Chapter 7 - CRISPR technology for genome editing

CRISPR/Cas9-based genome editing has yet to be reported in species of the Platyhelminthes. We tested this approach by targeting omega-1 (ω1) of Schistosoma mansoni as proof of principle. This secreted ribonuclease is crucial for Th2 polarization and granuloma formation. Schistosome eggs were exposed to Cas9 complexed with guide RNA complementary to ω1 by electroporation or by transduction with lentiviral particles. Some eggs were also transfected with a single stranded donor template. Sequences of amplicons from gene-edited parasites exhibited Cas9-catalyzed mutations including homology directed repaired alleles, and other analyses revealed depletion of ω1 transcripts and the ribonuclease. Gene-edited eggs failed to polarize Th2 cytokine responses in macrophage/T-cell co-cultures, while the volume of pulmonary granulomas surrounding ω1-mutated eggs following tail-vein injection into mice was vastly reduced. Knock-out of ω1 and the diminished levels of these cytokines following exposure showcase the novel application of programmed gene editing for functional genomics in schistosomes.

478. Highly Efficient Targeted Gene Addition in CD34+ Hematopoietic Stem/Progenitor Cells by Combining ZFN mRNA and AAV6 Donor Delivery

Combined effects of carbon ion radiation and PARP inhibitor on non-small cell lung carcinoma cells: Insights into DNA repair pathways and cell death mechanisms

Numerous examples of successful 'genome editing' now exist. Genome editing uses engineered nucleases as powerful tools to target specific DNA sequences to edit genes precisely in the genomes of both model and crop plants, as well as a variety of other organisms. The DNA-binding domains of zinc finger (ZF) proteins were the first to be used as genome editing tools, in the form of designed ZF nucleases (ZFNs). More recently, transcription activator-like effector nucleases (TALENs), as well as the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system, which utilizes RNA-DNA interactions, have proved useful. A key step in genome editing is the generation of a double-stranded DNA break that is specific to the target gene. This is achieved by custom-designed endonucleases, which enable site-directed mutagenesis via a non-homologous end-joining (NHEJ) repair pathway and/or gene targeting via homologous recombination (HR) to occur efficiently at specific sites in the genome. This review provides an overview of recent advances in genome editing technologies in plants, and discusses how these can provide insights into current plant molecular biology research and molecular breeding technology.

Effects of various environments on epigenetic settings and chromosomal damage

Stearoyl-CoA desaturase 1 (SCD1) is a fatty acid desaturase catalyzing cis-double-bond formation in the Δ9 position to produce monounsaturated fatty acids essential for the synthesis of milk fat. Previous studies using RNAi methods have provided support for a role of SCD1 in goat mammary epithelial cells (GMEC); however, RNAi presents several limitations that might preclude a truthful understanding of the biological function of SCD1. To explore the function of SCD1 on fatty acid metabolism in GMEC, we used CRISPR-Cas9-mediated SCD1 knockout through non-homologous end-joining (NHEJ) and homology-directed repair (HDR) pathways in GMEC. We successfully introduced nucleotide deletions and mutations in the SCD1 gene locus through the NHEJ pathway and disrupted its second exon via insertion of an EGFP-PuroR segment using the HDR pathway. In clones derived from the latter, gene- and protein-expression data indicated that we obtained a monoallelic SCD1 knockout. A T7EN1-mediated assay revealed no off-targets in the surveyed sites. The contents of triacylglycerol and cholesterol and the desaturase index were significantly decreased as a consequence of SCD1 knockout. The deletion of SCD1 decreased the expression of other genes involved in de novo fatty acid synthesis, including SREBF1 and FASN, as well the fatty acid transporters FABP3 and FABP4. The downregulation of these genes partly explains the decrease of intracellular triacylglycerols. Our results indicate a successful SCD1 knockout in goat mammary cells using CRISPR-Cas9. The demonstration of the successful use of CRISPR-Cas9 in GMEC is an important step to producing transgenic goats to study mammary biology in vivo.

Complex regulatory circuits determine whether DNA double-strand breaks (DSBs) are repaired by nonhomologous end-joining (NHEJ) or homology-directed repair (HDR) pathways, a carefully balanced equilibrium of which is critical for genome stability. In this issue of EMBO Reports, Deng etal [1] report that a novel p53-suppressed long noncoding RNA (lncRNA), PRLH1, interacts with and stabilizes the E3 ubiquitin ligase RNF169 to stimulate HDR-mediated DSB repair and proliferation of p53-deficient cancer cells. These findings suggest a new regulatory principle in modulating DSB repair pathway selection that may contribute to tumorigenesis.

Three-dimensional (3D) stem cell differentiation cultures recently emerged as a novel model system for investigating human embryonic development and disease progression in vitro, complementing existing animal and two-dimensional (2D) cell culture models. Organoids, the 3D self-organizing structures derived from pluripotent or somatic stem cells, can recapitulate many aspects of structural organization and functionality of their in vivo organ counterparts, thus holding great promise for biomedical research and translational applications. Importantly, faithful recapitulation of disease and development processes relies on the ability to modify the genomic contents in organoid cells. The revolutionary genome engineering technologies, CRISPR/Cas9 in particular, enable investigators to generate various reporter cell lines for prompt validation of specific cell lineages as well as to introduce disease-associated mutations for disease modeling. In this review, we provide historical overviews, and discuss technical considerations, and potential future applications of genome engineering in 3D organoid models.

Versatile and precise gene-targeting strategies for functional studies in mammalian cell lines.

Ewing sarcoma is an aggressive mesenchymal malignancy commonly affecting children and young adolescents. The molecular basis of this neoplasia is well reported with the formation of the EWSR1/FLI1 fusion gene being the most common genetic finding. However, this fusion gene has not been targeted therapeutically nor is being used as a prognostic marker. Its relevance regarding the molecular steps leading to Ewing sarcoma genesis are yet to be defined. The generation of the oncogenic EWSR1/FLI1 fusion gene, can be attributed to the simultaneous introduction of two DNA double-strand breaks (DSBs). The scope of this study is to detect any association between DNA repair deficiency and the clinicopathological aspects of Ewing's sarcoma disease. We have conducted an expression analysis of 35 patients diagnosed with Ewing sarcoma concerning the genes involved in non-homologous end joining (NHEJ) and homologous recombination (HR) repair pathways. We have analyzed the expression levels of 6 genes involved in NHEJ (XRCC4, XRCC5, XRCC6, POLλ, POLμ) and 9 genes involved in HR (RAD51, RAD52, RAD54, BRCA1, BRCA2, FANCC, FANCD, DNTM1, BRIT1) using real time PCR. Age, sex, location of primary tumor, tumor size, KI67, mitotic count, invasion of adjacent tissues and treatment were the clinicopathological parameters included in the statistical analysis. Our results show that both these DNA repair pathways are deregulated in Ewing sarcoma. In addition, low expression of the xrcc4 gene has been associated with better overall survival probability (p=0.032). Our results, even though retrospective and in a small number of patients, highlight the importance of DSBs repair and propose a potential therapeutic target for this type of sarcoma.

RNA-Guided Genome Editing in Plants Using a CRISPR–Cas System

Non-homologous end joining (NHEJ) is a principal pathway of DNA double-strand break (DSB) repair in mammalian cells,1 but is also involved in assembly of antigen receptor genes and telomere maintenance.2,3 Loss of NHEJ function can result in chromosome instability promoting malignant transformation, in particular when other cellular safeguards are compromised. Inherited NHEJ defects underlie radiosensitive severe combined immunodeficiency. NHEJ is initiated by binding of Ku70–Ku80 heterodimers to both ends of a DNA break, followed by recruitment of catalytic subunits of DNA-dependent protein kinase (DNA-PKcs) to form juxtaposed DNA-PK holoenzymes which join the ends in a synaptic complex. Cohesive blunt DNA ends can be joined directly by the XFL-XRCC4-DNA ligase IV complex, but in many cases DNA ends contain damaged bases or DNA backbone sugars that require processing before ligation. End processing may involve nucleases such as Artemis, DNA polymerases and polynucleotide kinase among other enzymes.4,5

<div>Abstract<p>Previous evidence indicates that HPV integration status may be associated with cervical cancer development and progression. However, host genetic variation within genes that may play important roles in the viral integration process is understudied. The aim of this study was to examine the association between HPV16 and HPV18 viral integration status and single nucleotide polymorphisms (SNPs) in non-homologous end-joining (NHEJ) DNA repair pathway genes on cervical dysplasia. Women enrolled in two large trials of optical technologies for cervical cancer detection and positive for HPV16 or HPV18 were selected for HPV integration analysis and genotyping. Associations between SNPs and cytology (normal, low-grade, or high-grade lesions) were evaluated. Among women with cervical dysplasia, polytomous logistic regression models were used to evaluate the effect of each SNP on viral integration status. Of the 710 women evaluated (149 HSIL, 251 LSIL, 310 Normal), 395 (55.6%) were positive for HPV16 and 192 (27%) were positive for HPV18. Tag-SNPs in 13 DNA repair genes, including RAD50, WRN, and XRCC4, were significantly associated with cervical dysplasia. HPV16 integration status was differential across cervical cytology, but overall, most participants had a mix of both episomal and integrated HPV16. Four tag-SNPs in the XRCC4 gene were found to be significantly associated with HPV16 integration status. Our findings indicate that host genetic variation in NHEJ DNA repair pathway genes, specifically XRCC4, are significantly associated with HPV integration, and that these genes may play an important role in determining cervical cancer development and progression.</p></div>

Rad9 plays roles in both cell cycle checkpoint control and DNA repair. It can interact with the components of multiple DNA repair pathways and regulate their functions. Non-homologous end-joining (NHEJ) repair pathway is predominantly used in vertebrates for the repair of DNA double strand break (DSB). Proper activation of DNA-dependent protein kinase (DNA-PK), composed of Ku70, Ku80, and DNA-dependent protein kinase catalytic subunit (DNA-PKcs), is essential for NHEJ repair. In this study, we found that Rad9 interacts with Ku70 physically and functionally. Deletion of Rad9 gene, knockdown of Rad9 expression or removal of Rad9 protein led to inefficient DNA end-joining repair. Furthermore, loss of Rad9 impaired the DNA damage-induced binding of Ku70 to the chromatin and attenuated the DNA damage-induced kinase activity of DNA-PKcs. Taken together, our data unveil a novel functional interplay between Rad9 and the NHEJ protein Ku70, indicating a role of Rad9 in NHEJ repair through modulating the activation of the DNA-PKcs/Ku70/Ku80 complex.

487. Therapeutic Genome Editing by Combined Viral and Non-Viral Delivery of CRISPR System Components to the Mouse Liver