogenic ends of DNA molecules at chromosomal breaks, and quantifying their contributions, our study supports the development of tailor-made strategies toward predictable engineering of crop plants. Show less
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... Show moreG 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. elegans. These two mutant libraries enable effective deorphanization of chemoreceptors, and characterization of receptors for neuropeptides in various cellular processes. Mutating a set of closely related GPCRs in a single strain permits the assignment of functions to GPCRs with functional redundancy. Our analyses identify a neuropeptide that interacts with three receptors in hypoxia-evoked locomotory responses, unveil a collection of regulators in pathogen-induced immune responses, and define receptors for the volatile food-related odorants. These results establish our GPCR and neuropeptide mutant libraries as valuable resources for the C. elegans community to expedite studies of GPCR signaling in multiple contexts. Show less
Schimmel, J.; Munoz Subirana, N.; Kool, H.; Schendel, R. van; Vlies, S. van der; Kamp, J.A.; ... ; Tijsterman, M. 2023
Gene editing through repair of CRISPR-Cas9-induced chromosomal breaks offers a means to correct a wide range of genetic defects. Directing repair to produce desirable outcomes by modulating DNA... Show moreGene editing through repair of CRISPR-Cas9-induced chromosomal breaks offers a means to correct a wide range of genetic defects. Directing repair to produce desirable outcomes by modulating DNA repair path-ways holds considerable promise to increase the efficiency of genome engineering. Here, we show that inhibition of non-homologous end joining (NHEJ) or polymerase theta-mediated end joining (TMEJ) can be exploited to alter the mutational outcomes of CRISPR-Cas9. We show robust inhibition of TMEJ activity at CRISPR-Cas9-induced double-strand breaks (DSBs) using ART558, a potent polymerase theta (PolW) inhib-itor. Using targeted sequencing, we show that ART558 suppresses the formation of microhomology-driven deletions in favor of NHEJ-specific outcomes. Conversely, NHEJ deficiency triggers the formation of large kb-sized deletions, which we show are the products of mutagenic TMEJ. Finally, we show that combined chemical inhibition of TMEJ and NHEJ increases the efficiency of homology-driven repair (HDR)-mediated precise gene editing. Our work reports a robust strategy to improve the fidelity and safety of genome engi-neering. Show less
Schendel, R. van; Schimmel, J.; Tijsterman, M. 2022
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... Show moreWith 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.g. Illumina and PacBio). SIQ facilitates their interpretation by establishing mutational profiles, with a focus on event classification such as deletions, single-nucleotide variations, (templated) insertions and tandem duplications. SIQ results can be directly analysed and visualized via SIQPlotteR, an interactive web tool that we made freely available. Using insightful tornado plot visualizations as outputs, we illustrate that SIQ readily identifies sequence- and repair pathway-specific mutational signatures in a variety of model systems, such as nematodes, plants and mammalian cell culture. Show less
Kralemann, L.E.M.; Pater, B.S. de; Shen, H.; Kloet, S.L.; Schendel, R. van; Hooykaas, P.J.J.; Tijsterman, M. 2022
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... Show moreAgrobacterium 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. Here, using newly developed technology that yields hundreds of T-DNA integrations in somatic tissue of Arabidopsis thaliana, we uncover two redundant mechanisms for the genomic capture of the T-DNA 5' end. Different from capture of the 3' end of the T-DNA, which is the exclusive action of polymerase theta-mediated end joining (TMEJ), 5' attachment is accomplished either by TMEJ or by canonical non-homologous end joining (cNHEJ). We further find that TMEJ needs MRE11, whereas cNHEJ requires TDP2 to remove the 5' end-blocking protein VirD2. As a consequence, T-DNA integration is severely impaired in plants deficient for both MRE11 and TDP2 (or other cNHEJ factors). In support of MRE11 and cNHEJ specifically acting on the 5' end, we demonstrate rescue of the integration defect of double-deficient plants by using T-DNAs that are capable of forming telomeres upon 3' capture. Our study provides a mechanistic model for how Agrobacterium exploits the plant's own DNA repair machineries to transform it. Show less
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... Show moreThe 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. Defects in RNA processing, due to viable THO complex or PNN-1 mutations, induce a shift in DNA repair in dividing and non-dividing tissues. Loss of SMG-1, an ATM/ATR-like kinase central to RNA surveillance by nonsense-mediated decay (NMD), restores DNA repair and radio-resistance in THO-deficient animals. Mechanistically, we find SMG-1 and its downstream target SMG-2/UPF1, but not NMD per se, to suppress DNA repair by non-homologous end-joining in favour of single strand annealing. We postulate that moonlighting proteins create short-circuits in vivo, allowing aberrant RNA to redirect DNA repair. Show less
Kralemann, L.E.M.; Pater, S. de; Shen, H.X.; Kloet, S.L.; Schendel, R. van; Hooykaas, P.J.J.; Tijsterman, M. 2022
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... Show moreAgrobacterium 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. Here, using newly developed technology that yields hundreds of T-DNA integrations in somatic tissue of Arabidopsis thaliana, we uncover two redundant mechanisms for the genomic capture of the T-DNA 5' end. Different from capture of the 3' end of the T-DNA, which is the exclusive action of polymerase theta-mediated end joining (TMEJ), 5' attachment is accomplished either by TMEJ or by canonical non-homologous end joining (cNHEJ). We further find that TMEJ needs MRE11, whereas cNHEJ requires TDP2 to remove the 5' end-blocking protein VirD2. As a consequence, T-DNA integration is severely impaired in plants deficient for both MRE11 and TDP2 (or other cNHEJ factors). In support of MRE11 and cNHEJ specifically acting on the 5' end, we demonstrate rescue of the integration defect of double-deficient plants by using T-DNAs that are capable of forming telomeres upon 3' capture. Our study provides a mechanistic model for how Agrobacterium exploits the plant's own DNA repair machineries to transform it. Show less
Hoijer, I.; Emmanouilidou, A.; Ostlund, R.; Schendel, R. van; Bozorgpana, S.; Tijsterman, M.; ... ; Ameur, A. 2022
CRISPR-Cas9 can introduce unintended off-target effects. Here authors show that unintended mutations produced by in vivo of zebrafish can be inherited by their off-spring.CRISPR-Cas9 genome editing... Show moreCRISPR-Cas9 can introduce unintended off-target effects. Here authors show that unintended mutations produced by in vivo of zebrafish can be inherited by their off-spring.CRISPR-Cas9 genome editing has potential to cure diseases without current treatments, but therapies must be safe. Here we show that CRISPR-Cas9 editing can introduce unintended mutations in vivo, which are passed on to the next generation. By editing fertilized zebrafish eggs using four guide RNAs selected for off-target activity in vitro, followed by long-read sequencing of DNA from >1100 larvae, juvenile and adult fish across two generations, we find that structural variants (SVs), i.e., insertions and deletions >= 50 bp, represent 6% of editing outcomes in founder larvae. These SVs occur both at on-target and off-target sites. Our results also illustrate that adult founder zebrafish are mosaic in their germ cells, and that 26% of their offspring carries an off-target mutation and 9% an SV. Hence, pre-testing for off-target activity and SVs using patient material is advisable in clinical applications, to reduce the risk of unanticipated effects with potentially large implications. Show less
Kamp, J.A.; Lemmens, B.B.L.G.; Romeijn, R.J.; Changoer, S.C.; Schendel, R. van; Tijsterman, M. 2021
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... Show moreDNA 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. single-strand annealing (SSA), polymerase theta-mediated end-joining (TMEJ) and residual ill-defined microhomology-mediated end-joining (MMEJ) activity. Here, we identify C. elegans Helicase Q (HELQ-1) as being essential for MMEJ as well as for SSA. We also find HELQ-1 to be crucial for the synthesis-dependent strand annealing (SDSA) mode of homologous recombination (HR). Loss of HELQ-1 leads to increased genome instability: patchwork insertions arise at deletion junctions due to abortive rounds of polymerase theta activity, and tandem duplications spontaneously accumulate in genomes of helq-1 mutant animals as a result of TMEJ of abrogated HR intermediates. Our work thus implicates HELQ activity for all DSB repair modes guided by complementary base pairs and provides mechanistic insight into mutational signatures common in HR-defective cancers.Microhomology-mediated end-joining (MMEJ) is a poorly defined mutagenic DNA break repair pathway. Here the authors show that the helicase HELQ is essential for polymerase theta-independent MMEJ, single-strand annealing and homologous recombination through synthesis dependent strand annealing in C. elegans. Show less
Kamp, J.A.; Lemmens, B.B.L.G.; Romeijn, R.J.; Changoer, S.C.; Schendel, R. van; Tijsterman, M. 2021
Facioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy clinically characterized by weakness in the facial, shoulder girdle and upper a muscles. FSHD is caused by chromatin... Show moreFacioscapulohumeral muscular dystrophy (FSHD) is an inherited myopathy clinically characterized by weakness in the facial, shoulder girdle and upper a muscles. FSHD is caused by chromatin relaxation of the D4Z4 macrosatellite repeat, mostly by a repeat contraction, facilitating ectopic expression of DUX4 in skeletal muscle. Genetic diagnosis for FSHD is generally based on the sizing and haplotyping of the D4Z4 repeat on chromosome 4 by Southern blotting (SB), molecular combing or single-molecule optical mapping, which is usually straight forward but can be complicated by atypical rearrangements of the D4Z4 repeat. One of these rearrangements is a D4Z4 proximally extended deletion (DPED) allele, where not only the D4Z4 repeat is partially deleted, but also sequences immediately proximal to the repeat are lost, which can impede accurate diagnosis in all genetic methods. Previously, we identified several DPED alleles in FSHD and estimated the size of the proximal deletions by a complex pulsed-field gel electrophoresis and SB strategy. Here, using the next-generation sequencing, we have defined the breakpoint junctions of these DPED alleles at the base pair resolution in 12 FSHD families and 4 control individuals facilitating a PCR-based diagnosis of these DPED alleles. Our resultsshow that half of the DPED alleles are derivates of an ancient founder allele. For some DPED alleles, we found that genetic elements are deleted such as DUX4c, FRG2, DBE-T and myogenic enhancers necessitating re-evaluation of their role in FSHD pathogenesis. Show less
Schimmel, J.; Muñoz-Subirana, N.; Kool, H.; Schendel, R. van; Tijsterman, M. 2021
Error-prone repair of DNA double-strand breaks have been implied to cause cancer-associated genome alterations, but the mechanism of their formation remains unclear. Here the authors find that DNA... Show moreError-prone repair of DNA double-strand breaks have been implied to cause cancer-associated genome alterations, but the mechanism of their formation remains unclear. Here the authors find that DNA polymerase alpha primase plays part in tandem duplication formation at CRISPR/Cas9-induced complementary 3 ' ssDNA protrusions.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 alpha (Pol alpha)-primase in tandem duplication formation at breaks having complementary 3 ' ssDNA protrusions. By including so-called primase deserts in CRISPR/Cas9-induced DNA break configurations, we reveal that fill-in synthesis preferentially starts at the 3 ' tip, and find this activity to be dependent on 53BP1, and the CTC1-STN1-TEN1 (CST) and Shieldin complexes. This axis generates near-blunt ends specifically at DNA breaks with 3 ' overhangs, which are subsequently repaired by non-homologous end-joining. Our study provides a mechanistic explanation for a mutational signature abundantly observed in the genomes of species and cancer cells. Show less
Schendel, R. van; Romeijn, R.; Buijs, H.; Tijsterman, M. 2021
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... Show moreDuring 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 Caenorhabditis elegans genome close to replication-impeding G-quadruplexes, we show that de novo DNA synthesis downstream of the blocked fork suppresses DNA loss. We next identify the pol alpha-primase complex to limit deletion mutagenesis, a conclusion substantiated by whole-genome analysis of animals carrying mutated POLA2/DIV-1. We subsequently identify a new role for the 9-1-1 checkpoint clamp in protecting Okazaki fragments from resection by EXO1. Together, our results provide a mechanistic model for controlling the fate of replication intermediates at sites of stalled replication. Show less
Kamp, J.A.; Schendel, R. van; Dilweg, I.W.; Tijsterman, M. 2020
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... Show moreFailure 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. We here show that the mutational landscape of BRCA1 deficiency in C. elegans closely resembles that of BRCA1-deficient tumours. We identify polymerase theta-mediated end-joining (TMEJ) to be responsible: knocking out polq-1 suppresses the accumulation of deletions and tandem duplications in brc-1 and brd-1 animals. We find no additional back-up repair in HR and TMEJ compromised animals; non-homologous end-joining does not affect BRCAness. The notion that TMEJ acts as an alternative to HR, promoting the genome alteration of HR-deficient cells, supports the idea that polymerase theta is a promising therapeutic target for HR-deficient tumours. Show less
Bostelen, I. van; Schendel, R. van; Romeijn, R.; Tijsterman, M. 2020
Author summaryResearch in the fields of DNA repair and mutagenesis has led to enormous insight into the mechanisms responsible for maintaining genetic integrity. However, which processes drive de... Show moreAuthor summaryResearch in the fields of DNA repair and mutagenesis has led to enormous insight into the mechanisms responsible for maintaining genetic integrity. However, which processes drive de novo mutations and will thus contribute to inherited diseases are still unclear. One process thought to underlie spontaneous mutagenesis is replication of damaged DNA by specialised so-called "Translesion synthesis" polymerases, which have the ability to replicate across damaged bases, but are not very accurate. To address the impact of TLS or the lack thereof on genome integrity, we have knocked out all TLS enzymes that are encoded by the C. elegans genome, individually and in combination, and monitored mutation accumulation during prolonged culturing of these animals without external sources of DNA damage. We found that TLS is not the major driver of spontaneous mutagenesis in this organism, however, it protects the genome from harmful small deletions that result from mutagenic repair of DNA breaks. We also found that, contrary to what was expected, TLS activity is not essential for reproduction in a multicellular organism with the tissue complexity and genome size of C. elegans.Bases within DNA are frequently damaged, producing obstacles to efficient and accurate DNA replication by replicative polymerases. Translesion synthesis (TLS) polymerases, via their ability to catalyze nucleotide additions to growing DNA chains across DNA lesions, promote replication of damaged DNA, thus preventing checkpoint activation, genome instability and cell death. In this study, we used C. elegans to determine the contribution of TLS activity on long-term stability of an animal genome. We monitored and compared the types of mutations that accumulate in REV1, REV3, POLH1 and POLK deficient animals that were grown under unchallenged conditions. We also addressed redundancies in TLS activity by combining all deficiencies. Remarkably, animals that are deficient for all Y-family polymerases as well as animals that have lost all TLS activity are viable and produce progeny, demonstrating that TLS is not essential for animal life. Whole genome sequencing analyses, however, reveal that TLS is needed to prevent genomic scars from accumulating. These scars, which are the product of polymerase theta-mediated end joining (TMEJ), are found overrepresented at guanine bases, consistent with TLS suppressing DNA double-strand breaks (DSBs) from occurring at replication-blocking guanine adducts. We found that in C. elegans, TLS across spontaneous damage is predominantly error free and anti-clastogenic, and thus ensures preservation of genetic information. Show less
Schimmel, J.; Schendel, R. van; Dunnen, J.T. den; Tijsterman, M. 2019
A recognized source of disease-causing genome alterations is erroneous repair of broken chromosomes, which can be executed by two distinct mechanisms: non-homologous end joining (NHEJ) and the... Show moreA recognized source of disease-causing genome alterations is erroneous repair of broken chromosomes, which can be executed by two distinct mechanisms: non-homologous end joining (NHEJ) and the recently discovered polymerase theta-mediated end joining (TMEJ) pathway. While TMEJ has previously been considered to act as an altemative mechanism backing up NHEJ, recent work points to a role for TMEJ in the repair of replication-associated DNA breaks that are excluded from repair through homologous recombination. Because of its mode of action, TMEJ is intrinsically mutagenic and sometimes leaves behind a recognizable genomic scar when joining chromosome break ends (i.e., 'templated insertions'). This review article focuses on the intriguing observation that this polymerase theta signature is frequently observed in disease alleles, arguing for a prominent role of this double-strand break repair pathway in genome diversification and disease-causing spontaneous mutagenesis in humans. Show less
Schimmel, J.; Kool, H.; Schendel, R. van; Tijsterman, M. 2017
