Frequency Of Homologous Recombination Research Articles

Defective transfer of parental histone decreases frequency of homologous recombination by increasing free histone pools in budding yeast.

Recycling of parental histones is an important step in epigenetic inheritance. During DNA replication, DNA polymerase epsilon subunit DPB3/DPB4 and DNA replication helicase subunit MCM2 are involved in the transfer of parental histones to the leading and lagging strands, respectively. Single Dpb3 deletion (dpb3Δ) or Mcm2 mutation (mcm2-3A), which each disrupts one parental histone transfer pathway, leads to the other's predominance. However, the biological impact of the two histone transfer pathways on chromatin structure and DNA repair remains elusive. In this study, we used budding yeast Saccharomyces cerevisiae to determine the genetic and epigenetic outcomes from disruption of parental histone H3-H4 tetramer transfer. We found that a dpb3Δ mcm2-3A double mutant did not exhibit the asymmetric parental histone patterns caused by a single dpb3Δ or mcm2-3A mutation, suggesting that the processes by which parental histones are transferred to the leading and lagging strands are independent. Surprisingly, the frequency of homologous recombination was significantly lower in dpb3Δ, mcm2-3Aand dpb3Δ mcm2-3A mutants, likely due to the elevated levels of free histones detected in the mutant cells. Together, these findings indicate that proper transfer of parental histones during DNA replication is essential for maintaining chromatin structure and that lower homologous recombination activity due to parental histone transfer defects is detrimental to cells.

A gln-tRNA-based CRISPR/Cas9 knockout system enables the functional characterization of genes in the genetically recalcitrant brassica anthracnose fungus Colletotrichum higginsianum

Colletotrichum higginsianum causes anthracnose disease in brassicas. The availability of the C. higginsianum genome has paved the way for the genome-wide exploration of genes associated with virulence/pathogenicity. However, delimiting the biological functions of these genes remains an arduous task due to the recalcitrance of C. higginsianum to genetic manipulations. Here, we report a CRISPR/Cas9-based system that can knock out the genes in C. higginsianum with a staggering 100% homologous recombination frequency (HRF). The system comprises two vectors: pCas9-Ch_tRp-sgRNA, in which a C. higginsianum glutaminyl-tRNA drives the expression of sgRNA, and pCE-Zero-HPT carrying a donor DNA cassette containing the marker gene HPT flanked by homology arms. Upon co-transformation of the C. higginsianum protoplasts, pCas9-Ch_tRp-sgRNA causes a DNA double-strand break in the targeted gene, followed by homology-directed replacement of the gene with HPT by pCE-Zero-HPT, thereby generating loss-of-function mutants. Using the system, we generated the knockout mutants of two effector candidates (ChBas3 and OBR06881) with a 100% HRF. Interestingly, the ΔChBas3 and ΔOBR06881 mutants did not seem to affect the C. higginsianum infection of Arabidopsis thaliana. Altogether, the CRISPR/Cas9 system developed in the study enables the targeted deletion of genes, including effectors, in C. higginsianum, thus determining their biological functions.

INFLUENCE OF KU70 GENE KNOCKOUT ON TRANSFORMATION FREQUENCY OF PENICILLIUM VERRUCULOSUM MYCELIAL FUNGI

To increase the frequency of homologous recombination (HR) during the transformation of the industrial strain Penicillium verruculosum 221-151 (VKM F-3972D), the ku70 gene encoding the Ku70, which binds at the sites of double-stranded DNA breaks and is involved in the repair process by the non-homologous end joint (NHEJ), was knocked out by the CRISPR/CAS9 method. Presumably, the new host strain, P. verruculosum ΔniaDΔku70, should have had an increased frequency of homologous recombination during transformation in comparison with the host strain P. verruculosum ΔniaD due to the integrative insertion of the expression cassette only by the HR mechanism. The pep1 gene encoding homologous aspartate protease was chosen as a marker. However, it was shown that the knockout of the ku70 gene led to a dramatic decrease in the frequency of co-transformation in the P. verruculosum ΔniaDΔku70 strain compared to the P. verruculosum ΔniaD strain at the same load of exogenous DNA (3 μg). The number of copies of the pep1 gene in recombinant strains of the P. verruculosum Pep1 (with a native Ku70) series ranged from 3 to 28 copies, which indicated the predominance of the non-homologous recombination mechanism.

DNA damage response and repair gene mutations are associated with tumor mutational burden and outcomes to platinum-based chemotherapy/immunotherapy in advanced NSCLC patients

BackgroundDNA damage response and repair (DDR) genes are crucial for maintaining the integrity of the genome. This study aims to explore the correlation of DDR gene mutations with TMB, clinical characteristics, and outcomes to platinum-based chemotherapy and platinum-based chemotherapy/immunotherapy in non-small cell lung cancer (NSCLC) without EGFR and ALK alterations.MethodsTumor tissue from 49 patients with stage III or IV NSCLC who were without EGFR and ALK alterations were analyzed using targeted next-generation sequencing (NGS). Among them, 13 patients received first-line platinum-based chemotherapy, 32 patients received first-line platinum-based chemotherapy/immunotherapy.ResultsIn these NSCLC patients without EGFR and ALK alterations, the frequently mutated genes included TP53, KMT2D and KRAS, the most frequently mutated DDR gene was FANCG, DDR gene mutations were detected in 20 patients. The mutation frequency of homologous recombination (HR) pathway was significantly higher in lung squamous cell carcinoma (LUSC) than that in lung adenocarcinoma (LUAD) (30.8% vs. 5.7%). Among DDR positive patients, a lower percentage exhibited metastasis. Patients with DDR gene mutations, cell-cycle checkpoint pathway mutations, and BER pathway mutations had significantly higher TMB compared to those without corresponding mutations. In the patients receiving platinum-based chemotherapy/immunotherapy, the disease control rate was significantly lower in the DDR-positive group compared with that in the DDR-negative group (55.6% vs. 100.0%). Among LUAD patients receiving platinum-based chemotherapy/immunotherapy, we observed a worse overall survival (OS) in DDR-positive group, as well as poorer progression-free survival(PFS)and OS in BER-positive and FANCG mutated group.ConclusionsDDR gene mutations are associated with tumor metastasis, TMB, and outcomes to platinum-based chemotherapy/immunotherapy in advanced NSCLC patients.

Recyclable CRISPR/Cas9-mediated gene disruption and deletions in Histoplasma.

Histoplasma is a primary fungal pathogen with the ability to infect otherwise healthy mammalian hosts, causing systemic and sometimes life-threatening disease. Thus far, molecular genetic manipulation of this organism has utilized RNA interference, random insertional mutagenesis, and a homologous recombination protocol that is highly variable and often inefficient. Targeted gene manipulations have been challenging due to poor rates of homologous recombination events in Histoplasma. Interrogation of the virulence strategies of this organism would be highly accelerated by a means of efficiently generating targeted mutations. We have developed a recyclable CRISPR/Cas9 system that can be used to introduce gene disruptions in Histoplasma with high efficiency, thereby allowing disruption of multiple genes.

Multi-host infection and phylogenetically diverse lineages shape the recombination and gene pool dynamics of Staphylococcus aureus.

Staphylococcus aureus can infect and adapt to multiple host species. However, our understanding of the genetic and evolutionary drivers of its generalist lifestyle remains inadequate. This is particularly important when considering local populations of S. aureus, where close physical proximity between bacterial lineages and between host species may facilitate frequent and repeated interactions between them. Here, we aim to elucidate the genomic differences between human- and animal-derived S. aureus from 437 isolates sampled from disease cases in the northeast region of the United States. Multi-locus sequence typing revealed the existence of 75 previously recognized sequence types (ST). Our population genomic analyses revealed heterogeneity in the accessory genome content of three dominant S. aureus lineages (ST5, ST8, ST30). Genes related to antimicrobial resistance, virulence, and plasmid types were differentially distributed among isolates according to host (human versus non-human) and among the three major STs. Across the entire population, we identified a total of 1,912 recombination events that occurred in 765 genes. The frequency and impact of homologous recombination were comparable between human- and animal-derived isolates. Low-frequency STs were major donors of recombined DNA, regardless of the identity of their host. The most frequently recombined genes (clfB, aroA, sraP) function in host infection and virulence, which were also frequently shared between the rare lineages. Taken together, these results show that frequent but variable patterns of recombination among co-circulating S. aureus lineages, including the low-frequency lineages, that traverse host barriers shape the structure of local gene pool and the reservoir of host-associated genetic variants. Our study provides important insights to the genetic and evolutionary factors that contribute to the ability of S. aureus to colonize and cause disease in multiple host species. Our study highlights the importance of continuous surveillance of S. aureus circulating in different ecological host niches and the need to systematically sample from them. These findings will inform development of effective measures to control S. aureus colonization, infection, and transmission across the One Health continuum.

Dicer mutants and rdr6 mutant are impaired in an intergenerational increase in homologous recombination frequency in response to the infection with oilseed rape mosaic virus

Previous reports demonstrated that viral infection of Arabidopsis and tobacco plants resulted in a local and systemic increase in the homologous recombination frequency (HRF). Furthermore, the progeny of infected plants also exhibited a higher spontaneous frequency of recombination. Arabidopsis plants are known to produce DNA virus-derived small interfering RNAs (siRNAs) in response to Oilseed rape mosaic virus (ORMV) infection; dcl4 and rdr6 mutants are affected in this process and as a result, in viral suppression. Previous research also showed that siRNA biogenesis mutants had a lower frequency of homologous recombination and some of them were even impaired in the intergenerational response to abiotic stress. Therefore, we hypothesized that an increase in the HRF in response to infection might be impaired in Dicer-like (dcl) mutants, and that they might also be required for intergenerational changes. We infected single mutants, dcl2, dcl3, dcl4, rdr6, a double mutant dcl2 dcl3 and a triple mutant dcl2 dcl3 dcl4 with ORMV and tested the HRF in the systemic leaves of infected plants as well as in the progeny of infected plants. A somatic increase in the HRF was impaired in rdr6 and dcl2 dcl3 mutants, while it was not affected in single mutants. All tested mutants were impaired in the intergenerational increase in the HRF. Thus, different proteins in the RdDM pathway are needed to modulate homologous recombination frequency in response to the oilseed rape mosaic virus infection in somatic tissues and in the progeny.

Split-marker-mediated genome editing improves homologous recombination frequency in the CTG clade yeast Candida intermedia.

Genome editing toolboxes are essential for the exploration and exploitation of non-conventional yeast species as cell factories, as they facilitate both genome studies and metabolic engineering. The non-conventional yeast Candida intermedia is a biotechnologically interesting species due to its capacity to convert a wide range of carbon sources, including xylose and lactose found in forestry and dairy industry waste and side-streams, into added-value products. However, possibilities of genetic manipulation have so far been limited due to lack of molecular tools for this species. We describe here the development of a genome editing method for C. intermedia, based on electroporation and gene deletion cassettes containing the Candida albicans NAT1 dominant selection marker flanked by 1,000 base pair sequences homologous to the target loci. Linear deletion cassettes targeting the ADE2 gene originally resulted in <1% targeting efficiencies, suggesting that C. intermedia mainly uses non-homologous end joining for integration of foreign DNA fragments. By developing a split-marker based deletion technique for C. intermedia, we successfully improved the homologous recombination rates, achieving targeting efficiencies up to 70%. For marker-less deletions, we also employed the split-marker cassette in combination with a recombinase system, which enabled the construction of double deletion mutants via marker recycling. Overall, the split marker technique proved to be a quick and reliable method for generating gene deletions in C. intermedia, which opens the possibility to uncover and enhance its cell factory potential.

A Multi-plex Protein Expression System for Production of Complex Enzyme Formulations in Trichoderma reesei.

Historically, heterologous protein production has been challenging using the hyper-cellulolytic fungus, Trichoderma reesei. T. reesei is known for poor transformation efficiency, low homologous recombination frequency, and marginal screening systems for identification of successful transformants. In this work, we have applied the 2A-peptide multi-gene expression system to co-express four enzymes, which include three cellulases, a cellobiohydrolase (CBH1), an endoglucanase (EG1), and a β-D-glucosidase (BGL1); as well as the enhanced Green Fluorescent Protein, (eGFP), used here as a marker for monitoring expression levels. We designed a new chassis vector, pTrEno-4X-2A for this work. Expression of these cellulase enzymes was confirmed by real-time quantitative reverse transcription PCR and immunoblot analysis. The activity of each of the cellulases was assessed using pNP glycosides and the chromogenic substrate, AZCL-HE-cellulose, which confirmed the functionality of the enzymes. Expression and activity of these enzymes was proportional to the level of eGFP fluorescence, thereby validating the reliability of this screening technique. Although all three cellulase proteins were successfully expressed, their expression levels varied significantly. Specifically, up to an 18-fold difference was observed between the first and the third gene within the 2A-peptide construct, based on protein quantitation. The availability of this new screening tool is expected to greatly impact multi-enzyme applications, such as production of complex commercial enzyme formulations and the study of metabolic pathway enzymes, especially those destined for cell free applications.

Analysis of BRCT5 domain-containing proteins reveals a new component of DNA damage repair in Arabidopsis.

The integrity of plant genetic information is constantly challenged by various internal and external factors. Therefore, plants use a sophisticated molecular network to identify, signal and repair damaged DNA. Here, we report on the identification and analysis of four uncharacterized Arabidopsis BRCT5 DOMAIN CONTAINING PROTEINs (BCPs). Proteins with the BRCT5 domain are frequently involved in the maintenance of genome stability across eukaryotes. The screening for sensitivity to induced DNA damage identified BCP1 as the most interesting candidate. We show that BCP1 loss of function mutants are hypersensitive to various types of DNA damage and accumulate an increased number of dead cells in root apical meristems upon DNA damage. Analysis of publicly available sog1 transcriptomic and SOG1 genome-wide DNA binding data revealed that BCP1 is inducible by gamma radiation and is a direct target of this key DNA damage signaling transcription factor. Importantly, bcp1 plants showed a reduced frequency of somatic homologous recombination in response to both endogenous and induced DNA damage. Altogether, we identified a novel plant-specific DNA repair factor that acts downstream of SOG1 in homology-based repair.

Capripoxviruses, leporipoxviruses, and orthopoxviruses: Occurrences of recombination.

Poxviruses are double-stranded DNA viruses with several members displaying restricted host ranges. They are genetically stable with low nucleotide mutation rates compared to other viruses, due to the poxviral high-fidelity DNA polymerase. Despite the low accumulation of mutations per replication cycle, poxvirus genomes can recombine with each other to generate genetically rearranged viruses through recombination, a process directly associated with replication and the aforementioned DNA polymerase. Orthopoxvirus replication is intimately tethered to high frequencies of homologous recombination between co-infecting viruses, duplicated sequences of the same virus, and plasmid DNA transfected into poxvirus-infected cells. Unfortunately, the effect of these genomic alterations on the cellular context for all poxviruses across the family Poxviridae remains elusive. However, emerging sequence data on currently circulating and archived poxviruses, such as the genera orthopoxviruses and capripoxviruses, display a wide degree of divergence. This genetic variability cannot be explained by clonality or genetic drift alone, but are probably a result of significant genomic alterations, such as homologous recombination, gene loss and gain, or gene duplications as the major selection forces acting on viral progeny. The objective of this review is to cross-sectionally overview the currently available findings on natural and laboratory observations of recombination in orthopoxviruses, capripoxviruses, and leporipoxviruses, as well as the possible mechanisms involved. Overall, the reviewed available evidence allows us to conclude that the current state of knowledge is limited in terms of the relevance of genetic variations across even a genus of poxviruses as well as fundamental features governing and precipitating intrinsic gene flow and recombination events.

Multigenerational Exposure to Heat Stress Induces Phenotypic Resilience, and Genetic and Epigenetic Variations in Arabidopsis thaliana Offspring.

Plants are sedentary organisms that constantly sense changes in their environment and react to various environmental cues. On a short-time scale, plants respond through alterations in their physiology, and on a long-time scale, plants alter their development and pass on the memory of stress to the progeny. The latter is controlled genetically and epigenetically and allows the progeny to be primed for future stress encounters, thus increasing the likelihood of survival. The current study intended to explore the effects of multigenerational heat stress in Arabidopsis thaliana. Twenty-five generations of Arabidopsis thaliana were propagated in the presence of heat stress. The multigenerational stressed lineage F25H exhibited a higher tolerance to heat stress and elevated frequency of homologous recombination, as compared to the parallel control progeny F25C. A comparison of genomic sequences revealed that the F25H lineage had a three-fold higher number of mutations [single nucleotide polymorphisms (SNPs) and insertions and deletions (INDELs)] as compared control lineages, suggesting that heat stress induced genetic variations in the heat-stressed progeny. The F25H stressed progeny showed a 7-fold higher number of non-synonymous mutations than the F25C line. Methylome analysis revealed that the F25H stressed progeny showed a lower global methylation level in the CHH context than the control progeny. The F25H and F25C lineages were different from the parental control lineage F2C by 66,491 and 80,464 differentially methylated positions (DMPs), respectively. F25H stressed progeny displayed higher frequency of methylation changes in the gene body and lower in the body of transposable elements (TEs). Gene Ontology analysis revealed that CG-DMRs were enriched in processes such as response to abiotic and biotic stimulus, cell organizations and biogenesis, and DNA or RNA metabolism. Hierarchical clustering of these epimutations separated the heat stressed and control parental progenies into distinct groups which revealed the non-random nature of epimutations. We observed an overall higher number of epigenetic variations than genetic variations in all comparison groups, indicating that epigenetic variations are more prevalent than genetic variations. The largest difference in epigenetic and genetic variations was observed between control plants comparison (F25C vs. F2C), which clearly indicated that the spontaneous nature of epigenetic variations and heat-inducible nature of genetic variations. Overall, our study showed that progenies derived from multigenerational heat stress displayed a notable adaption in context of phenotypic, genotypic and epigenotypic resilience.

Plant Gene Modification by BAC Recombineering.

Recombineering approaches exploiting the bacteriophage λ Red recombination functions are widely used for versatile modification of eukaryotic genes carried by bacterial artificial chromosomes (BACs) in E. coli. Whereas BAC transformation provides a simple means for integration of modified genes into the genomes of animal cells to generate knock-in and knockout lines, successful application of this strategy is hampered by low frequency of homologous recombination in higher plants. However, plant cells can be transformed at a high frequency using the transferred DNA (T-DNA) of Agrobacterium, which is stably and randomly integrated into the plant genome. The function of plant genes that are modified by recombineering and transferred by Agrobacterium T-DNA vectors into plant cells can thus be suitably studied using genetic complementation of knockout mutations induced by either T-DNA insertions or genome editing with T-DNA-based Crisp/Cas9 constructs. Here we describe two recombineering protocols for modification and transfer of plant genes from BACs into Agrobacterium T-DNA plant transformation vectors. The first protocol uses a conditional suicide ccdB gene cassette to assist the genetic complementation assays by generation of point mutations, deletions, and insertions at any gene position. The second "turbo"-recombineering protocol exploits various I-SceI insertion cassettes for fusing of fluorescent protein tags to the plant gene products to facilitate the characterization of their in vivo interacting partners by affinity purification, mass spectrometry, and cellular localization studies.

Targeted Gene Knockouts by Protoplast Transformation in the Moss Physcomitrella patens.

Targeted gene knockout is particularly useful for analyzing gene functions in plant growth, signaling, and development. By transforming knockout cassettes consisting of homologous sequences of the target gene into protoplasts, the classical gene targeting method aims to obtain targeted gene replacement, allowing for the characterization of gene functions in vivo. The moss Physcomitrella patens is a known model organism for a high frequency of homologous recombination and thus harbors a remarkable rate of gene targeting. Other moss features, including easy to culture, dominant haploidy phase, and sequenced genome, make gene targeting prevalent in Physcomitrella patens. However, even gene targeting was powerful to generate knockouts, researchers using this method still experienced technical challenges. For example, obtaining a good number of targeted knockouts after protoplast transformation and regeneration disturbed the users. Off-target mutations such as illegitimate random integration mediated by nonhomologous end joining and targeted insertion wherein one junction on-target but the other end off-target is commonly present in the knockouts. Protoplast fusion during transformation and regeneration was also a problem. This review will discuss the advantages and technical challenges of gene targeting. Recently, CRISPR-Cas9 is a revolutionary technology and becoming a hot topic in plant gene editing. In the second part of this review, CRISPR-Cas9 technology will be focused on and compared to gene targeting regarding the practical use in Physcomitrella patens. This review presents an updated perspective of the gene targeting and CRISPR-Cas9 techniques to plant biologists who may consider studying gene functions in the model organism Physcomitrella patens.

Establishing an Efficient Genetic Manipulation System for Sulfated Echinocandin Producing Fungus Coleophoma empetri.

Micafungin is an important echinocandin antifungal agent for the treatment of invasive fungal infections. In industry, micafungin is derived from the natural product FR901379, which is a non-ribosomal cyclic hexapeptide produced by the filamentous fungus Coleophoma empetri. The difficulty of genetic manipulation in C. empetri restricts the clarification of FR901379 biosynthetic mechanism. In this work, we developed an efficient genetic manipulation system in the industrial FR901379-producing strain C. empetri MEFC009. Firstly, a convenient protoplast-mediated transformation (PMT) method was developed. Secondly, with this transformation method, the essential genetic elements were verified. Selectable markers hph, neo, and nat can be used for the transformation, and promotors Ppgk, PgpdA, and PgpdAt are functional in C. empetri MEFC009. Thirdly, the frequency of homologous recombination was improved from 4 to 100% by deleting the ku80 gene, resulting in an excellent chassis cell for gene-targeting. Additionally, the advantage of this genetic manipulation system was demonstrated in the identification of the polyketide synthase (PKS) responsible for the biosynthesis of dihydroxynapthalene (DHN)-melanin. This genetic manipulation system will be a useful platform for the research of FR901379 and further genome mining of secondary metabolites in C. empetri.

Frequency of homologous recombination associated gene mutations in Japanese patients with ovarian cancer

<h3>Objectives:</h3> Homologous recombination deficiency (HRD) is a well-known biomarker of PARP inhibitor sensitivity. The Cancer Genome Atlas (TCGA) has clarified that about 50% of high-grade serous ovarian cancer shows HRD. However, the frequency of HRD in Japanese patients with ovarian cancer remains unclear. The aim of JGOG3025 study (NCT03159572) is to identify the frequency of HR-associated gene mutations in ovarian cancer, especially non-serous histologic types. <h3>Methods:</h3> The JGOG3025 study is a collaborative prospective observational study involving 65 study sites throughout Japan. We recruited 996 patients who were clinically diagnosed with ovarian cancer before surgery from March 2017 to March 2019, and 711 patients were eligible for JGOG3025 criteria. After central pathological review, we used frozen tumor tissues to extract DNA and performed targeted sequencing for 51 HR-associated genes in 711 ovarian cancers (298 high-grade serous, 189 clear cell, 142 endometrioid, 15 mucinous, 4 low-grade serous, and 63 others). <h3>Results:</h3> To focus on pathogenic mutations of HR-associated genes which led to HRD, we used only loss-of-function (LOF) mutations (nonsense, readthrough, and frameshift) to perform subsequent analysis. At least one HR-associated gene was mutated in 105 high-grade serous (35.2%), 21 clear cell (11.1%), 36 endometrioid (25.4%), 1 low-grade serous (25.0%) carcinomas. Although 81.8% of ovarian cancers with <i>BRCA1</i> LOF mutation were diagnosed as high-grade serous type, one case of mucinous carcinoma harbored <i>BRCA1</i> c.1961delA. On the other hand, 71.4% of ovarian cancers harboring <i>BRCA2</i> LOF mutation were high-grade serous type. Mutant allele frequencies of <i>BRCA1</i> and <i>RAD51D</i> were apparently higher than that of other HR-associated genes, suggesting allelic imbalance. <h3>Conclusions:</h3> Our findings demonstrated that mutations in HR-associated genes were detected in not only high-grade serous but also non-serous histologic type ovarian cancers. To clarify the frequency of HRD in Japanese patients with ovarian cancer, we need to assess the pathogenicity of missense mutations and investigate the correlation between HR associated gene mutations and HRD score based on copy number assay.

BRCA2 Promotes Spontaneous Homologous Recombination In Vivo.

Simple SummaryInherited mutation of either BRCA1 or BRCA2 is associated with familial breast and ovarian cancer. As a tumor suppressor, BRCA2 functions to maintain genome stability but we do not know the genomic impact of this deficiency on normal tissues in normal contexts, rather than cancer cells that carry other gene mutations. Numerous murine models of BRCA2 have been developed to investigate BRCA2 tumor suppressor function but have been met with difficulties due to the lethality of most of these models during embryogenesis. To circumvent embryonic lethality here, we employ the cre conditional system combined with an assay for measuring changes in stability of a large tandem DNA repeat by homology-directed recombination. The relevance of this assay is that it can measure DNA recombination events in a normal growing/developing tissue. Here, we show that BRCA2 is required for homologous recombination in a normal developing tissue long before cancer arises.Background: BRCA2 is known to be a tumor suppressor involved in homologous recombination repair and presumed to prevent genome instability in normal tissues prior to the development of tumors. Typical assessment of BRCA2 deficiency on the genome involves cell-based models using cancer cells with mixed genetic contexts, but the role in normal tissue in vivo has not been clearly demonstrated. Methods: Using conditional deletion of Brca2 exon 11, the region containing all eight BRC repeats, in the retinal pigment epithelium and the pink-eyed unstable mouse model, we evaluate the frequency of DNA deletion events. Results: In the current study, we show that conditional loss of Brca2 exon 11 results in a decreased frequency of spontaneous homologous recombination compared to wild-type mice. Of note, we observe no apparent concomitant increase in events that indicate single-strand annealing by the pink-eyed unstable mouse model. Conclusions: Therefore, our results demonstrate that BRCA2, as expected, is required for high-fidelity homologous recombination DNA repair in normal tissues, here in a tissue undergoing normal proliferation through normal development.

Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species.

Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species.

Cloning of human ABCB11 gene in E. coli required the removal of an intragenic Pribnow-Schaller Box before it’s Insertion into genomic safe harbor AAVS1 site using CRISPR–Cas9

Background: Genomic safe harbors are sites in the genome which are safe for gene insertion such that the inserted gene will function properly, and the disruption of the genomic location doesn’t cause any foreseeable risk to the host. The AAVS1 site is the genetic location which is disrupted upon integration of adeno associated virus (AAV) and is considered a ‘safe-harbor’ in human genome because about one-third of humans are infected with AAV and so far there is no apodictic evidence that AAV is pathogenic or disruption of AAVS1 causes any disease in man. Therefore, we chose to target the AAVS1 site for the insertion of ABCB11, a bile acid transporter which is defective in progressive familial intra hepatic cholestasis type-2 (PFIC-2), a lethal disease of children where cytotoxic bile salts accumulate inside hepatocytes killing them and eventually the patient. Methods: We used the CRISPR Cas9 a genome editing system to insert the ABCB11 gene at AAVS1 site in human cell-lines. Results: We found that human ABCB11 sequence has a “Pribnow- Schaller Box” which allows its expression in bacteria and expression of ABCB11 protein which is toxic to E. coli; the removal of this was required for successful cloning. We inserted ABCB11 at AAVS1 site in HEK 293T using CRISPR-Cas9 tool. We also found that the ABCB11 protein has similarity with E. coli endotoxin (lipid A) transporter MsbA. Conclusions: We inserted ABCB11 at AAVS1 site using CRISPR-Cas9; however, the frequency of homologous recombination was very low for this approach to be successful in vivo.

Cloning of human ABCB11 gene in E. coli required the removal of an intragenic Pribnow-Schaller Box before it's Insertion into genomic safe harbor AAVS1 site using CRISPR-Cas9.

Background: Genomic safe harbors are sites in the genome which are safe for gene insertion such that the inserted gene will function properly, and the disruption of the genomic location doesn't cause any foreseeable risk to the host. The AAVS1 site is the genetic location which is disrupted upon integration of adeno associated virus (AAV) and is considered a 'safe-harbor' in human genome because about one-third of humans are infected with AAV and so far there is no apodictic evidence that AAV is pathogenic or disruption of AAVS1 causes any disease in man. Therefore, we chose to target the AAVS1 site for the insertion of ABCB11, a bile acid transporter which is defective in progressive familial intra hepatic cholestasis type-2 (PFIC-2), a lethal disease of children where cytotoxic bile salts accumulate inside hepatocytes killing them and eventually the patient. Methods: We used the CRISPR Cas9 a genome editing system to insert the ABCB11 gene at AAVS1 site in human cell-lines. Results: We found that human ABCB11 sequence has a "Pribnow- Schaller Box" which allows its expression in bacteria and expression of ABCB11 protein which is toxic to E. coli; the removal of this was required for successful cloning. We inserted ABCB11 at AAVS1 site in HEK 293T using CRISPR-Cas9 tool. We also found that the ABCB11 protein has similarity with E. coli endotoxin (lipid A) transporter MsbA. Conclusions: We inserted ABCB11 at AAVS1 site using CRISPR-Cas9; however, the frequency of homologous recombination was very low for this approach to be successful in vivo.