Heavy Metal Exposure Influences Double Strand Break DNA Repair Outcomes

Heavy metals such as cadmium, arsenic and nickel are classified as carcinogens. Although the precise mechanism of carcinogenesis is undefined, heavy metal exposure can contribute to genetic damage by inducing double strand breaks (DSBs) as well as inhibiting critical proteins from different DNA repair pathways. Here we take advantage of two previously published culture assay systems developed to address mechanistic aspects of DNA repair to evaluate the effects of heavy metal exposures on competing DNA repair outcomes. Our results demonstrate that exposure to heavy metals significantly alters how cells repair double strand breaks. The effects observed are both specific to the particular metal and dose dependent. Low doses of NiCl₂ favored resolution of DSBs through homologous recombination (HR) and single strand annealing (SSA), which were inhibited by higher NiCl₂ doses. In contrast, cells exposed to arsenic trioxide preferentially repaired using the “error prone” non-homologous end joining (alt-NHEJ) while inhibiting repair by HR. In addition, we determined that low doses of nickel and cadmium contributed to an increase in mutagenic recombination-mediated by Alu elements, the most numerous family of repetitive elements in humans. Sequence verification confirmed that the majority of the genetic deletions were the result of Alu-mediated non-allelic recombination events that predominantly arose from repair by SSA. All heavy metals showed a shift in the outcomes of alt-NHEJ repair with a significant increase of non-templated sequence insertions at the DSB repair site. Our data suggest that exposure to heavy metals will alter the choice of DNA repair pathway changing the genetic outcome of DSBs repair.     

Involvement of Nucleotide Excision and Mismatch Repair Mechanisms in Double Strand Break Repair

Living organisms are constantly threatened by environmental DNA-damaging agents, including UV and ionizing radiation (IR). Repair of various forms of DNA damage caused by IR is normally thought to follow lesion-specific repair pathways with distinct enzymatic machinery. DNA double strand break is one of the most serious kinds of damage induced by IR, which is repaired through double strand break (DSB) repair mechanisms, including homologous recombination (HR) and non-homologous end joining (NHEJ). However, recent studies have presented increasing evidence that various DNA repair pathways are not separated, but well interlinked. It has been suggested that non-DSB repair mechanisms, such as Nucleotide Excision Repair (NER), Mismatch Repair (MMR) and cell cycle regulation, are highly involved in DSB repairs. These findings revealed previously unrecognized roles of various non-DSB repair genes and indicated that a successful DSB repair requires both DSB repair mechanisms and non-DSB repair systems. One of our recent studies found that suppressed expression of non-DSB repair genes, such as XPA, RPA and MLH1, influenced the yield of IR induced micronuclei formation and/or chromosome aberrations, suggesting that these genes are highly involved in DSB repair and DSB-related cell cycle arrest, which reveals new roles for these gene products in the DNA repair network. In this review, we summarize current progress on the function of non-DSB repair-related proteins, especially those that participate in NER and MMR pathways, and their influence on DSB repair. In addition, we present our developing view that the DSB repair mechanisms are more complex and are regulated by not only the well known HR/NHEJ pathways, but also a systematically coordinated cellular network.Key Words: Ionizing radiation (IR), DNA damage, DSB repair, NER, MMR and cell cycle.     

Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems

Single-stranded DNA oligonucleotides (ssODNs) can direct the repair of a single base mutation in human genes. While the regulation of this gene editing reaction has been partially elucidated, the low frequency with which repair occurs has hampered development toward clinical application. In this work a CRISPR/Cas9 complex is employed to induce double strand DNA breakage at specific sites surrounding the nucleotide designated for exchange. The result is a significant elevation in ssODN-directed gene repair, validated by a phenotypic readout. By analysing reaction parameters, we have uncovered restrictions on gene editing activity involving CRISPR/Cas9 complexes. First, ssODNs that hybridize to the non-transcribed strand direct a higher level of gene repair than those that hybridize to the transcribed strand. Second, cleavage must be proximal to the targeted mutant base to enable higher levels of gene editing. Third, DNA cleavage enables a higher level of gene editing activity as compared to single-stranded DNA nicks, created by modified Cas9 (Nickases). Fourth, we calculated the hybridization potential and free energy levels of ssODNs that are complementary to the guide RNA sequences of CRISPRs used in this study. We find a correlation between free energy potential and the capacity of single-stranded oligonucleotides to inhibit specific DNA cleavage activity, thereby indirectly reducing gene editing activity. Our data provide novel information that might be taken into consideration in the design and usage of CRISPR/Cas9 systems with ssODNs for gene editing.     

CRISPR/Cas9 editing of carotenoid genes in tomato

CRISPR/Cas9 technology is rapidly spreading as genome editing system in crop breeding. The efficacy of CRISPR/Cas9 in tomato was tested on Psy1 and CrtR-b2, two key genes of carotenoid biosynthesis. Carotenoids are plant secondary metabolites that must be present in the diet of higher animals because they exert irreplaceable functions in important physiological processes. Psy1 and CrtR-b2 were chosen because their impairment is easily detectable as a change of fruit or flower color. Two CRISPR/Cas9 constructs were designed to target neighboring sequences on the first exon of each gene. Thirty-four out of forty-nine (69%) transformed plants showed the expected loss-of-function phenotypes due to the editing of both alleles of a locus. However, by including the seven plants edited only at one of the two homologs and showing a normal phenotype, the editing rate reaches the 84%. Although none chimeric phenotype was observed, the cloning of target region amplified fragments revealed that in the 40% of analyzed DNA samples were present more than two alleles. As concerning the type of mutation, it was possible to identify 34 new different alleles across the four transformation experiments. The sequence characterization of the CRISPR/Cas9-induced mutations showed that the most frequent repair errors were the insertion and the deletion of one base. The results of this study prove that the CRISPRCas9 system can be an efficient and quick method for the generation of useful mutations in tomato to be implemented in breeding programs.     

Small Ubiquitin-like Modifier (SUMO) Isoforms and Conjugation-independent Function in DNA Double-strand Break Repair Pathways

Small ubiquitin-like modifier (SUMO) proteins act in DNA double-strand break (DSB) repair, but the pathway specificity of the three major isoforms has not been defined. In experiments in which we depleted the endogenous SUMO protein by RNAi, we found that SUMO1 functioned in all subpathways of either homologous recombination (HR) or non-homologous end joining (NHEJ), whereas SUMO2/3 was required for the major NHEJ pathway, called conservative NHEJ, but dispensable in other DSB repair pathways. To our surprise, we found that depletion of UBC9, the unique SUMO E2 enzyme, had no effect in HR or alternative NHEJ (Alt-NHEJ) but was required for conservative NHEJ. Consistent with this result, both non-conjugatable mutant and wild-type SUMO1 proteins functioned similarly in HR and Alt-NHEJ. These results detail the functional roles of specific SUMO isoforms in DSB repair in mammalian cells and reveal that SUMO1 functions in HR or Alt-NHEJ as a free protein and not as a protein conjugate.     

CRISPR/Cas9-mediated correction of human genetic disease

The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas) protein 9 system(CRISPR/Cas9) provides a powerful tool for targeted genetic editing. Directed by programmable sequence-specific RNAs,this system introduces cleavage and double-stranded breaks at target sites precisely. Compared to previously developed targeted nucleases, the CRISPR/Cas9 system demonstrates several promising advantages, including simplicity, high specificity,and efficiency. Several broad genome-editing studies with the CRISPR/Cas9 system in different species in vivo and ex vivo have indicated its strong potential, raising hopes for therapeutic genome editing in clinical settings. Taking advantage of non-homologous end-joining(NHEJ) and homology directed repair(HDR)-mediated DNA repair, several studies have recently reported the use of CRISPR/Cas9 to successfully correct disease-causing alleles ranging from single base mutations to large insertions. In this review, we summarize and discuss recent preclinical studies involving the CRISPR/Cas9-mediated correction of human genetic diseases.     

Loss of GGN Leads to Pre-Implantation Embryonic Lethality and Compromised Male Meiotic DNA Double Strand Break Repair in the Mouse

The integrity of male germ cell genome is critical for the correct progression of spermatogenesis, successful fertilization, and proper development of the offspring. Several DNA repair pathways exist in male germ cells. However, unlike somatic cells, key components of such pathways remain largely unidentified. Gametogenetin (GGN) is a testis-enriched protein that has been shown to bind to the DNA repair protein FANCL via yeast-two-hybrid assays. This finding and its testis-enriched expression pattern raise the possibility that GGN plays a role in DNA repair during spermatogenesis. Herein we demonstrated that the largest isoform GGN1 interacted with components of DNA repair machinery in the mouse testis. In addition to FANCL, GGN1 interacted with the critical component of the Fanconi Anemia (FA) pathway FANCD2 and a downstream component of the BRCA pathway, BRCC36. To define the physiological function of GGN, we generated a Ggn null mouse line. A complete loss of GGN resulted in embryonic lethality at the very earliest period of pre-implantation development, with no viable blastocysts observed. This finding was consistent with the observation that Ggn mRNA was also expressed in lower levels in the oocyte and pre-implantation embryos. Moreover, pachytene spermatocytes of the Ggn heterozygous knockout mice showed an increased incidence of unrepaired DNA double strand breaks (DSBs). Together, our results suggest that GGN plays a role in male meiotic DSB repair and is absolutely required for the survival of pre-implantation embryos.     

Small Rad51 and Dmc1 Complexes Often Co-occupy Both Ends of a Meiotic DNA Double Strand Break

The Eukaryotic RecA-like proteins Rad51 and Dmc1 cooperate during meiosis to promote recombination between homologous chromosomes by repairing programmed DNA double strand breaks (DSBs). Previous studies showed that Rad51 and Dmc1 form partially overlapping co-foci. Here we show these Rad51-Dmc1 co-foci are often arranged in pairs separated by distances of up to 400 nm. Paired co-foci remain prevalent when DSBs are dramatically reduced or when strand exchange or synapsis is blocked. Super-resolution dSTORM microscopy reveals that individual foci observed by conventional light microscopy are often composed of two or more substructures. The data support a model in which the two tracts of ssDNA formed by a single DSB separate from one another by distances of up to 400 nm, with both tracts often bound by one or more short (about 100 nt) Rad51 filaments and also by one or more short Dmc1 filaments.     

Genetic chimerism of CRISPR/Cas9-mediated rice mutants

The CRISPR/Cas9 technology is useful for genome editing to generate targeted mutants. Based on this genome editing technology, it was attempted to generate the rice mutant which lacks JAZ9 activity to understand its function in stress response. The sequence of guide RNA for the recognition of target site was obtained from CRISPR-PLANT website (http://www.genome.arizona.edu/crispr) to minimize off-target effect and was recombined into the CRISPR/Cas9 binary vector pRGEB31. Embryonic calli regenerated from the mature seeds (O. sativa L. cv. Nakdong) were co-cultivated with transformed Agrobacterium tumefaciens LBA4404, and 26 individual transgenic plants were obtained through the hygromycin selection process. Nucleotide sequence analysis showed that most of T₀ plants carried both edited and unedited wt sequence of JAZ9, suggesting genetic chimerism of T₀ plants. Even though 2 individual lines carried homozygous mutation on JAZ9, they were also chimeric due to biallelic mutation. The relative ratio between edited and unedited wt sequence was variable among individual lines. Expression level of Cas9 is correlated with the frequency of genome editing frequency. In some plants, the enrichment ratio changed along with developmental stage. The nucleotide sequence analysis revealed that Cas9-mediated A/T addition occurred at -3 nucleotide position from protospacer adjacent motif (PAM), whereas G addition at -5 nucleotide position from the PAM. Further analysis of T₁ transgenic plants showed that the genome editing patterns were similar between T₀ plants and their T₁ sibling plants. These suggested that earlier selection of T₀ plants with homozygous mutation is an efficient way to obtain homozygous mutants in T₁ generation.     

The expanding footprint of CRISPR/Cas9 in the plant sciences

CRISPR/Cas9 has evolved and transformed the field of biology at an unprecedented pace. From the initial purpose of introducing a site specific mutation within a genome of choice, this technology has morphed into enabling a wide array of molecular applications, including site-specific transgene insertion and multiplexing for the simultaneous induction of multiple cleavage events. Efficiency, specificity, and flexibility are key attributes that have solidified CRISPR/Cas9 as the genome-editing tool of choice by scientists from all areas of biology. Within the field of plant biology, several CRISPR/Cas9 technologies, developed in other biological systems, have been successfully implemented to probe plant gene function and to modify specific crop traits. It is anticipated that this trend will persist and lead to the development of new applications and modifications of the CRISPR technology, adding to an ever-expanding collection of genome-editing tools. We envision that these tools will bestow plant researchers with new utilities to alter genome complexity, engineer site-specific integration events, control gene expression, generate transgene-free edited crops, and prevent or cure plant viral disease. The successful implementation of such utilities will represent a new frontier in plant biotechnology.     

Optimization of Genome Engineering Approaches with the CRISPR/Cas9 System

Designer nucleases such as TALENS and Cas9 have opened new opportunities to scarlessly edit the mammalian genome. Here we explored several parameters that influence Cas9-mediated scarless genome editing efficiency in murine embryonic stem cells. Optimization of transfection conditions and enriching for transfected cells are critical for efficiently recovering modified clones. Paired gRNAs and wild-type Cas9 efficiently create programmed deletions, which facilitate identification of targeted clones, while paired gRNAs and the Cas9D10A nickase generated smaller targeted indels with lower chance of off-target mutagenesis. Genome editing is also useful for programmed introduction of exogenous DNA sequences at a target locus. Increasing the length of the homology arms of the homology-directed repair template strongly enhanced targeting efficiency, while increasing the length of the DNA insert reduced it. Together our data provide guidance on optimal design of scarless gene knockout, modification, or knock-in experiments using Cas9 nuclease.     

Exploiting the CRISPR/Cas9 PAM Constraint for Single-Nucleotide Resolution Interventions

CRISPR/Cas9 is an enabling RNA-guided technology for genome targeting and engineering. An acute DNA binding constraint of the Cas9 protein is the Protospacer Adjacent Motif (PAM). Here we demonstrate that the PAM requirement can be exploited to specifically target single-nucleotide heterozygous mutations while exerting no aberrant effects on the wild-type alleles. Specifically, we target the heterozygous G13A activating mutation of KRAS in colorectal cancer cells and we show reversal of drug resistance to a MEK small-molecule inhibitor. Our study introduces a new paradigm in genome editing and therapeutic targeting via the use of gRNA to guide Cas9 to a desired protospacer adjacent motif.     

CRISPR/Cas9:一种新型的基因组定点编辑技术

CRISPR/Cas9系统是原核生物抵御病毒或质粒等外来遗传物质入侵的一种获得性免疫系统,主要由非特异性的Cas9核酸酶和起识别作用的cr RNA所组成。相较于传统的基因组编辑技术,基于CRISPR/Cas9系统的基因组定点编辑技术具有快速、简单、高效等优点,并且几乎可以用于任何物种的基因编辑。尽管CRISPR/Cas9系统的基因组特异性还有待进一步确认,但该系统在基因组编辑方面的简便性和有效性必将促进生物学的研究和人类疾病基因治疗方面的发展。     

CRISPR/Cas9:基因编辑的新时代

基因编辑技术是通过核酸内切酶对基因组DNA进行定向改造的技术,可以实现对特定DNA碱基的缺失、替换等,常用的四种基因编辑工具分别是:巨型核酸酶、锌指核酸酶、转录激活因子样效应物核酸酶以及CRISPR/Cas9系统.其中CRISPR/Cas9系统作为一种新型的基因组编辑技术具有组成简单、特异性好、切割效率高的优点.该文对...     

CRISPR/Cas9技术在感染性疾病中的应用

成簇的规律间隔的短回文重复序列及其相关蛋白9〔clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein 9(Cas9),CRISPR/Cas9〕是一种新兴的基因编辑技术,与以前的三大基因编辑技术——归巢核酸内切酶、锌指核酸酶和转录激活因子样效应物核酸酶技术相比,其在靶向特异性、操作简便性、治疗彻底性、应用广泛性等方面具有更大的优势和发展潜力。艾滋病、乙型肝炎、疟疾等感染性疾病的治疗一直是医学上的重大难题,科学家正努力尝试利用CRISPR/Cas9技术解决这些医学难题。本文主要综述了CRISPR/Cas9技术在这些感染性疾病中应用的研究进展。