Inhibition of nonhomologous end joining to increase the specificity of CRISPR/Cas9 genome editing

DNA repair, one of the fundamental processes occurring in a cell, safeguards the genome and maintains its integrity. Among various DNA lesions, double-strand breaks are considered to be the most deleterious, as they can lead to potential loss of genetic information, if not repaired. Non-homologous end joining (NHEJ) and homologous recombination are two major double-strand break repair pathways. SCR7, a DNA ligase IV inhibitor, was recently identified and characterized as a potential anticancer compound. Interestingly, SCR7 was shown to have several applications, owing to its unique property as an NHEJ inhibitor. Here, we focus on three main areas of research in which SCR7 is actively being used, and discuss one of the applications, i.e. genome editing via CRISPR/Cas, in detail. In the past year, different studies have shown that SCR7 significantly increases the efficiency of precise genome editing by inhibiting NHEJ, and favouring the error-free homologous recombination pathway, both in vitro and in vivo. Overall, we discuss the current applications of SCR7 to shed light on the unique property of the small molecule of having distinct applications in normal and cancer cells, when used at different cellular concentrations.

RNA-Guided Genome Editing

Ciliates have evolved highly complex and intricately controlled pathways to ensure the precise and complete removal of all genomic sequences not required for vegetative growth. At the same time, they retain a reference copy of all their genetic information for future generations. This chapter describes how different ciliates use RNA-mediated DNA comparison processes to form new somatic nuclei from germline nuclei. While these processes vary in their precise mechanisms, they all use RNA to target genomic DNA sequences—either for retention or elimination. They also all consist of more than one individual pathway acting cooperatively—the two subsets of small RNAs in Paramecium and the guide RNAs and Piwi-interacting RNAs in Oxytricha—to ensure a strong belt-and-braces approach to consistent and precise somatic nucleus development. Nonetheless, this genome comparison approach to somatic nucleus development provides an elegant method for trans-generational environmental adaptation. Conceptually, it is easy to imagine how somatic changes that occur during vegetative growth could be transferred to meiotic offspring, while an unaltered germline genome is retained. Further research in this area will have far-reaching implications for the trans-generational adaptation of more distantly related eukaryotes, such as humans.

Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line

This project was carried out during CD’s BBSRC Eastbio PIPS placement at Marine Scotland. The authors are grateful to Dr Milena Monte (University of Aberdeen) for help with the FACS analysis. The authors wish to thank Dr Filippo Del Bene (Neuronal Circuit Development, Institut Curie) and Dr Wenbiao Chen (School of Medicine, Vanderbilt University) for the Addgene plasmids, #61051 and #47929, respectively, and Prof. Nancy C. Reich Marshall (Department of Molecular Genetics and Microbiology, Stony Brooks University) for the plasmid pmEGFP-N1.

Development of an Efficient Genome Editing Method by CRISPR/Cas9 in a Fish Cell Line

This project was carried out during CD’s BBSRC Eastbio PIPS placement at Marine Scotland. The authors are grateful to Dr Milena Monte (University of Aberdeen) for help with the FACS analysis. The authors wish to thank Dr Filippo Del Bene (Neuronal Circuit Development, Institut Curie) and Dr Wenbiao Chen (School of Medicine, Vanderbilt University) for the Addgene plasmids, #61051 and #47929, respectively, and Prof. Nancy C. Reich Marshall (Department of Molecular Genetics and Microbiology, Stony Brooks University) for the plasmid pmEGFP-N1.

Genome editing in zebrafish: a practical overview

Zebrafish is a powerfulmodel for the study of vertebrate development, being amenable to a wide range of genetic and other manipulations to probe themolecular basis of development and its perturbation in disease. Over recent years, genome editing approaches have become increasingly used as an efficient and sophisticated approach to precisely engineer the zebrafish genome, which has further enhanced the utility of this organism. This review provides a practical overview of genome editing and its application in zebrafish research, including alternate strategies for introducing and screening for specific genetic changes.

Examination of Endogenous Rotund Expression and Function in Developing Drosophila Olfactory System Using CRISPR-Cas9-Mediated Protein Tagging.

The zinc-finger protein Rotund (Rn) plays a critical role in controlling the development of the fly olfactory system. However, little is known about its molecular function in vivo. Here, we added protein tags to the rn locus using CRISPR-Cas9 technology in Drosophila to investigate its subcellular localization and the genes that it regulates . We previously used a reporter construct to show that rn is expressed in a subset of olfactory receptor neuron (ORN) precursors and it is required for the diversification of ORN fates. Here, we show that tagged endogenous Rn protein is functional based on the analysis of ORN phenotypes. Using this method, we also mapped the expression pattern of the endogenous isoform-specific tags in vivo with increased precision. Comparison of the Rn expression pattern from this study with previously published results using GAL4 reporters showed that Rn is mainly present in early steps in antennal disc patterning, but not in pupal stages when ORNs are born. Finally, using chromatin immunoprecipitation, we showed a direct binding of Rotund to a previously identified regulatory element upstream of the bric-a-brac gene locus in the developing antennal disc.

The eukaryotic way to defend and edit genomes by sRNA-targeted DNA deletion

While there is currently burgeoning interest in the application of the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) to genome editing, it is perhaps not widely appreciated that this is the second discovery of a small RNA (sRNA)-targeted DNA-deletion system. The first sRNA-targeted DNA-deletion system to be discovered, which we call IES/Ias (internal eliminated sequence/IES-associated genes) to contrast with CRISPR/Cas, is found in ciliates, and, like CRISPR/Cas, is thought to serve as a form of immune defense against invasive DNAs. The manner in which the ciliate IES/Ias system functions is distinct from that of the CRISPR/Cas system in archaea and bacteria, and arose independently through a synthesis of RNA interference-derived and DNA-specific molecular components. Despite the major differences between CRISPR/Cas and IES/Ias, both systems face similar conceptual challenges in targeting invasive DNAs. In this review, we focus on the discovery, effects, function, and evolutionary consequences of the IES/Ias system.

Galactofuranose in Mycoplasma mycoides is important for membrane integrity and conceals adhesins but does not contribute to serum resistance.

Mycoplasma mycoides subsp. capri (Mmc) and subsp. mycoides (Mmm) are important ruminant pathogens worldwide causing diseases such as pleuropneumonia, mastitis and septicaemia. They express galactofuranose residues on their surface, but their role in pathogenesis has not yet been determined. The M. mycoides genomes contain up to several copies of the glf gene, which encodes an enzyme catalysing the last step in the synthesis of galactofuranose. We generated a deletion of the glf gene in a strain of Mmc using genome transplantation and tandem repeat endonuclease coupled cleavage (TREC) with yeast as an intermediary host for the genome editing. As expected, the resulting YCp1.1-Δglf strain did not produce the galactofuranose-containing glycans as shown by immunoblots and immuno-electronmicroscopy employing a galactofuranose specific monoclonal antibody. The mutant lacking galactofuranose exhibited a decreased growth rate and a significantly enhanced adhesion to small ruminant cells. The mutant was also 'leaking' as revealed by a β-galactosidase-based assay employing a membrane impermeable substrate. These findings indicate that galactofuranose-containing polysaccharides conceal adhesins and are important for membrane integrity. Unexpectedly, the mutant strain showed increased serum resistance.

Chromosome fusions triggered by noncoding RNA

Chromosomal fusions are common in normal and cancer cells and can produce aberrant gene products that promote transformation. The mechanisms driving these fusions are poorly understood, but recurrent fusions are widespread. This suggests an underlying mechanism, and some authors have proposed a possible role for RNA in this process. The unicellular eukaryote Oxytricha trifallax displays an exorbitant capacity for natural genome editing, when it rewrites its germline genome to form a somatic epigenome. This developmental process provides a powerful model system to directly test the influence of small noncoding RNAs on chromosome fusion events during somatic differentiation. Here we show that small RNAs are capable of inducing chromosome fusions in four distinct cases (out of four tested), including one fusion of three chromosomes. We further show that these RNA-mediated chromosome fusions are heritable over multiple sexual generations and that transmission of the acquired fusion is associated with endogenous production of novel piRNA molecules that target the fused junction. We also demonstrate the capacity of a long noncoding RNA (lncRNA) to induce chromosome fusion of two distal germline loci. These results underscore the ability of short-lived, aberrant RNAs to act as drivers of chromosome fusion events that can be stably transmitted to future generations.

Human resource management, innovation and performance:looking across levels

The importance of innovation can hardly be exaggerated, given that landmark change has defined human progress in our technological age. The business pages of popular journals are replete with a dazzling array of inventions that have overturned existing ways of working and fundamentally changed human experience — from agricultural drones that offer farmers new ways to increase crop yield to genome editing that provides powerful insights into genetically baffling brain disorders. Innovation has become a topical theme within organisations, too, with no shortage of advice and suggestions often targeted at business leaders about how to craft an innovation strategy or increase the number and quality of ideas with a view to enriching organisational life. The quote at the start of this chapter bears testament to the sheer effort of moving away from familiar, habitual practices in the direction of less-certain, risky future terrain. Setting aside what has gone before to move in new directions requires determination, resilience and courage at a personal level. Often overlooked, though, are the multi-level dynamics that this entails.

Functional Evaluation of Causal Mutations Identified in Human Genetic Studies

Human genetics has been experiencing a wave of genetic discoveries thanks to the development of several technologies, such as genome-wide association studies (GWAS), whole-exome sequencing, and whole genome sequencing. Despite the massive genetic discoveries of new variants associated with human diseases, several key challenges emerge following the genetic discovery. GWAS is known to be good at identifying the locus associated with the patient phenotype. However, the actually causal variants responsible for the phenotype are often elusive. Another challenge in human genetics is that even the causal mutations are already known, the underlying biological effect might remain largely ambiguous. Functional evaluation plays a key role to solve these key challenges in human genetics both to identify causal variants responsible for the phenotype, and to further develop the biological insights from the disease-causing mutations.

We adopted various methods to characterize the effects of variants identified in human genetic studies, including patient genetic and phenotypic data, RNA chemistry, molecular biology, virology, and multi-electrode array and primary neuronal culture systems. Chapter 1 is a broader introduction for the motivation and challenges for functional evaluation in human genetic studies, and the background of several genetics discoveries, such as hepatitis C treatment response, in which we performed functional characterization.

Chapter 2 focuses on the characterization of causal variants following the GWAS study for hepatitis C treatment response. We characterized a non-coding SNP (rs4803217) of IL28B (IFNL3) in high linkage disequilibrium (LD) with the discovery SNP identified in the GWAS. In this chapter, we used inter-disciplinary approaches to characterize rs4803217 on RNA structure, disease association, and protein translation.

Chapter 3 describes another avenue of functional characterization following GWAS focusing on the novel transcripts and proteins identified near the IL28B (IFNL3) locus. It has been recently speculated that this novel protein, which was named IFNL4, may affect the HCV treatment response and clearance. In this chapter, we used molecular biology, virology, and patient genetic and phenotypic data to further characterize and understand the biology of IFNL4. The efforts in chapter 2 and 3 provided new insights to the candidate causal variant(s) responsible for the GWAS for HCV treatment response, however, more evidence is still required to make claims for the exact causal roles of these variants for the GWAS association.

Chapter 4 aims to characterize a mutation already known to cause a disease (seizure) in a mouse model. We demonstrate the potential use of multi-electrode array (MEA) system for the functional characterization and drug testing on mutations found in neurological diseases, such as seizure. Functional characterization in neurological diseases is relatively challenging and available systematic tools are relatively limited. This chapter shows an exploratory research and example to establish a system for the broader use for functional characterization and translational opportunities for mutations found in neurological diseases.

Overall, this dissertation spans a range of challenges of functional evaluations in human genetics. It is expected that the functional characterization to understand human mutations will become more central in human genetics, because there are still many biological questions remaining to be answered after the explosion of human genetic discoveries. The recent advance in several technologies, including genome editing and pluripotent stem cells, is also expected to make new tools available for functional studies in human diseases.

Genetic medicines for CF: hype versus reality

Since identification of the CFTR gene over 25 years ago, gene therapy for cystic fibrosis (CF) has been actively developed. More recently gene therapy has been joined by other forms of “genetic medicines” including mRNA delivery, as well as genome editing and mRNA repair-based strategies. Proof-of-concept that gene therapy can stabilize the progression of CF lung disease has recently been established in a Phase IIb trial. An early phase study to assess the safety and explore efficacy of CFTR mRNA repair is ongoing, while mRNA delivery and genome editing-based strategies are currently at the pre-clinical phase of development. This review has been written jointly by some of those involved in the various CF “genetic medicine” fields and will summarize the current state-of-the-art, as well as discuss future developments. Where applicable, it highlights common problems faced by each of the strategies, and also tries to highlight where a specific strategy may have an advantage on the pathway to clinical translation. We hope that this review will contribute to the ongoing discussion about the hype versus reality of genetic medicine-based treatment approaches in CF.

Genetic Modification of Disease Resistance: Fungal and Oomycete Pathogens

Development of recombinant DNA technology allowed scientists to manipulate plant genomes, making it possible to study genes and exploit them to modify novel agronomic traits. Here, we review the current and future potential of genetic modification (GM) strategies used to increase the resistance of plants to oomycete and fungal pathogens. Numerous resistance genes (R-genes) have been cloned, and under laboratory conditions, transgenic plants have given promising results against some important plant pathogens. However, only a few have so far been deployed as commercial crop plants.GMof plants to disrupt pathogenicity, such as by inhibiting or degrading pathogenicity factors, especially by necrotrophic pathogens, has also been exploited. The potential to engineer plants for the production of antimicrobial peptides or to modify defense-signaling pathways have been successfully demonstrated under laboratory conditions. The most promising current technology is genome editing, which allows researchers to edit DNA sequences directly in their endogenous environment. The potential of this approach is discussed in detail and examples where broad-spectrum resistance has been achieved are given.

Decision tools for bacterial blight resistance gene deployment in rice-based agricultural ecosystems

Attempting to achieve long-lasting and stable resistance using uniformly deployed rice varieties is not a sustainable approach. The real situation appears to be much more complex and dynamic, one in which pathogens quickly adapt to resistant varieties. To prevent disease epidemics, deployment should be customized and this decision will require interdisciplinary actions. This perspective article aims to highlight the current progress on disease resistance deployment to control bacterial blight in rice. Although the model system rice-Xanthomonas oryzae pv. oryzae has distinctive features that underpin the need for a case-by-case analysis, strategies to integrate those elements into a unique decision tool could be easily extended to other crops.

Targeted gene modification in Fragaria vesca mediated by CRISPR/Cas9 system

Genome editing is becoming an important biotechnological tool for gene function analysis and crop improvement, being the CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat-CRISPR associated protein 9) system the most widely used. The natural CRISPR/Cas9 system has been reduced to two components: a single-guide RNA (sgRNA) for target recognition via RNA-DNA base pairing, which is commonly expressed using a promoter for small-RNAs (U6 promoter), and the Cas9 endonuclease for DNA cleavage (1). To validate the CRISPR/Cas9 system in strawberry plants, we designed two sgRNAs directed against the floral homeotic gene APETALA3 (sgRNA-AP3#1 and sgRNA-AP3#2). This gene was selected because ap3 mutations induce clear developmental phenotypes in which petals and stamens are missing or partially converted to sepals and carpels respectively (2). In this work, we used two different U6 promoters to drive the sgRNA-AP3s expression: AtU6-26 from Arabidopsis (4), and a U6 promoter from Fragaria vesca (FvU6) (this work). We also tested two different coding sequences of Cas9: a human- (hSpCas9) (3) and a plant-codon optimized (pSpCas9) (this work). Transient expression experiments using both CRISPR/Cas9 systems (AtU6-26:sgRNA-AP3#1_35S:hSpCas9_AtU6-26:sgRNA-AP3#2 and FvU6:sgRNA-AP3#1_35S:pSpCas9_FvU6:sgRNA-AP3#2) were performed infiltrating Agrobacterium tumefaciens into F. vesca fruits. PCR amplification and sequencing analyses across the target sites showed a deletion of 188-189 bp corresponding to the region comprised between the two cutting sites of Cas9, confirming that the CRISPR/Cas9 system is functional in F. vesca. Remarkably, the two systems showed different mutagenic efficiency that could be related to differences in expression of the U6 promoters as well as differences in the Cas9 transcripts stability and translation. Stable transformants for both F. vesca (2n) and Fragaria X anannassa (8n) are currently being established to test whether is possible to obtain heritable homozygous mutants derived from CRISPR/Cas9 strategies in strawberry. Thus, our work offers a promising tool for genome editing and gene functional analysis in strawberry. This tool might represent a more efficient alternative to the sometimes inefficient RNAi silencing methods commonly used in this species.