Targeted Genome Editing Research Articles

CRISPR/Cas9-Mediated Knockout of Galactinol Synthase-Encoding Genes Reduces Raffinose Family Oligosaccharide Levels in Soybean Seeds.

Raffinose family oligosaccharides (RFOs) are major soluble carbohydrates in soybean seeds that cannot be digested by human and other monogastric animals. Hence, a major goal is to reduce RFO levels to improve the nutritional quality of soybean. In this study, we utilized a dual gRNAs CRISPR/Cas9 system to induce knockouts in two soybean galactinol synthase (GOLS) genes, GmGOLS1A and its homeolog GmGOLS1B. Genotyping of T0 plants showed that the construct design was efficient in inducing various deletions in the target sites or sequences spanning the two target sites of both GmGOLS1A and GmGOLS1B genes. A subset of induced alleles was successfully transferred to progeny and, at the T2 generation, we identified null segregants of single and double mutant genotypes without off-target induced mutations. The seed carbohydrate analysis of double mutant lines showed a reduction in the total RFO content of soybean seed from 64.7 mg/g dry weight to 41.95 mg/g dry weight, a 35.2% decrease. On average, the stachyose content, the most predominant RFO in soybean seeds, decreased by 35.4% in double mutant soybean, while the raffinose content increased by 41.7%. A slight decrease in verbascose content was also observed in mutant lines. Aside from changes in soluble carbohydrate content, some mutant lines also exhibited increased protein and fat contents. Otherwise, no difference in seed weight, seed germination, plant development and morphology was observed in the mutants. Our findings indicate that GmGOLS1A and GmGOLS1B contribute to the soybean oligosaccharide profile through RFO biosynthesis pathways, and are promising targets for future investigation, as well as crop improvement efforts. Our results also demonstrate the potential in using elite soybean cultivars for transformation and targeted genome editing.

Co-delivery of Sorafenib and CRISPR/Cas9 Based on Targeted Core-Shell Hollow Mesoporous Organosilica Nanoparticles for Synergistic HCC Therapy.

The rapid development of CRISPR/Cas9 systems has opened up tantalizing prospects to sensitize cancers to chemotherapy using efficient targeted genome editing, but safety concerns and possible off-target effects of viral vectors remain a major obstacle for clinical application. Thus, the construction of novel nonviral tumor-targeting nanodelivery systems has great potential for the safe application of CRISPR/Cas9 systems for gene-chemo-combination therapy. Here, we report a polyamidoamine-aptamer-coated hollow mesoporous silica nanoparticle for the co-delivery of sorafenib and CRISPR/Cas9. The core-shell nanoparticles had good stability, enabled ultrahigh drug loading, targeted delivery, and controlled-release of the gene-drug combination. The nanocomplex showed >60% EGFR-editing efficiency without off-target effects in all nine similar sites, regulating the EGFR-PI3K-Akt pathway to inhibit angiogenesis, and exhibited a synergistic effect on cell proliferation. Importantly, the co-delivery nanosystem achieved efficient EGFR gene therapy and caused 85% tumor inhibition in a mouse model. Furthermore, the nanocomplex showed high accumulation at the tumor site in vivo and exhibited good safety with no damage to major organs. Due to these properties, the nanocomplex provides a versatile delivery approach for efficient co-loading of gene-drug combinations, allowing for precise gene editing and synergistic inhibition of tumor growth without apparent side effects on normal tissues.

Improvement of Disease Resistance in Livestock: Application of Immunogenomics and CRISPR/Cas9 Technology.

Simple SummaryDisease resistance is the ability of animals to inhibit the growth of invading pathogens within the body, which is influenced by the interaction of the host immune system, host genetics, and the pathogens. Resistant animals can be produced by molecular breeding by introducing the genomic marker responsible for disease resistance or immunocompetence. Immunogenomics is an information science that enables the genome-scale investigation of host immune response to pathogenic infection thereby identification of the genomic marker for disease resistance. Once the genomic marker is determined, it could be implemented in producing disease resistance animals by applying the advanced reproductive biotechnology like genome editing. The technical ease and decreasing cost over time might enhance the application of genome editing techniques for producing disease resistance livestock.Disease occurrence adversely affects livestock production and animal welfare, and have an impact on both human health and public perception of food–animals production. Combined efforts from farmers, animal scientists, and veterinarians have been continuing to explore the effective disease control approaches for the production of safe animal-originated food. Implementing the immunogenomics, along with genome editing technology, has been considering as the key approach for safe food–animal production through the improvement of the host genetic resistance. Next-generation sequencing, as a cutting-edge technique, enables the production of high throughput transcriptomic and genomic profiles resulted from host-pathogen interactions. Immunogenomics combine the transcriptomic and genomic data that links to host resistance to disease, and predict the potential candidate genes and their genomic locations. Genome editing, which involves insertion, deletion, or modification of one or more genes in the DNA sequence, is advancing rapidly and may be poised to become a commercial reality faster than it has thought. The clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) [CRISPR/Cas9] system has recently emerged as a powerful tool for genome editing in agricultural food production including livestock disease management. CRISPR/Cas9 mediated insertion of NRAMP1 gene for producing tuberculosis resistant cattle, and deletion of CD163 gene for producing porcine reproductive and respiratory syndrome (PRRS) resistant pigs are two groundbreaking applications of genome editing in livestock. In this review, we have highlighted the technological advances of livestock immunogenomics and the principles and scopes of application of CRISPR/Cas9-mediated targeted genome editing in animal breeding for disease resistance.

Sequential Genome Editing and Induced Excision of the Transgene in N. tabacum BY2 Cells.

While plant cells in suspension are becoming a popular platform for expressing biotherapeutic proteins, the need to pre-engineer these cells to better comply with their role as host cell lines is emerging. Heterologous DNA and selectable markers are used for transformation and genome editing designated to produce improved host cell lines for overexpression of recombinant proteins. The removal of these heterologous DNA and selectable markers, no longer needed, can be beneficial since they limit additional gene stacking in subsequent transformations and may pose excessive metabolic burden on the cell machinery. In this study we developed an innovative stepwise methodology in which the CRISPR-Cas9 is used sequentially to target genome editing, followed by its own excision. The first step included a stable insertion of a CRISPR-Cas9 cassette, targeted to knockout the β(1,2)-xylosyltranferase (XylT) and the α(1,3)-fucosyltransferase (FucT) genes in Nicotiana tabacum L. cv Bright Yellow 2 (BY2) cell suspension. The second step included the excision of the inserted cassette of 14.3 kbp by induction of specific sgRNA designed to target the T-DNA boundaries. The genome editing step and the transgene removal step are achieved in one transformation run. This mechanism enables CRISPR genome editing and subsequently eliminating the introduced transgenes thus freeing the cells from foreign DNA no longer needed.

The development of genome editing tools as powerful techniques with versatile applications in biotechnology and medicine: CRISPR/Cas9, ZnF and TALE nucleases, RNA interference, and Cre/loxP

The huge progress in whole genome sequencing (genomic revolution) methods including next generation sequencing (NGS) techniques allows one to obtain data on genome sequences of all organisms, ranging from bacteria to plants to mammals, within hours to days (era of whole genome/exome sequencing) (Goodwin et al. in Nat Rev Genet 17:333–351, 2016; Levy and Myers in Annu Rev Genomics Hum Genet 17:95–115, 2016; Giani et al. in Comput Struct Biotechnol J 18:9–19, 2020). Today, within the era of functional genomics the highest goal is to transfer this huge amount of sequencing data into information of functional and clinical relevance (genome annotation project). The World Health Organization (WHO) estimates that more than 10,000 diseases in humans are monogenic, i.e., that these diseases are caused by mutations within single genes (Jackson et al. in Essays Biochem 62:643–723, 2018). NGS technologies are continuously improving while our knowledge on genetic mutations driving the development of diseases is also still emerging (Giani et al. in Comput Struct Biotechnol J 18:9–19, 2020). It would be desirable to have tools that allow one to correct these genetic mutations, so-called genome editing tools. Apart from applications in biotechnology, medicine, and agriculture, it is still not concisely understood in basic science how genotype influences phenotype. Firstly, the Cre/loxP system and RNA-based technologies for gene knockout or knockdown are explained. Secondly, zinc-finger (ZnF) nucleases and transcription activator-like effector nucleases (TALENs) are discussed as targeted genome editing systems. Thirdly, CRISPR/Cas is presented including outline of the discovery and mechanisms of this adaptive immune system in bacteria and archaea, structure and function of CRISPR/Cas9 and its application as a tool for genomic editing. Current developments and applications of CRISPR/Cas9 are discussed. Moreover, limitations and drawbacks of the CRISPR/Cas system are presented and questions on ethical concerns connected to application of genome editing tools are discussed.

CRISPR-ERA: A Webserver for Guide RNA Design of Gene Editing and Regulation.

The CRISPR/Cas9 system has been developed as a powerful technology for both targeted genome editing and gene regulation. However, the design of efficient single-guide RNAs (sgRNAs) remains challenging with the consideration of many criteria. In this section, we introduce how to design sgRNA sequences and build genome-wide sgRNA library using CRISPR-ERA, which is one of the state-of-the-art designer webserver tools for sgRNA design based on a set of sgRNA design rules summarized from published reports.

CRISPR based targeted genome editing of Chlamydomonas reinhardtii using programmed Cas9-gRNA ribonucleoprotein.

The clustered regularly interspaced short palindromic repeats (CRISPR) - Cas associated protein 9 (Cas9) system is very precise, efficient and relatively simple in creating genetic modifications at a predetermined locus in the genome. Genome editing with Cas9 ribonucleoproteins (RNPs) has reduced cytotoxic effects, off-target cleavage and increased on-target activity and the editing efficiencies. The unicellular algaChlamydomonas reinhardtii is an emerging model for studying the production of high-value products for industrial applications. Development of C. reinhardtii as an industrial biotechnology host can be achieved more efficiently through genetic modifications using genome editing tools. We made an attempt to target MAA7 gene that encodes the tryptophan synthase β-Subunit using CRISPR-Cas9 RNPs to demonstrate knock-out and knock-in through homology-dependent repair template at the target site. In this study, we have demonstrated targeted gene knock-out in C. reinhardtii using programmed RNPs. Targeted editing of MAA7 gene was confirmed by sequencing the clones that were resistant to 5-Fluoroindole (5-FI). Non-homologous end joining (NHEJ) repair mechanism led to insertion, deletion, and/or base substitution in the Cas9 cleavage vicinity, encoding non-functional MAA7 protein product (knock-out), conferring resistance to 5-FI. Here, we report an efficient protocol for developing knock-out mutants in Chlamydomonas using CRISPR-Cas9 RNPs. The high potential efficiency of editing may also eliminate the need to select mutants by phenotype. These research findings would be more likely applied to other green algae for developing green cell factories to produce high-value molecules.

Agrobacterium-Mediated Genetic Transformation, Transgenic Production, and Its Application for the Study of Male Reproductive Development in Rice.

Male sterility is an important agronomic trait for hybrid seed production that is usually characterized by functional defects in male reproductive organs/gametes. Recent advances in CRISPR-Cas9 genome editing technology allow for high editing efficacy and timesaving knockout mutations of endogenous candidate genes at specific sites. Additionally, Agrobacterium-mediated genetic transformation of rice is also a key method for gene modification, which has been widely adopted by many public and private laboratories. In this study, we applied CRISPR-Cas9 genome editing tools and successfully generated three male sterile mutant lines by targeted genome editing of OsABCG15 in a japonica cultivar. We used a modified Agrobacterium-mediated rice transformation method that could provide excellent means of genetic emasculation for hybrid seed production in rice. Transgenic plants can be obtained within 2-3 months and homozygous transformants were screened by genotyping using PCR amplification and Sanger sequencing. Basic phenotypic characterization of the male sterile homozygous line was performed by microscopic observation of the rice male reproductive organs, pollen viability analysis by iodine potassium iodide (I2-KI) staining semi-thin cross-sectioning of developing anthers.

CRISPR Gene Therapy: Applications, Limitations, and Implications for the Future.

A series of recent discoveries harnessing the adaptive immune system of prokaryotes to perform targeted genome editing is having a transformative influence across the biological sciences. The discovery of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated (Cas) proteins has expanded the applications of genetic research in thousands of laboratories across the globe and is redefining our approach to gene therapy. Traditional gene therapy has raised some concerns, as its reliance on viral vector delivery of therapeutic transgenes can cause both insertional oncogenesis and immunogenic toxicity. While viral vectors remain a key delivery vehicle, CRISPR technology provides a relatively simple and efficient alternative for site-specific gene editing, obliviating some concerns raised by traditional gene therapy. Although it has apparent advantages, CRISPR/Cas9 brings its own set of limitations which must be addressed for safe and efficient clinical translation. This review focuses on the evolution of gene therapy and the role of CRISPR in shifting the gene therapy paradigm. We review the emerging data of recent gene therapy trials and consider the best strategy to move forward with this powerful but still relatively new technology.

Abstract 498: Krueppel-like Factor 15 Modulation: A Crispr-based Re-establishment of Homeostatic Transcription in Diseased Mouse and Human Cardiomyocytes

An altered transcriptomic profile of cardiomyocytes is a hallmark of cardiac remodelling and heart failure. Re-activation of gene programs necessary for myocardial homeostasis and inhibition of deleterious signalling pathways, such as Wnt/beta-catenin signalling, is therefore therapeutically desirable, however challenging to achieve tissue-specifically. We identified Krueppel-like factor 15 (KLF15) as a cardiac specific inhibitor of Wnt/beta-catenin signalling, lost during heart failure progression in the mouse and human heart. To re-establish endogenous Klf15 expression, we designed a cardiomyocyte specific CRISPR activation mouse model expressing enzymatically inactive Cas9 (dCas9) fused to a transcriptional activator (VPR) under Myh6 promoter control. The dCas9VPR protein is directed by guide RNAs (gRNA), delivered by AAV9, to the promoter region of Klf15 and induces gene expression. We tested activation of Klf15 in the adult myocardium by injecting 15 weeks old Myh6-dCas9VPR mice systemically with AAV9 carrying Klf15 gRNAs and increasing Klf15 transcript levels 8 weeks later up to 1.6 fold compared to controls. Since we found a 0.5 fold change of Klf15 expression upon transverse aortic constriction compared to sham operated mice, we expect the CRISPR activation induced Klf15 activation to be sufficient to re-establish Klf15 expression. As a translational approach and since we demonstrated KLF15-WNT regulation in human cardiomyocytes, we integrated dCas9VPR into human induced pluripotent stem cells (hIPSC) by targeted genome editing at the AAVS1 safe harbour locus and confirmed dCas9VPR expression in hIPSC and in hIPSC-derived cardiomyocytes (hIPSC-CM). We tested KLF15 gRNAs in HEK293 cells and selected the most efficient candidate gRNAs to test Klf15 activation in dCas9VPR-IPSC resulting in an up to 1.7 fold activation compared to controls. For gene activation in dCas9VPR hIPSC-CM, we are currently testing lentivirus constructs to deliver up to 3 gRNAs simultaneously to tailor KLF15 expression. With these prerequisites, we aim to re-activate master regulators of normal cardiomyocyte function in failing cardiomyocytes at physiologically relevant levels, such as KLF15, to test potential therapeutic targets for the prevention of heart failure progression.

Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins

Targeted genome editing has a great therapeutic potential to treat disorders that require protein replacement therapy. To develop a platform independent of specific patient mutations, therapeutic transgenes can be inserted in a safe and highly transcribed locus to maximize protein expression. Here, we describe an ex vivo editing approach to achieve efficient gene targeting in human hematopoietic stem/progenitor cells (HSPCs) and robust expression of clinically relevant proteins by the erythroid lineage. Using CRISPR-Cas9, we integrate different transgenes under the transcriptional control of the endogenous α-globin promoter, recapitulating its high and erythroid-specific expression. Erythroblasts derived from targeted HSPCs secrete different therapeutic proteins, which retain enzymatic activity and cross-correct patients’ cells. Moreover, modified HSPCs maintain long-term repopulation and multilineage differentiation potential in transplanted mice. Overall, we establish a safe and versatile CRISPR-Cas9-based HSPC platform for different therapeutic applications, including hemophilia and inherited metabolic disorders.

A simple and highly efficient method for multi-allelic CRISPR-Cas9 editing in primary cell cultures.

BackgroundCRISPR‐Cas9‐based technologies have revolutionized experimental manipulation of mammalian genomes. None‐the‐less, limitations of the delivery and efficacy of these technologies restrict their application in primary cells.AimsTo create an optimized protocol for penetrant, reproducible, and fast targeted genome editing in cell cultures derived from primary cells, using patient‐derived glioblastoma stem‐like cells (GSCs) and human neural stem/progenitor cells (NSCs) for proof‐of‐concept experiments.Methods and resultsWe employed transient nucleofection of Cas9:sgRNA ribonucleoprotein complexes composed of chemically synthesized 2′‐O‐methyl 3′phosphorothioate‐modified sgRNAs and purified Cas9 protein. Insertion‐deletion mutation (indel) frequency and size distribution were measured via computational deconvolution of Sanger sequencing trace data. We found that this optimized technique routinely allows for >90% indel formation in only 3 days, without the need to create clonal lines for simple loss‐of‐function experiments. Using Western blotting, we observed near‐total protein loss of target genes in cell pools. Additionally, we found that this approach allows for the creation of targeted genomic deletions. Furthermore, by using RNA‐seq in edited NSCs to assess gene expression changes resulting from knockout of tumor suppressors commonly altered in glioblastoma, we also demonstrated the utility of this method for quickly creating a series of gene knockouts that allow for the study of oncogenic activities.ConclusionOur data suggest that this relatively simple method can be used for highly efficient and fast gene knockout, as well as for targeted genomic deletions, even in hyperdiploid cells (such as GSCs). This represents an extremely useful tool for the cancer research community when wishing to inactivate not only coding genes, but also non‐coding RNAs, UTRs, enhancers, and promoters. This method can be readily applied to diverse cell types by varying the nucleofection conditions.

CRISPR-Cas9 System for Plant Genome Editing: Current Approaches and Emerging Developments

Targeted genome editing using CRISPR-Cas9 has been widely adopted as a genetic engineering tool in various biological systems. This editing technology has been in the limelight due to its simplicity and versatility compared to other previously known genome editing platforms. Several modifications of this editing system have been established for adoption in a variety of plants, as well as for its improved efficiency and portability, bringing new opportunities for the development of transgene-free improved varieties of economically important crops. This review presents an overview of CRISPR-Cas9 and its application in plant genome editing. A catalog of the current and emerging approaches for the implementation of the system in plants is also presented with details on the existing gaps and limitations. Strategies for the establishment of the CRISPR-Cas9 molecular construct such as the selection of sgRNAs, PAM compatibility, choice of promoters, vector architecture, and multiplexing approaches are emphasized. Progress in the delivery and transgene detection methods, together with optimization approaches for improved on-target efficiency are also detailed in this review. The information laid out here will provide options useful for the effective and efficient exploitation of the system for plant genome editing and will serve as a baseline for further developments of the system. Future combinations and fine-tuning of the known parameters or factors that contribute to the editing efficiency, fidelity, and portability of CRISPR-Cas9 will indeed open avenues for new technological advancements of the system for targeted gene editing in plants.

Targeted gene deletion in Brettanomyces bruxellensis with an expression-free CRISPR-Cas9 system.

The ability to genetically manipulate microorganisms has been essential for understanding their biology and metabolism. Targeted genome editing relies on highly efficient homologous recombination, and while this is readily observed in the yeast Saccharomyces cerevisiae, most non-conventional yeast species do not display this trait and remain recalcitrant to targeted editing methods. CRISPR-based editing can bypass the requirement for high levels of native homologous recombination, enabling targeted modification to be more broadly implemented. While genetic transformation has been reported previously in Brettanomyces bruxellensis, a yeast with broad biotechnological potential and responsible for significant economic losses during the production of fermented beverages, targeted editing approaches have not been reported. Here, we describe the use of an expression-free CRISPR-Cas9 system, in combination with gene transformation cassettes tailored for B. bruxellensis, to provide the means for targeted gene deletion in this species. Deletion efficiency was shown to be dependent on homologous flanking DNA length, with higher targeting efficiencies observed with cassettes containing longer flanking regions. In a diploid strain, it was not possible to delete multiple alleles in one step, with heterozygous deletants only obtained when using DNA cassettes with long flanking regions. However, stepwise transformations (using two different marker genes) were successfully used to delete both wild-type alleles. Thus, the approach reported here will be crucial to understand the complex physiology of B. bruxellensis. Key points • The use of CRISPR-Cas9 enables targeted gene deletion in Brettanomyces bruxellensis. • Homozygous diploid deletions are possible with step-wise transformations. • Deletion of SSU1 confirmed the role of this gene in sulphite tolerance.

Targeted genome editing in tetraploid potato through transient TALEN expression by Agrobacterium infection.

Genome editing using site-specific nucleases, such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat-CRISPR-associated protein 9 (CRISPR-Cas9), is a powerful technology for crop breeding. For plant genome editing, the genome-editing reagents are usually expressed in plant cells from stably integrated transgenes within the genome. This requires crossing processes to remove foreign nucleotides from the genome to generate null segregants. However, in highly heterozygous plants such as potato, the progeny lines have different agronomic traits from the parent cultivar and do not necessarily become elite lines. Agrobacteria can transfer exogenous genes on T-DNA into plant cells. This has been used both to transform plants stably and to express the genes transiently in plant cells. Here, we infected potato, with Agrobacterium tumefaciens harboring TALEN-expression vector targeting sterol side chain reductase 2 (SSR2) gene and regenerated shoots without selection. We obtained regenerated lines with disrupted-SSR2 gene and without transgene of the TALEN gene, revealing that their disruption should be caused by transient gene expression. The strategy using transient gene expression by Agrobacterium that we call Agrobacterial mutagenesis, developed here should accelerate the use of genome-editing technology to modify heterozygous plant genomes.

Targeted mutagenesis in Arabidopsis thaliana using CRISPR‐Cas12b/C2c1

Cas12b/C2c1 is a newly identified class 2 CRISPR endonuclease that was recently engineered for targeted genome editing in mammals and rice. To explore the potential applications of the CRISPR-Cas12b system in the dicot Arabidopsis thaliana, we selected BvCas12b and BhCas12b v4 for analysis. We successfully used both endonucleases to induce mutations, perform multiplex genome editing, and create large deletions at multiple loci. No significant mutations were detected at potential off-target sites. Analysis of the insertion/deletion frequencies and patterns of mutants generated via targeted gene mutagenesis highlighted the potential utility of CRISPR-Cas12b systems for genome editing in Arabidopsis.

Abstract D133: Development of a racially/ethnically diverse collection of immortalized lung epithelial cell lines to model lung adenocarcinoma development and drug resistance across population groups

Abstract Introduction: The most common type of lung cancer is lung adenocarcinoma, arising in the alveolar epithelium (air sacs). Drugs that specifically target certain known driver mutations are available. Unfortunately, resistance usually develops, and the cancer returns. In some cases, second and even third line targeted therapies have been established. The availability of such therapies is dependent on being able to screen for drugs that can treat resistant cells and elucidating the molecular basis of resistance. Importantly, the susceptibility of cancer cells to different therapies may be affected by the genetic background and thus the race/ethnicity of the patient. In order to ensure that effective therapies are available for all population groups, we propose to develop a collection of immortalized alveolar epithelial cell lines that represent a diversity of racial/ethnic groups. These cell lines can be used to study driver mutations and development of drug resistance, and to screen for new therapies to combat resistance. Results: Human alveolar epithelial cells are difficult to immortalize; they become senescent or undergo epithelial to mesenchymal transition, turning into fibroblasts. By using Y27632 dihydrochloride, a highly potent ATP-competitive inhibitor of Rho– associated coiled-coil forming protein serine/threonine kinase pathway, in combination with a viral oncogene, we have developed a method to culture and immortalize alveolar epithelial cells from remnant human transplant lung. The cells maintain their epithelial phenotype and can form spheres expressing lung epithelial markers in three dimensional culture. We are now in the process of inserting known driver mutations into these cells through targeted genome editing using CRISPR/Cas9. We intend to derive 10 similar cell lines each, from white, Latino, black and Asian subjects. Future directions: We will introduce driver mutations into cell lines of different backgrounds and resulting cell lines will be treated with targeted therapies in vitro to derive resistant cells. The parental, mutated, and resistant cell lines will be characterized by exome/transcriptome sequencing, and copy number variation analysis to determine the consequences of driver mutations and the basis for acquired resistance. We will use high throughput screening to identify drugs that can target resistant cells, using USC’s advanced drug screening facility, focusing on drugs that are approved by the Food and Drug Administration to ensure the greatest and fastest benefit to people suffering from lung cancer. Numerous existing drugs may have failed in trials due to differences between patients, and their therapeutic effect may be underestimated. Conclusion: Our ability to immortalize alveolar epithelial cells allows us to establish a large collection of alveolar epithelial cell lines that represent the diverse genetic background of the population of the United States. This will ensure that any developed therapies will be effective on a diversity of patients. Citation Format: Evelyn Tran, Tuo Shi, Xiuwen Li, Amy Firth, Beiyun Zhou, Zea Borok, Ite A Offringa. Development of a racially/ethnically diverse collection of immortalized lung epithelial cell lines to model lung adenocarcinoma development and drug resistance across population groups [abstract]. In: Proceedings of the Twelfth AACR Conference on the Science of Cancer Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; 2019 Sep 20-23; San Francisco, CA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2020;29(6 Suppl_2):Abstract nr D133.

Acquiring Resistance Against a Retroviral Infection via CRISPR/Cas9 Targeted Genome Editing in a Commercial Chicken Line.

Genome editing technology provides new possibilities for animal breeding and aid in understanding host-pathogen interactions. In poultry, retroviruses display one of the most difficult pathogens to control by conventional strategies such as vaccinations. Avian leukosis virus subgroup J (ALV-J) is an oncogenic, immunosuppressive retrovirus that causes myeloid leukosis and other tumors in chickens. Severe economic losses caused by ALV-J remain an unsolved problem in many parts of the world due to inefficient eradication strategies and lack of effective vaccines. ALV-J attachment and entry are mediated through the specific receptor, chicken Na+/H+ exchanger type 1 (chNHE1). The non-conserved amino acid tryptophan 38 (W38) in chNHE1 is crucial for virus entry, making it a favorable target for the introduction of disease resistance. In this study, we obtained ALV-J-resistance in a commercial chicken line by precise deletion of chNHE1 W38, utilizing the CRISPR/Cas9-system in combination with homology directed repair. The genetic modification completely protected cells from infection with a subgroup J retrovirus. W38 deletion did neither have a negative effect on the development nor on the general health condition of the gene edited chickens. Overall, the generation of ALV-J-resistant birds by precise gene editing demonstrates the immense potential of this approach as an alternative disease control strategy in poultry.

Plant Genome Editing and the Relevance of Off-Target Changes.

Site-directed nucleases (SDNs) used for targeted genome editing are powerful new tools to introduce precise genetic changes into plants. Like traditional approaches, such as conventional crossing and induced mutagenesis, genome editing aims to improve crop yield and nutrition. Next-generation sequencing studies demonstrate that across their genomes, populations of crop species typically carry millions of single nucleotide polymorphisms and many copy number and structural variants. Spontaneous mutations occur at rates of ∼10-8 to 10-9 per site per generation, while variation induced by chemical treatment or ionizing radiation results in higher mutation rates. In the context of SDNs, an off-target change or edit is an unintended, nonspecific mutation occurring at a site with sequence similarity to the targeted edit region. SDN-mediated off-target changes can contribute to a small number of additional genetic variants compared to those that occur naturally in breeding populations or are introduced by induced-mutagenesis methods. Recent studies show that using computational algorithms to design genome editing reagents can mitigate off-target edits in plants. Finally, crops are subject to strong selection to eliminate off-type plants through well-established multigenerational breeding, selection, and commercial variety development practices. Within this context, off-target edits in crops present no new safety concerns compared to other breeding practices. The current generation of genome editing technologies is already proving useful to develop new plant varieties with consumer and farmer benefits. Genome editing will likely undergo improved editing specificity along with new developments in SDN delivery and increasing genomic characterization, further improving reagent design and application.

A CRISPR/Cas9-Based Mutagenesis Protocol for Brachypodium distachyon and Its Allopolyploid Relative, Brachypodium hybridum.

The CRISPR/Cas9 system enables precise genome editing and is a useful tool for functional genomic studies. Here we report a detailed protocol for targeted genome editing in the model grass Brachypodium distachyon and its allotetraploid relative B. hybridum, describing gRNA design, a transient protoplast assay to test gRNA efficiency, Agrobacterium-mediated transformation and the selection and analysis of regenerated plants. In B. distachyon, we targeted the gene encoding phytoene desaturase (PDS), which is a crucial enzyme in the chlorophyll biosynthesis pathway. The albino phenotype of mutants obtained confirmed the effectiveness of the protocol for functional gene analysis. Additionally, we targeted two genes related to cell wall maintenance, encoding a fasciclin-like arabinogalactan protein (FLA) and a pectin methylesterase (PME), also in B. distachyon. Two genes encoding cyclin-dependent kinases (CDKG1 and CDKG2), which may be involved in DNA recombination were targeted in both B. distachyon and B. hybridum. Cas9 activity induces mainly insertions or deletions, resulting in frameshift mutations that, may lead to premature stop codons. Because of the close phylogenetic relationship between Brachypodium species and key temperate cereals and forage grasses, this protocol should be easily adapted to target genes underpinning agronomically important traits.