Recent Advances In Genome Engineering Research Articles

Streamlined Generation of CRISPR/Cas9-Mediated Single-Cell Knockout Clones in Murine Cell Lines.

Clonal cell lines harboring loss-of-function mutations in genes of interest are crucial for studying the cellular functions of the encoded proteins. Recent advances in genome engineering have converged on the CRISPR/Cas9 technology to quickly and reliably generate frameshift mutations in the target genes across various cell lines and species. Although high on-target cleavage efficiencies can be obtained reproducibly, screening and identifying clones with loss-of-function alleles remains a major bottleneck. Here, we describe a single sgRNA strategy to generate CRISPR/Cas9-mediated frameshift mutations in target genes of mammalian cell lines that can be easily and cost-effectively identified. Given the proliferation of workhorse cell lines such as N2a cells and the resulting clonal expansion of the cell type, our protocol can facilitate the isolation of knockout clonal cell lines and their genetic validation within a period of down to 6-8 weeks.

Abstract NG03: Functional and computational approaches to uncover selection advantages of cancer aneuploidy

Abstract Aneuploidy, including the gain or loss of whole chromosomes or chromosome arms, is a near-universal feature of cancer. We previously applied methods that define chromosome arm aneuploidy to over 10,000 tumors in the Cancer Genome Atlas (TCGA). Cancer subtypes are often characterized by tumor specific patterns of chromosome arm copy number alterations and breakpoints; for example, squamous cell carcinomas (SCCs) from different tissues of origin are characterized by chromosome 3p (chr3p) loss and chromosome 3q (chr3q) gain. From the TCGA aneuploidy data, we developed an algorithm called BISCUT to distinguish peak regions of aneuploidy breakpoints on each chromosome arm. BISCUT identified loci affected by broad copy number alterations that provide fitness advantages or disadvantages both within individual cancer types and across cancers. Our analyses are consistent with selection being the primary driver of aneuploidy events in cancer. We next wanted to validate some BISCUT peaks without a known driver, to identify potential gene deletions that are beneficial in cancer cells. We focused on chromosome 8p (chr8p), as this arm is frequently deleted across cancer types, but no strong tumor suppressors have been identified. Recent advances in genome engineering allow generation of large chromosomal alterations and validation of findings from patient genomic data. For this study, we used our CRISPR-Cas9 arm-deletion system to delete chr8p in human immortalized epithelial cells. Cells with chr8p deletion showed lower amounts of cell death in culture. Knockdown of WRN, one of the two genes in the smallest chr8p BISCUT peak, was sufficient to reproduce this phenotype, suggesting that WRN haploinsufficiency may be beneficial to tumor development. As aneuploidy occurs in tissue-specific patterns, we also wanted to explore the role of chr3 arm aneuploidies in squamous cancers. We again used the CRISPR-Cas9 system to delete one copy of chr3p in a human immortalized lung epithelial cell line similar to the putative cell-of-origin in lung SCC. Consistent with patient data, expression of chr3p genes was decreased upon deletion, as well as increased expression of interferon response genes. Cells with chr3p deletion initially proliferated more slowly than their siblings. Interestingly, after several passages in culture, this proliferation defect was rescued in chr3p deleted cells. Genome sequencing and karyotype analyses suggested that this was partially the result of chr3 duplication, with cells transitioning to a state of chr3q gain. Culturing these cells as organoids also demonstrated that chr3p deletion and chr3q gain can affect basal cell differentiation, consistent with their high frequency in squamous cancers. Our genome engineering approach to model chromosome arm aneuploidies provides a robust model to validate drivers from aneuploidy events, which will be critical to address the gap in our understanding of aneuploidy in cancer. In addition, our methods have identified consequences of individual aneuploidy events, which will lead to new precision oncology targets for patients. Citation Format: Alison M. Taylor. Functional and computational approaches to uncover selection advantages of cancer aneuploidy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr NG03.

Recent advances in genome engineering by CRISPR technology

Recent advances in genome engineering by CRISPR technology

An update and review of arthropod vector sensory systems: Potential targets for behavioural manipulation by parasites and other disease agents.

For over a century, vector ecology has been a mainstay of vector-borne disease control. Much of this research has focused on the sensory ecology of blood-feeding arthropods (black flies, mosquitoes, ticks, etc.) with terrestrial vertebrate hosts. Of particular interest are the cues and sensory systems that drive host seeking and host feeding behaviours as they are critical for a vector to locate and feed from a host. An important yet overlooked component of arthropod vector ecology are the phenotypic changes observed in infected vectors that increase disease transmission. While our fundamental understanding of sensory mechanisms in disease vectors has drastically increased due to recent advances in genome engineering, for example, the advent of CRISPR-Cas9, and high-throughput "big data" approaches (genomics, proteomics, transcriptomics, etc.), we still do not know if and how parasites manipulate vector behaviour. Here, we review the latest research on arthropod vector sensory systems and propose key mechanisms that disease agents may alter to increase transmission.

Rise of synthetic yeast: Charting courses to new applications

Microbes have long provided us with important capabilities, and the genome engineering of microbes has greatly empowered research and applications in biotechnology. This is especially true with the emergence of synthetic biology and recent advances in genome engineering to control microbial behavior. A fully synthetic, rationally designed genome promises opportunities for unprecedented control of cellular function. As a eukaryotic workhorse for research and industrial use, yeast is an organism at the forefront of synthetic biology; the tools and engineered cellular platform being delivered by the Sc2.0 consortium are enabling a new era of bespoke biology. This issue highlights recent advances delivered by this consortium, but hurdles remain to maximize the impact of engineered eukaryotic cells more broadly.

Abstract LE03-01: Functional and computational approaches to uncover selection advantages of cancer aneuploidy

Abstract Aneuploidy, including the gain or loss of whole chromosomes or chromosome arms, is a near-universal feature of cancer. We previously applied methods that define chromosome arm aneuploidy to over 10,000 tumors in the Cancer Genome Atlas (TCGA). Cancer subtypes are often characterized by tumor specific patterns of chromosome arm copy number alterations and breakpoints; for example, squamous cell carcinomas (SCCs) from different tissues of origin are characterized by chromosome 3p (chr3p) loss and chromosome 3q (chr3q) gain. From the TCGA aneuploidy data, we developed an algorithm called BISCUT to distinguish peak regions of aneuploidy breakpoints on each chromosome arm. BISCUT identified loci affected by broad copy number alterations that provide fitness advantages or disadvantages both within individual cancer types and across cancers. Our analyses are consistent with selection being the primary driver of aneuploidy events in cancer. Recent advances in genome engineering allow generation of large chromosomal alterations and validation of findings from patient genomic data. We used the CRISPR-Cas9 system to delete one copy of chr3p in a human immortalized lung epithelial cell line similar to the putative cell-of-origin in lung SCC. Consistent with patient data, expression of chr3p genes was decreased upon deletion, as well as increased expression of interferon response genes. Cells with chr3p deletion initially proliferated more slowly than their siblings. Interestingly, after several passages in culture, this proliferation defect was rescued in chr3p deleted cells. Genome sequencing and karyotype analyses suggested that this was partially the result of chr3 duplication, with cells transitioning to a state of chr3q gain. Isolated cells with chr3q gain show deficiencies in squamous differentiation in organoid culture. Our genome engineering approach to model chromosome arm aneuploidies provides a robust model that will address a gap in our understanding of aneuploidy in cancer. Citation Format: Alison Marie Taylor. Functional and computational approaches to uncover selection advantages of cancer aneuploidy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LE03-01.

Advances in Adoptive Cell Therapy Using Induced Pluripotent Stem Cell-Derived T Cells.

Adoptive cell therapy (ACT) using chimeric antigen receptor (CAR) T cells holds impressive clinical outcomes especially in patients who are refractory to other kinds of therapy. However, many challenges hinder its clinical applications. For example, patients who undergo chemotherapy usually have an insufficient number of autologous T cells due to lymphopenia. Long-term ex vivo expansion can result in T cell exhaustion, which reduces the effector function. There is also a batch-to-batch variation during the manufacturing process, making it difficult to standardize and validate the cell products. In addition, the process is labor-intensive and costly. Generation of universal off-the-shelf CAR T cells, which can be broadly given to any patient, prepared in advance and ready to use, would be ideal and more cost-effective. Human induced pluripotent stem cells (iPSCs) provide a renewable source of cells that can be genetically engineered and differentiated into immune cells with enhanced anti-tumor cytotoxicity. This review describes basic knowledge of T cell biology, applications in ACT, the use of iPSCs as a new source of T cells and current differentiation strategies used to generate T cells as well as recent advances in genome engineering to produce next-generation off-the-shelf T cells with improved effector functions. We also discuss challenges in the field and future perspectives toward the final universal off-the-shelf immunotherapeutic products.

Abstract 2135: Functional models of chromosome arm aneuploidies in lung squamous cell carcinoma

Abstract Aneuploidy, which we define as whole chromosome or chromosome arm copy number imbalance, is a near-universal characteristic of human cancers. Cancer subtypes are often characterized by tumor specific patterns of chromosome arm copy number alterations; for example, squamous cell carcinomas (SCCs) from different tissues of origin (including lung, head and neck, esophagus, and bladder) have a pattern of chromosome 3p loss and chromosome 3q gain. Although these alterations are frequent, they are not well understood due to difficulty in modeling specific aneuploidy alterations in the matching cell type. However, recent advances in genome engineering allow generation of large chromosomal alterations. We used the CRISPR-Cas9 system to delete one copy of chromosome 3p in human immortalized lung epithelial cells most similar to upper airway basal cells. Deletion of chromosome 3p was validated by whole genome sequencing and karyotyping. Consistent with patient data, expression of 3p genes was decreased upon deletion, as well as increased expression of interferon response genes. Phenotypic characterization revealed that cells with chromosome 3p deletion initially proliferated more slowly than their siblings. These chromosome 3p deleted cells had increased G1 arrest but did not undergo increased apoptosis or cell death. Interestingly, after several passages in culture, the proliferation defect was rescued in chromosome 3p deleted cells. Genome sequencing and karyotype analyses found that evidence of chromosome 3 duplication, transitioning the cell to a state of chromosome 3q gain. We isolated sibling cells with chromosome 3p deletion or chromosome 3q gain and demonstrated that duplication of chromosome 3 could indeed rescue proliferation rates. With our cellular model of chromosome arm-level aneuploidy, we uncovered a selection mechanism that allowed aneuploidy tolerance in vitro, and a possible explanation for joint alteration of both arms of chromosome 3. In conclusion, our genome engineering approach to model chromosome arm-level deletions provides a robust model that will address a gap in our understanding of aneuploidy in cancer. Citation Format: Alison Marie Taylor, Sejal Jain, Juliann Shih, Andrew D. Cherniack, Rameen Beroukhim, Matthew Meyerson. Functional models of chromosome arm aneuploidies in lung squamous cell carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2135.

Versatile Labeling and Detection of Endogenous Proteins Using Tag-Assisted Split Enzyme Complementation.

Recent advances in genome engineering have expanded our capabilities to study proteins in their natural states. In particular, the ease and scalability of knocking-in small peptide tags has enabled high throughput tagging and analysis of endogenous proteins. To improve enrichment capacities and expand the functionality of knock-ins using short tags, we developed the tag-assisted split enzyme complementation (TASEC) approach, which uses two orthogonal small peptide tags and their cognate binders to conditionally drive complementation of a split enzyme upon labeled protein expression. Using this approach, we have engineered and optimized the tag-assisted split HaloTag complementation system (TA-splitHalo) and demonstrated its versatile applications in improving the efficiency of knock-in cell enrichment, detection of protein-protein interaction, and isolation of biallelic gene edited cells through multiplexing.

Abstract A44: Functional models of chromosome arm-level aneuploidies in cancer

Abstract Aneuploidy, whole-chromosome or chromosome arm copy number imbalance, is a near-universal characteristic of human cancers. We previously applied methods that define chromosome arm-level aneuploidy and a global cancer aneuploidy score to 10,522 tumors of 33 types in The Cancer Genome Atlas (TCGA). Aneuploidy level was correlated with TP53 mutation, somatic mutation rate, and expression of proliferation genes. Overall aneuploidy was anti-correlated with expression of immune signaling genes, due to decreased leukocyte infiltrates in high-aneuploidy samples. In contrast, some individual chromosome arm alterations correlated with higher expression of immune signaling genes, including deletion of chromosome 3p, 8p, and 17p. Cancer subtypes are often characterized by tumor specific patterns of arm-level copy number alterations; for example, squamous cell carcinomas (SCCs) from different tissues of origin (including lung, esophagus, and bladder) have a pattern of chromosome 3p loss and chromosome 3q gain. Although yeast and mammalian models of whole-chromosome aneuploidies have been extensively investigated, chromosome arm-level aneuploidies have rarely been modeled. However, recent advances in genome engineering and targeting of endonucleases allow generation of large chromosomal alterations. We used the CRISPR-Cas9 system to delete one copy of chromosome 3p in human immortalized lung epithelial cells, validated in 8 clones by whole-genome sequencing. Consistent with patient data, expression of 3p genes was decreased upon deletion, as well as increased expression of interferon response genes. Phenotypic characterization revealed that cells with chromosome 3p deletion initially proliferated more slowly than their siblings. These chromosome 3p-deleted cells had increased G1 arrest but did not undergo increased apoptosis or cell death. Interestingly, after several passages in culture, the proliferation defect was rescued in chromosome 3p-deleted cells; genome sequencing and karyotype analyses suggested that this was partially the result of chromosome 3 duplication. With our cellular model of chromosome arm-level aneuploidy, we uncovered a selection mechanism that allowed aneuploidy tolerance in vitro. We have also grown these cells in spheroid and organoid culture systems to assess the effects of chromosome 3 arm-level aneuploidies on cytokine production and squamous differentiation and are using these systems to identify how chromosome 3p deletion affects drug sensitivity. In conclusion, our genome engineering approach to model chromosome arm-level deletions provides a robust model that will address a gap in our understanding of aneuploidy in cancer. Citation Format: Alison M. Taylor, Sejal Jain, Juliann Shih, Andrew D. Cherniack, Rameen Beroukhim, Matthew Meyerson. Functional models of chromosome arm-level aneuploidies in cancer [abstract]. In: Proceedings of the AACR Special Conference on the Evolving Landscape of Cancer Modeling; 2020 Mar 2-5; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2020;80(11 Suppl):Abstract nr A44.

Pipeline for the Generation and Characterization of Transgenic Human Pluripotent Stem Cells Using the CRISPR/Cas9 Technology

Recent advances in genome engineering based on the CRISPR/Cas9 technology have revolutionized our ability to manipulate genomic DNA. Its use in human pluripotent stem cells (hPSCs) has allowed a wide range of mutant cell lines to be obtained at an unprecedented rate. The combination of these two groundbreaking technologies has tremendous potential, from disease modeling to stem cell-based therapies. However, the generation, screening and molecular characterization of these cell lines remain a cumbersome and multi-step endeavor. Here, we propose a pipeline of strategies to efficiently generate, sub-clone, and characterize CRISPR/Cas9-edited hPSC lines in the function of the introduced mutation (indels, point mutations, insertion of large constructs, deletions).

Phenotypic chemical and mutant screening of zebrafish larvae using an on-demand response to electric stimulation.

Behavioral responses of zebrafish larvae to environmental cues are important functional readouts that should be evoked on-demand and studied phenotypically in behavioral, genetical and developmental investigations. Very recently, it was shown that zebrafish larvae execute a voluntary and oriented movement toward the positive electrode of an electric field along a microchannel. Phenotypic characterization of this response was not feasible due to larva's rapid movement along the channel. To overcome this challenge, a microfluidic device was introduced to partially immobilize the larva's head while leaving its mid-body and tail unrestrained in a chamber to image motor behaviors in response to electric stimulation, hence achieving quantitative phenotyping of the electrically evoked movement in zebrafish larvae. The effect of electric current on the tail-beat frequency and response duration of 5-7days postfertilization zebrafish larvae was studied. Investigations were also performed on zebrafish exposed to neurotoxin 6-hydroxydopamine and larvae carrying a pannexin1a (panx1a) gene knockout, as a proof of principle applications to demonstrate on-demand movement behavior screening in chemical and mutant assays. We demonstrated for the first time that 6-hydroxydopamine leads to electric response impairment, levodopa treatment rescues the response and panx1a is involved in the electrically evoked movement of zebrafish larvae. We envision that our technique is broadly applicable as a screening tool to quantitatively examine zebrafish larvae's movements in response to physical and chemical stimulations in investigations of Parkinson's and other neurodegenerative diseases, and as a tool to combine recent advances in genome engineering of model organisms to uncover the biology of electric response.

Emerging Genome Engineering Tools in Crop Research and Breeding.

Recent advances in genome engineering are revolutionizing crop research and plant breeding. The ability to make specific modifications to a plant's genetic material creates opportunities for rapid development of elite cultivars with desired traits. The plant genome can be altered in several ways, including targeted introduction of nucleotide changes, deleting DNA segments, introducing exogenous DNA fragments and epigenetic modifications. Targeted changes are mediated by sequence specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspersed short palindromic repeats)-Cas (CRISPR associated protein) systems. Recent advances in engineering chimeric Cas nucleases fused to base editing enzymes permit for even greater precision in base editing and control over gene expression. In addition to gene editing technologies, improvement in delivery systems of exogenous DNA into plant cells have increased the rate of successful gene editing events. Regeneration of fertile plants containing the desired edits remains challenging; however, manipulation of embryogenesis-related genes such as BABY BOOM (BBM) has been shown to facilitate regeneration through tissue culture, often a major hurdle in recalcitrant cultivars. Epigenome reprogramming for improved crop performance is another possibility for future breeders, with recent studies on MutS HOMOLOG 1 (MSH1) demonstrating epigenetic-dependent hybrid vigor in several crops. While these technologies offer plant breeders new tools in creating high yielding, better adapted crop varieties, constantly evolving government policy regarding the cultivation of plants containing transgenes may impede the widespread adoption of some of these techniques. This chapter summarizes advances in genome editing tools and discusses the future of these techniques for crop improvement.

The influence of genetics on therapeutic developments in pulmonary alveolar proteinosis.

Pulmonary alveolar proteinosis (PAP) is characterized by the massive accumulation of lipoproteinaceous material within alveoli, which results in progressive respiratory failure. The abnormalities in surfactant clearance are caused by defective pulmonary macrophages, whose terminal differentiation is GM-CSF-dependent. In hereditary PAP, the rupture of GM-CSF signaling is because of mutations in the GM-CSF receptor genes. This review focus on the innovative technologies of gene-correction proposed for the development of new therapeutic strategies, for hereditary PAP patients. Hematopoietic stem cell gene therapy has been successfully experimented in murine models to restore the expression of the GM-CSF receptor, however, a therapeutic approach based on bone marrow transplantation requires a preconditioning, which could be hazardous in PAP patients, who are highly susceptible to pulmonary infections. Gene-corrected pulmonary macrophages, administered directly to the lung, could represent an improved approach. Finally, patient-derived induced pluripotent stem cells seem to be promising to overcome the limited availability of primary patient cells and to generate gene-corrected macrophages, able to recover pulmonary surfactant clearance. WLL is the gold standard therapy for PAP. However, its use in hereditary PAP is limited by the difficulty of performing this technique in paediatric patients and by its purely symptomatic efficacy. The recent advances in genome engineering could provide efficacious strategies for clinical application.

Abstract 3002: Genome engineering approaches to generate models of chromosome arm-level cancer aneuploidy

Abstract Aneuploidy, whole chromosome or chromosome arm copy number imbalance, is a near-universal characteristic of human cancers. Although yeast and mammalian models of whole chromosome aneuploidies have been extensively investigated, chromosome arm-level aneuploidies have rarely been modeled. Cancer subtypes are often characterized by tumor-specific patterns of these arm-level copy number alterations; for example, squamous cell carcinomas (SCCs) from different tissues of origin (including lung, esophagus, and bladder) have a pattern of chromosome 3p loss and chromosome 3q gain. Our analysis of 495 lung SCCs found chromosome 3p deletion to be the most frequent genomic alteration, occurring in almost 80% of the tumors and covering the entire length of the chromosome arm. Over two-thirds of chromosome 3p genes showed significantly decreased expression in these samples. Without models of chromosome arm-level alterations, the phenotypic effects of specific aneuploidies in cancer, such as 3p deletion, remain unknown. However, recent advances in genome engineering and targeting of endonucleases allow new approaches to generate chromosomal alterations. Here, we used the CRISPR-Cas9 system to delete one copy of chromosome 3p in vitro. We successfully isolated almost 90 clones of immortalized lung epithelial cells with deletion of the 3p arm, with 8 validated by whole-genome sequencing. Consistent with patient data, expression of 3p genes was also decreased upon deletion. Phenotypic characterization revealed that cells with chromosome 3p deletion initially proliferated more slowly than their siblings. These chromosome 3p-deleted cells had increased G1 arrest, but did not undergo increased apoptosis or cell death. Interestingly, after several passages in culture, the proliferation defect was rescued in chromosome 3p-deleted cells; genome sequencing and karyotype analyses suggested that this was the result of chromosome 3 duplication. With our cellular model of chromosome arm-level aneuploidy, we uncovered a possible selection mechanism that allows aneuploidy tolerance in vitro. We used genome engineering to model chromosome arm-level deletions, providing a robust model that will address a gap in our understanding of aneuploidy in cancer. Citation Format: Alison Marie Taylor, Xiaoyang Zhang, Juliann Shih, Gavin Ha, Galen F. Gao, Ashton C. Berger, Andrew D. Cherniack, Rameen Beroukhim, Matthew Meyerson. Genome engineering approaches to generate models of chromosome arm-level cancer aneuploidy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3002.

Overview of Rodent Zic Genes.

The five murine Zic genes encode multifunctional transcriptional regulator proteins important for a large number of processes during embryonic development. The genes and proteins are highly conserved with respect to the orthologous human genes, an attribute evidently mirrored by functional conservation, since the murine and human genes mutate to give the same phenotypes. Each ZIC protein contains a zinc finger domain that participates in both protein-DNA and protein-protein interactions. The ZIC proteins are capable of interacting with the key transcriptional mediators of the SHH, WNT and NODAL signalling pathways as well as with components of the transcriptional machinery and chromatin-modifying complexes. It is possible that this diverse range of protein partners underlies characteristics uncovered by mutagenesis and phenotyping of the murine Zic genes. These features include redundant and unique roles for ZIC proteins, regulatory interdependencies amongst family members and pleiotropic Zic gene function. Future investigations into the complex nature of the Zic gene family activity should be facilitated by recent advances in genome engineering and functional genomics.

Genetically engineered mouse models for studying radiation biology.

Genetically engineered mouse models (GEMMs) are valuable research tools that have transformed our understanding of cancer. The first GEMMs generated in the 1980s and 1990s were knock-in and knock-out models of single oncogenes or tumor suppressors. The advances that made these models possible catalyzed both technological and conceptual shifts in the way cancer research was conducted. As a result, dozens of mouse models of cancer exist today, covering nearly every tissue type. The advantages inherent to GEMMs compared to in vitro and in vivo transplant models are compounded in preclinical radiobiology research for several reasons. First, they accurately and robustly recapitulate primary cancers anatomically, histopathologically, and genetically. Reliable models are a prerequisite for predictive preclinical studies. Second, they preserve the tumor microenvironment, including the immune, vascular, and stromal compartments, which enables the study of radiobiology at a systems biology level. Third, they provide exquisite control over the genetics and kinetics of tumor initiation, which enables the study of specific gene mutations on radiation response and functional genomics in vivo. Taken together, these facets allow researchers to utilize GEMMs for rigorous and reproducible preclinical research. In the three decades since the generation of the first GEMMs of cancer, advancements in modeling approaches have rapidly progressed and expanded the mouse modeling toolbox with techniques such as in vivo short hairpin RNA (shRNA) knockdown, inducible gene expression, site-specific recombinases, and dual recombinase systems. Our lab and many others have utilized these tools to study cancer and radiobiology. Recent advances in genome engineering with CRISPR/Cas9 technology have made GEMMs even more accessible to researchers. Here, we review current and future approaches to mouse modeling with a focus on applications in preclinical radiobiology research.

Viral Vectors for Plant Genome Engineering.

Recent advances in genome engineering (GE) has made it possible to precisely alter DNA sequences in plant cells, providing specifically engineered plants with traits of interest. Gene targeting efficiency depends on the delivery-method of both sequence-specific nucleases and repair templates, to plant cells. Typically, this is achieved using Agrobacterium mediated transformation or particle bombardment, both of which transform only a subset of cells in treated tissues. The alternate in planta approaches, stably integrating nuclease-encoding cassettes and repair templates into the plant genome, are time consuming, expensive and require extra regulations. More efficient GE reagents delivery methods are clearly needed if GE is to become routine, especially in economically important crops that are difficult to transform. Recently, autonomously replicating virus-based vectors have been demonstrated as efficient means of delivering GE reagents in plants. Both DNA viruses (Bean yellow dwarf virus, Wheat dwarf virus and Cabbage leaf curl virus) and RNA virus (Tobacco rattle virus) have demonstrated efficient gene targeting frequencies in model plants (Nicotiana benthamiana) and crops (potato, tomato, rice, and wheat). Here we discuss the recent advances using viral vectors for plant genome engineering, the current limitations and future directions.

DOCK6 inactivation highlights ISGylation as RHO-GTPase balancer

The recent advances in genome-engineering by technologies such as CRISPR/Cas9 now enable an unprecedented level of genome manipulation to support the analysis of phenotypes arising from gene disrup...

Applications of CRISPR Genome Engineering in Cell Biology.

Recent advances in genome engineering are starting a revolution in biological research and translational applications. The clustered regularly interspaced short palindromic repeats (CRISPR)-associated RNA-guided endonuclease CRISPR associated protein 9 (Cas9) and its variants enable diverse manipulations of genome function. In this review, we describe the development of Cas9 tools for a variety of applications in cell biology research, including the study of functional genomics, the creation of transgenic animal models, and genomic imaging. Novel genome engineering methods offer a new avenue to understand the causality between the genome and phenotype, thus promising a fuller understanding of cell biology.