CRISPR-Cas9 Genome Editing Tools Research Articles

CRISPR-Cas9 mediated understanding of plants' abiotic stress-responsive genes to combat changing climatic patterns.

Multiple abiotic stresses like extreme temperatures, water shortage, flooding, salinity, and exposure to heavy metals are confronted by crop plants with changing climatic patterns. Prolonged exposure to these adverse environmental conditions leads to stunted plant growth and development with significant yield loss in crops. CRISPR-Cas9 genome editing tool is being frequently employed to understand abiotic stress-responsive genes. Noteworthy improvements in CRISPR-Cas technology have been made over the years, including upgradation of Cas proteins fidelity and efficiency, optimization of transformation protocols for different crop species, base and prime editing, multiplex gene-targeting, transgene-free editing, and graft-based heritable CRISPR-Cas9 approaches. These developments helped to improve the knowledge of abiotic stress tolerance in crops that could potentially be utilized to develop knock-out varieties and over-expressed lines to tackle the adverse effects of altered climatic patterns. This review summarizes the mechanistic understanding of heat, drought, salinity, and metal stress-responsive genes characterized so far using CRISPR-Cas9 and provides data on potential candidate genes that can be exploited by modern-day biotechnological tools to develop transgene-free genome-edited crops with better climate adaptability. Furthermore, the importance of early-maturing crop varieties to withstand abiotic stresses is also discussed in this review.

A versatile CRISPR-Cas13d platform for multiplexed transcriptomic regulation and metabolic engineering in primary human T cells

CRISPR technologies have begun to revolutionize Tcell therapies; however, conventional CRISPR-Cas9 genome-editing tools are limited in their safety, efficacy, and scope. To address these challenges, we developed multiplexed effector guide arrays (MEGA), a platform for programmable and scalable regulation of the Tcell transcriptome using the RNA-guided, RNA-targeting activity of CRISPR-Cas13d. MEGA enables quantitative, reversible, and massively multiplexed gene knockdown in primary human Tcells without targeting or cutting genomic DNA. Applying MEGA to a model of CAR Tcell exhaustion, we robustly suppressed inhibitory receptor upregulation and uncovered paired regulators of Tcell function through combinatorial CRISPR screening. We additionally implemented druggable regulation of MEGA to control CAR activation in a receptor-independent manner. Lastly, MEGA enabled multiplexed disruption of immunoregulatory metabolic pathwaysto enhance CAR Tcell fitness and anti-tumor activity invitro and invivo. MEGA offers a versatile synthetic toolkit for applications in cancer immunotherapy and beyond.

CRISPR gene technology—the next best thing in medicine

CRISPR gene technology—the next best thing in medicine

Establishment of a genome editing tool using CRISPR-Cas9 ribonucleoprotein complexes in the non-model plant pathogen Sphaerulina musiva.

CRISPR-Cas9 is a versatile genome editing system widely used since 2013 to introduce site-specific modifications into the genomes of model and non-model species. This technology is used in various applications, from gene knock-outs, knock-ins, and over-expressions to more precise changes, such as the introduction of nucleotides at a targeted locus. CRISPR-Cas9 has been demonstrated to be easy to establish in new species and highly efficient and specific compared to previous gene editing strategies such as Zinc finger nucleases and transcription activator-like effector nucleases. Grand challenges for emerging CRISPR-Cas9 tools in filamentous fungi are developing efficient transformation methods for non-model organisms. In this paper, we have leveraged the establishment of CRISPR-Cas9 genome editing tool that relies on Cas9/sgRNA ribonucleoprotein complexes (RNPs) in the model species Trichoderma reesei and developed the first protocol to efficiently transform the non-model species, Sphaerulina musiva. This fungal pathogen constitutes a real threat to the genus Populus, a foundational bioenergy crop used for biofuel production. Herein, we highlight the general considerations to design sgRNAs and their computational validation. We also describe the use of isolated protoplasts to deliver the CRISPR-Cas9 RNP components in both species and the screening for targeted genome editing events. The development of engineering tools in S. musiva can be used for studying genes involved in diverse processes such as secondary metabolism, establishment, and pathogenicity, among many others, but also for developing genetic mitigation approaches. The approach described here provides guidance for potential development of transformation systems in other non-model spore-bearing ascomycetes.

Synthetic Biology Toolbox, Including a Single-Plasmid CRISPR-Cas9 System to Biologically Engineer the Electrogenic, Metal-Resistant Bacterium Cupriavidus metallidurans CH34.

Cupriavidus metallidurans CH34 exhibits extraordinary metabolic versatility, including chemolithoautotrophic growth; degradation of BTEX (benzene, toluene, ethylbenzene, xylene); high resistance to numerous metals; biomineralization of gold, platinum, silver, and uranium; and accumulation of polyhydroxybutyrate (PHB). These qualities make it a valuable host for biotechnological applications such as bioremediation, bioprocessing, and the generation of bioelectricity in microbial fuel cells (MFCs). However, the lack of genetic tools for strain development and studying its fundamental physiology represents a bottleneck to boosting its commercial applications. In this study, inducible and constitutive promoter libraries were built and characterized, providing the first comprehensive list of biological parts that can be used to regulate protein expression and optimize the CRISPR-Cas9 genome editing tools for this host. A single-plasmid CRISPR-Cas9 system that can be delivered by both conjugation and electroporation was developed, and its efficiency was demonstrated by successfully targeting the pyrE locus. The CRISPR-Cas9 system was next used to target candidate genes encoding type IV pili, hypothesized by us to be involved in extracellular electron transfer (EET) in this organism. Single and double deletion strains (ΔpilA, ΔpilE, and ΔpilAE) were successfully generated. Additionally, the CRISPR-Cas9 tool was validated for constructing genomic insertions (ΔpilAE::gfp and ΔpilAE::λPrgfp). Finally, as type IV pili are believed to play an important role in extracellular electron transfer to solid surfaces, C. metallidurans CH34 ΔpilAE was further studied by means of cyclic voltammetry using disposable screen-printed carbon electrodes. Under these conditions, we demonstrated that C. metallidurans CH34 could generate extracellular currents; however, no difference in the intensity of the current peaks was found in the ΔpilAE double deletion strain when compared to the wild type. This finding suggests that the deleted type IV pili candidate genes are not involved in extracellular electron transfer under these conditions. Nevertheless, these experiments revealed the presence of different redox centers likely to be involved in both mediated electron transfer (MET) and direct electron transfer (DET), the first interpretation of extracellular electron transfer mechanisms in C. metallidurans CH34.

Identification of Novel Genes for Cell Fusion during Osteoclast Formation.

Osteoclasts are derived from hematopoietic stem cells. Monocyte preosteoclasts obtain resorbing activity via cell–cell fusion to generate multinucleated cells. However, the mechanisms and molecules involved in the fusion process are poorly understood. In this study, we performed RNA sequencing with single nucleated cells (SNCs) and multinucleated cells (MNCs) to identify the fusion-specific genes. The SNCs and MNCs were isolated under the same conditions during osteoclastogenesis with the receptor activator of nuclear factor-κB ligand (RANKL) administration. Based on this analysis, the expression of seven genes was found to be significantly increased in MNCs but decreased in SNCs, compared to that in bone marrow-derived macrophages (BMMs). We then generated knockout macrophage cell lines using a CRISPR-Cas9 genome-editing tool to examine their function during osteoclastogenesis. Calcrl-, Marco-, or Ube3a-deficient cells could not develop multinucleated giant osteoclasts upon RANKL stimulation. However, Tmem26-deficient cells fused more efficiently than control cells. Our findings demonstrate that Calcrl, Marco, and Ube3a are novel determinants of osteoclastogenesis, especially with respect to cell fusion, and highlight potential targets for osteoporosis therapy.

An Archaea-specific c-type cytochrome maturation machinery is crucial for methanogenesis in Methanosarcina acetivorans

c-Type cytochromes (cyt c) are proteins that undergo post-translational modification to covalently bind heme, which allows them to facilitate redox reactions in electron transport chains across all domains of life. Genomic evidence suggests that cyt c are involved in electron transfer processes among the Archaea, especially in members that produce or consume the potent greenhouse gas methane. However, neither the maturation machinery for cyt c in Archaea nor their role in methane metabolism has ever been functionally characterized. Here, we have used CRISPR-Cas9 genome editing tools to map a distinct pathway for cyt c biogenesis in the model methanogenic archaeon Methanosarcina acetivorans, and have also identified substrate-specific functional roles for cyt c during methanogenesis. Although the cyt c maturation machinery from M. acetivorans is universally conserved in the Archaea, our evolutionary analyses indicate that different clades of Archaea acquired this machinery through multiple independent horizontal gene transfer events from different groups of Bacteria. Overall, we demonstrate the convergent evolution of a novel Archaea-specific cyt c maturation machinery and its physiological role during methanogenesis, a process which contributes substantially to global methane emissions.

CRISPR-based gene disruption and integration of high-avidity, WT1-specific T cell receptors improve antitumor T cell function.

T cell receptor (TCR)-based therapy has the potential to induce durable clinical responses in patients with cancer by targeting intracellular tumor antigens with high sensitivity and by promoting T cell survival. However, the need for TCRs specific for shared oncogenic antigens and the need for manufacturing protocols able to redirect T cell specificity while preserving T cell fitness remain limiting factors. By longitudinal monitoring of T cell functionality and dynamics in 15 healthy donors, we isolated 19 TCRs specific for Wilms' tumor antigen 1 (WT1), which is overexpressed by several tumor types. TCRs recognized several peptides restricted by common human leukocyte antigen (HLA) alleles and displayed a wide range of functional avidities. We selected five high-avidity HLA-A*02:01-restricted TCRs, three that were specific to the less explored immunodominant WT137-45 and two that were specific to the noncanonical WT1-78-64 epitopes, both naturally processed by primary acute myeloid leukemia (AML) blasts. With CRISPR-Cas9 genome editing tools, we combined TCR-targeted integration into the TCR α constant (TRAC) locus with TCR β constant (TRBC) knockout, thus avoiding TCRαβ mispairing and maximizing TCR expression and function. The engineered lymphocytes were enriched in memory stem T cells. A unique WT137-45-specific TCR showed antigen-specific responses and efficiently killed AML blasts, acute lymphoblastic leukemia blasts, and glioblastoma cells in vitro and in vivo in the absence of off-tumor toxicity. T cells engineered to express this receptor are being advanced into clinical development for AML immunotherapy and represent a candidate therapy for other WT1-expressing tumors.

Elucidation of CRISPR-Cas9 application in novel cellular immunotherapy.

Novel cellular immunotherapy with engineered T cells has improved cancer treatment and established therapeutic promises to prevent tumor formation in clinical studies. Due to certain restrictions and difficulties, CAR and TCR T-cells therapies were inadequate at points. CRISPR Cas9 genome-editing tool has significant potential for these two cell-based therapies. As a specialized gene-editing technique, CRISPR Cas9 is used to repair genetic alternations with minimal damage. It is used as an adjunct to immunotherapy to stimulate a more robust immune response. CRISPR has long outpaced other target-specific genome editing methods such as ZFNs and TALEN because of its high efficiency, competence in targeting, and stable operating conditions. CRISPR can overcome the two major drawbacks of universal CAR T cells: allorejection and graft-vs-host disease. TCR-based T cell treatment can reduce inappropriate binding between endogenous and transgenic TCR, resulting in a reduction of severe toxicity. The CAR and TCR T based cell therapies uphold an excellent future for tumor malignancies. This article has elucidated the administration of CRISPR Cas9 in novel cellular immunotherapy, CAR, and TCR T cell therapy. However, this article did not fail to observe this technology's ethical concerns, limitations, and challenges. Furthermore, the article compares CRISPR-mediated allogeneic CAR T cell to TCR-T cell therapy.

CRISPRroots: on- and off-target assessment of RNA-seq data in CRISPR-Cas9 edited cells.

The CRISPR-Cas9 genome editing tool is used to study genomic variants and gene knockouts, and can be combined with transcriptomic analyses to measure the effects of such alterations on gene expression. But how can one be sure that differential gene expression is due to a successful intended edit and not to an off-target event, without performing an often resource-demanding genome-wide sequencing of the edited cell or strain? To address this question we developed CRISPRroots: CRISPR–Cas9-mediated edits with accompanying RNA-seq data assessed for on-target and off-target sites. Our method combines Cas9 and guide RNA binding properties, gene expression changes, and sequence variants between edited and non-edited cells to discover potential off-targets. Applied on seven public datasets, CRISPRroots identified critical off-target candidates that were overlooked in all of the corresponding previous studies. CRISPRroots is available via https://rth.dk/resources/crispr.

Engineering Resistance Against Viruses in Field Crops Using CRISPR- Cas9.

Food security is threatened by various biotic stresses that affect the growth and production of agricultural crops. Viral diseases have become a serious concern for crop plants as they incur huge yield losses. The enhancement of host resistance against plant viruses is a priority for the effective management of plant viral diseases. However, in the present context of the climate change scenario, plant viruses are rapidly evolving, resulting in the loss of the host resistance mechanism. Advances in genome editing techniques, such as CRISPR-Cas9 [clustered regularly interspaced palindromic repeats-CRISPR-associated 9], have been recognized as promising tools for the development of plant virus resistance. CRISPR-Cas9 genome editing tool is widely preferred due to high target specificity, simplicity, efficiency, and reproducibility. CRISPR-Cas9 based virus resistance in plants has been successfully achieved by gene targeting and cleaving the viral genome or altering the plant genome to enhance plant innate immunity. In this article, we have described the CRISPR-Cas9 system, mechanism of plant immunity against viruses and highlighted the use of the CRISPR-Cas9 system to engineer virus resistance in plants. We also discussed prospects and challenges on the use of CRISPR-Cas9-mediated plant virus resistance in crop improvement.

Simplified All-In-One CRISPR-Cas9 Construction for Efficient Genome Editing in Cryptococcus Species.

Cryptococcus neoformans and Cryptococcus deneoformans are opportunistic fungal pathogens found worldwide that are utilized to reveal mechanisms of fungal pathogenesis. However, their low homologous recombination frequency has greatly encumbered genetic studies. In preliminary work, we described a ‘suicide’ CRISPR-Cas9 system for use in the efficient gene editing of C. deneoformans, but this has not yet been used in the C. neoformans strain. The procedures involved in constructing vectors are time-consuming, whether they involve restriction enzyme-based cloning of donor DNA or the introduction of a target sequence into the gRNA expression cassette via overlap PCR, as are sophisticated, thus impeding their widespread application. Here, we report the optimized and simplified construction method for all-in-one CRISPR-Cas9 vectors that can be used in C. neoformans and C. deneoformans strains respectively, named pNK003 (Genbank: MW938321) and pRH003 (Genbank: KX977486). Taking several gene manipulations as examples, we also demonstrate the accuracy and efficiency of the new simplified all-in-one CRISPR-Cas9 genome editing tools in both Serotype A and Serotype D strains, as well as their ability to eliminate Cas9 and gDNA cassettes after gene editing. We anticipate that the availability of new vectors that can simplify and streamline the technical steps for all-in-one CRISPR-Cas9 construction could accelerate genetic studies of the Cryptococcus species.

CRISPeering: Bioengineering the Host Cells through CRISPRCas9 Genome Editing System as the Next-generation of Cell Factories.

Nowadays, the CRISPR-Cas9 genome editing system has become a popular bioengineering-based tool for various applications. Owing to its high-target specificity, efficiency, versatility, and simplicity, it has gained attention as a robust tool for molecular biology research, which unveils the biological functions of unexplored genes and engineers the metabolic pathways. Chinese hamster ovary (CHO) cells and Escherichia coli are regarded as the most commonly used expression platforms for industrial- scale production of recombinant proteins. The emergence of the CRISPR-Cas9 genome editing system promotes the current status of expression hosts towards controllable and predictable strains. This paper presents the current status of expression hosts for biopharmaceutical production. Some major accomplishments in the utilization of the CRISPR-Cas9 genome editing tool in the different prokaryotic and eukaryotic systems are discussed, and more importantly, the future directions of this newly arrived technology to make the next-generation cell factories with improved or novel properties are suggested. Moreover, the challenges faced in recent patents in this field are also discussed. The CRISPR-Cas9 genome-editing tool has been adopted to be utilized in some major expression platforms. CRISPeering has been successfully employed for genome editing in different prokaryotic and eukaryotic host cells. The emergence of systems metabolic engineering, systems biology, and synthetic biology fortify the current situation of the CRISPR-Cas9 genome editing system.

Understanding the role of Beclin1 in mouse embryonic stem cell differentiation through CRISPR-Cas9-mediated gene editing

Autophagy is a vacuolar pathway for the regulated degradation and recycling of cellular components. Beclin1, a Bcl2-interacting protein, is a well-studied autophagy regulator. Homozygous loss of Beclin1 in mice leads to early embryonic lethality. However, the role of Beclin1 in regulating the pluripotency of embryonic stem cells and their differentiation remains poorly explored. To study this, we generated Beclin1-Knockout (KO) mouse embryonic stem cells (mESCs) using the CRISPR-Cas9 genome-editing tool. Interestingly, Beclin1-KO mESCs did not show any change in the expression of pluripotency marker genes. Beclin1-KO mESCs also displayed active autophagy, suggesting the presence of Beclin1-independent autophagy in mESCs. However, loss of Beclin1 resulted in compromised differentiation of mESCs in vitro and in vivo due to misregulated expression of transcription factors. Our results suggest that Beclin1 may play an autophagy-independent role in regulating the differentiation of mESCs.

Nobel Prize in Chemistry 2020: The great breakthrough from frontier research in microbiology

The 2020 Nobel Prize in Chemistry was awarded to Emmanuelle Charpentier and Jennifer A. Doudna for their joint development of the highly efficient CRISPR-Cas9 genome editing tools, which have taken the life sciences into a new era. The CRISPR-Cas system was first discovered in bacteria in 1987. After more than 20 years of arduous exploration of these enigmatic systems that were found in more than 90% archaea and about half bacteria, their antiviral function was finally confirmed in 2007. This has inspired more researchers to focus on the CRISPR-Cas systems, of which the general anti-virus mechanisms were also uncovered within the next few years, that the crRNA containing the virus sequence information guides the Cas protein(s) to recognize the corresponding virus and cleave their DNA or RNA. Among those pioneers in this field, Charpentier and Doudna mainly focused on the type II CRISPR-Cas9 system, which is much simpler than the wide-spread type I systems. In 2012, Doudna and Charpentier had elucidated the fine structure of the anti-virus effector complex of the CRISPR-Cas9 system, which contains a single Cas9 protein and two RNA components (crRNA and tracrRNA). It was this work that led to the breakthrough in developing the CRISPR-Cas9 genome editing tools. Significantly, they innovatively connected the tracrRNA and crRNA into a single RNA chimera (sgRNA), greatly simplifying the genome editing tools to only two components, the sgRNA and the single Cas9 protein. Compared with the genome editing tools developed in the past (Meganuclease, ZFN and TALEN), CRISPR-Cas9 has significant advantages such as easy programming, high efficiency and a wide application range. These merits make CRISPR-Cas9 so far the most widely used genome editing technique. In basic research, CRISPR-Cas9 genome editing has been used to elucidate the functions of unknown genes or pathways. In application, this technique has greatly improved the efficiency of animal and plant breeding, and made it possible to cure some genetic diseases and even cancers. Moreover, based on this CRISPR-Cas9 platform, other advanced genome editing tools have also been developed, such as base editing and prime editing, etc. It is also believed that, in addition to CRISPR-Cas systems, there are still many other undiscovered antiviral systems in microbes, as well as a large number of unknown nucleic acid recognition and processing elements, which are expected to optimize or upgrade the current genome editing tools, or even innovate entirely new genome editing tools. The CRISPR-Cas genome editing tools were developed from the bacterial anti-virus immune systems, thus the 2020 Nobel Prize in Chemistry is essentially a reward to the frontier research in microbiology. In this article, we will briefly introduce i) the timeline for the development of genome editing tools; ii) the historical background of the CRISPR-Cas research; iii) the breakthrough works for the birth of CRISPR-Cas9 genome editing technology. The future frontier research in microbiology and big data mining to further optimize or innovate genome editing tools is also prospected.

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.

Detection of preQ0 deazaguanine modifications in bacteriophage CAjan DNA using Nanopore sequencing reveals same hypermodification at two distinct DNA motifs.

In the constant evolutionary battle against mobile genetic elements (MGEs), bacteria have developed several defense mechanisms, some of which target the incoming, foreign nucleic acids e.g. restriction-modification (R-M) or CRISPR-Cas systems. Some of these MGEs, including bacteriophages, have in turn evolved different strategies to evade these hurdles. It was recently shown that the siphophage CAjan and 180 other viruses use 7-deazaguanine modifications in their DNA to evade bacterial R-M systems. Among others, phage CAjan genome contains a gene coding for a DNA-modifying homolog of a tRNA-deazapurine modification enzyme, together with four 7-cyano-7-deazaguanine synthesis genes. Using the CRISPR-Cas9 genome editing tool combined with the Nanopore Sequencing (ONT) we showed that the 7-deazaguanine modification in the CAjan genome is dependent on phage-encoded genes. The modification is also site-specific and is found mainly in two separate DNA sequence contexts: GA and GGC. Homology modeling of the modifying enzyme DpdA provides insight into its probable DNA binding surface and general mode of DNA recognition.

The Improvement of CRISPR-Cas9 System With Ubiquitin-Associated Domain Fusion for Efficient Plant Genome Editing.

Genome editing technology represented by CRISPR-Cas9 had been widely used in many biological fields such as gene function analysis, gene therapy, and crop improvement. However, in the face of the complexity of the eukaryotic genome, the CRISPR-Cas9 genome editing tools have shown an unstable editing efficiency with large variability at different target sites. It was important to further improve the editing efficiency of the CRISPR-Cas9 system among the whole genome. In this study, based on the previous single transcription unit genome editing system (STU-SpCas9), using the ubiquitin-associated domain (UBA) to enhance the stability of Cas9 protein, we constructed three Cas9-UBA fusion systems (SpCas9-SD01, SpCas9-SD02, and SpCas9-SD03). Four different target sites of rice OsPDS, OsDEP1 and OsROC5 genes were chosen to evaluate the genome editing efficiency in rice protoplasts and stable transformed rice plants. The results showed that the fusion of UBA domains did not affect the cleavage mode of Cas9 protein, and effectively increase the editing efficiency of STU-SpCas9 at the target sites. This new CRISPR-Cas9-UBA system provided a new strategy and tool for improving the genome editing efficiency of CRISPR-Cas9 in plants.

NCAPG Is a Promising Therapeutic Target Across Different Tumor Types.

BackgroundWith the advent of CRISPR-Cas9 genome editing tool in gene therapy, identification of aberrantly expressed genes is of great value across various cancer types. Since a large number of patients may benefit from molecular targeted gene therapy. The purpose of this study was to identify aberrantly expressed genes across various cancer types, analyze prospective mechanisms and their correlation with survival outcomes.ResultsNCAPG was highly expressed in The Cancer Genome Atlas (TCGA) database, which includes the transcriptomes of 6,647 cancer and 647 normal tissue samples from 16 cancer types. Furthermore, a predicted NCAPG overexpression rate was also observed at the protein level in 16 tumor types. Importantly, high NCAPG level was significantly associated with unfavorable survival in various cancer types such as hepatocellular carcinoma (HCC), breast, lung or ovarian cancer. The multivariate analyses demonstrated that NCAPG, TNM, and Barcelona Clinic Liver Cancer (BCLC) staging were independent risk factors for mortality of patients with HCC. Moreover, functional and pathway enrichment analysis suggested that NCAPG was closely correlated with the pathways of cell cycle, cellular senescence, and mismatch repair. By weighted gene co-expression network analysis (WGCNA), we identified NCAPG as a hub gene in the turquoise module mostly related to the survival time of HCC samples.ConclusionTo our knowledge, this study represents a comprehensive RNA-Seq analysis of several tumor types, revealing NCAPG as a promising molecular target. NCAPG overexpression may play important roles in carcinogenesis and progression of tumors via regulating tumor-related pathways, thereby broadening the understanding of the pathogenic mechanisms and highlighting the possibility of developing novel targeted therapeutics.

Introduction to the Special Issue on CRISPR.

Introduction to the Special Issue on CRISPR George Q. Daley As i was finalizing this introduction to the Special Issue on CRISPR genome editing for Perspectives in Biology and Medicine, news broke that the Chinese scientist He Jiankui had been sentenced in Chinese court to three years in prison for "illegal medical practice" for his role in the creation of the world's first genome-edited babies. This official reprimand reinforced the worldwide condemnation and censure that followed He's announcement in November 2018 that his team at the Southern University of Science and Technology in Shenzhen, China, had used the CRISPR-Cas9 genome-editing tools on human embryos created via in vitro fertilization. He produced and brought to term two babies (and later a third) carrying mutations in the CCR5 gene, a critical co-receptor for HIV, in hopes of rendering these babies immune to infection with the virus that causes AIDS. The work was criticized as premature practice of an unproven and controversial biomedical technology, and vilified for its flouting of widely promulgated international guidelines. Numerous expert committees that had called for restraint, further research to understand the potential risks, and wider social dialogue to determine which, if any, of the many possible genetic changes that could be introduced into the human germline might one day be [End Page 1] deemed permissible. Scientists, bioethicists, and laypeople who had previously been at odds over whether genome editing of human embryos for the purpose of altering an individual's entire genetic constitution—and by virtue of passage through the reproductive germline, that of all of their offspring—should ever be practiced, were virtually united that the world's first experiment in human germline genome editing had been a gross violation of medical and bioethical norms. While the imprisonment of the first practitioner of human germline genome editing will likely dampen enthusiasm that might have been building in some quarters, an IVF clinic in Russia has boldly announced their intentions to create genome-edited babies, and application of this technology, however premature, may be inevitable. This volume is an important exercise in the critical dialogue that is essential if the potential application of this technology is ever to be managed for maximal benefit and minimal harm. Gene editing is not a new technology, nor is the genome editing of humans a novel concept barely entertained prior to the discovery of the CRISPR-Cas9 genome-editing toolkit. For more than a century and a half, dating from the time of Mendel's discovery of the rules of heredity, the later definition of the gene as units responsible for the generational passage of hereditary traits, and ultimately the discovery of DNA as the molecular vehicle of such inheritance, farmers and scientists have been engaged—wittingly, or unwittingly—in the manipulation of genetic traits through selective breeding or experimental interventions. The potential that scientists might one day prove capable of altering and defining human traits either chemically or genetically in the context of in vitro fertilization and embryo manipulation has long been the subject of critical commentary and fiction. Perhaps most notable was the publication in 1932 by Aldous Huxley of Brave New World, a dystopian view of a future society in which highly mechanized in vitro embryo manipulation produced babies preordained into specific castes of social strata. The modern era of gene editing began, arguably, in the Stanford laboratory of Paul Berg, who in 1972 published the creation of the world's first hybrid DNA molecule, a link between genes of the monkey tumor virus SV40 and a virus that infects bacteria, called lambda phage (Jackson, Symons, and Berg 1972). A natural next step was injecting this novel gene construct into the bacterial host Escherichia coli, a denizen of the human intestine. But Berg deferred this experiment because of concerns about the potential risks of propagating a monkey tumor virus within the human gut, and together with other scientists, he advocated for restrictions on certain types of recombinant DNA research (such as inserting toxins, antibiotic resistance, or cancer genes into bacteria) until safety concerns could be better addressed. Berg and colleagues organized a conference of scientists to address ways to ensure the...