Perform Gene Editing Research Articles

A novel method of Francisella infection of epithelial cells using HeLa cells expressing fc gamma receptor

BackgroundFrancisella tularensis, the causative agent of tularemia, is a facultative intracellular bacterium. Although the life cycle of this bacterium inside phagocytic cells (e.g., macrophages, neutrophils) has been well analyzed, the difficulty of gene silencing and editing genes in phagocytic cells makes it difficult to analyze host factors important for the infection. On the other hand, epithelial cell lines, such as HeLa, have been established as cell lines that are easy to perform gene editing. However, the infection efficiency of Francisella into these epithelial cells is extremely low.MethodsIn order to facilitate the molecular biological analysis of Francisella infection using epithelial cells, we constructed an efficient infection model of F. tularensis subsp. novicida (F. novicida) in HeLa cells expressing mouse FcγRII (HeLa-FcγRII), and the system was applied to evaluate the role of host GLS1 on Francisella infection.ResultsAs a result of colony forming unit count, HeLa-FcγRII cells uptake F. novicida in a serum-dependent manner and demonstrated an approximately 100-fold increase in intracellular bacterial infection compared to parental HeLa cells. Furthermore, taking advantage of the gene silencing capability of HeLa-FcγRII cells, we developed GLS1, a gene encoding glutaminase, knockdown cells using lentiviral sh RNA vector and assessed the impact of GLS1 on F. novicida infection. LDH assay revealed that GLS1-knockdown HeLa-FcγRII cells exhibited increased cytotoxicity during infection with F. novicida compared with control HeLa-FcγRII cells. Furthermore, the cell death was inhibited by the addition of ammonia, the metabolite produced through glutaminase activity. These results suggest that ammonia plays an important role in the proliferation of F. novicida.ConclusionsIn this report, we proposed a new cell-based infection system for Francisella infection using HeLa-FcγRII cells and demonstrated its effectiveness. This system has the potential to accelerate cell-based infection assays, such as large-scale genetic screening, and to provide new insights into Francisella infection in epithelial cells, which has been difficult to analyze in phagocytic cells.

Targeted Integration of siRNA against Porcine Cytomegalovirus (PCMV) Enhances the Resistance of Porcine Cells to PCMV.

In the world's first pig-to-human cardiac cytomegalovirus (PCMV), xenotransplant and elevated levels of porcine key factors contributing to patient mortality were considered. This has renewed attention on PCMV, a virus widely prevalent in pigs. Currently, there are no effective drugs or vaccines targeting PCMV, and its high detection difficulty poses challenges for prevention and control research. In this study, antiviral small hairpin RNA (shRNA) was selected and inserted into the Rosa26 and miR-17-92 loci of pigs via a CRISPR/Cas9-mediated knock-in strategy. Further in vitro viral challenge experiments demonstrated that these genetically edited pig cells could effectively limit PCMV replication. Through this process, we constructed a PCMV-infected cell model, validated partial viral interference sites, enhanced gene knock-in efficiency, performed gene editing at two different gene loci, and ultimately demonstrated that RNA interference (RNAi) technology combined with CRISPR/Cas9 has the potential to generate pig cells with enhanced antiviral infection capabilities. This opens up possibilities for the future production of pig populations with antiviral functionalities.

Disrupting Protein Expression with Double-Clicked sgRNA-Cas9 Complexes: A Modular Approach to CRISPR Gene Editing.

CRISPR-Cas9 is currently the most versatile technique to perform gene editing in living organisms. In this approach, the Cas9 endonuclease is guided toward its DNA target sequence by the guide RNA (gRNA). Chemical synthesis of a functional single gRNA (sgRNA) is nontrivial because of the length of the RNA strand. Recently we demonstrated that a sgRNA can be stitched together from three smaller fragments through a copper-catalyzed azide-alkyne cycloaddition, making the process highly modular. Here we further advance this approach by leveraging this modulator platform by incorporating chemically modified nucleotides at both ends of the modular sgRNA to increase resistance against ribonucleases. Modified nucleotides consisted of a 2'-O-Me group and a phosphorothioate backbone in varying number at both the 5'- and 3'-ends of the sgRNA. It was observed that three modified nucleotides at both ends of the sgRNA significantly increased the success of Cas9 in knocking out a gene of interest. Using these chemically stabilized sgRNAs facilitates multigene editing at the protein level, as demonstrated by successful knockout of both Siglec-3 and Siglec-7 using two fluorophores in conjunction with fluorescence-activated cell sorting. These results demonstrate the versatility of this modular platform for assembling sgRNAs from small, chemically modified strands to simultaneously disrupt the gene expression of two proteins.

Twin Research and Human Genetics - News, Views & Comments: Memoirs and Autobiographies by Twins: Research Angles and Human Interest/Twin Research Reviews: Fetal Reduction in Twin Pregnancy; Twins' Personality and Military Service; Growth Restriction in Twins; Advances in Conjoined Twin Separation/Media Reports: Update on Scientist Who Performed Gene Editing on Twins; Twins From 33-Year-Old Embryos; Twins' Outcomes From Dietary Differences;

Twins' memoirs and autobiographies both enlighten and entertain. These works, often overlooked by researchers, may suggest new avenues for investigation, such as nonshared environmental events that propel twins in different directions. Of course, MZ twins' generally parallel experiences and DZ twins generally criss-crossing paths are the bases of fascinating life stories. The following sections examined recent research on fetal reduction in twin pregnancy, twins' personality and military service, growth restriction in twins, and advances in conjoined twin separation. This article closes with reports of a scientist who performed gene editing on twins, a twin conception from 33-year-old embryos, twins' physical outcomes from dietary differences, fraternal twins with the world's largest height difference and the Twin Home Experts who conquer rat infestation in New York.

Beyond Busulfan: Building a Better Conditioner for Bone Marrow Transplantation

Beyond Busulfan: Building a Better Conditioner for Bone Marrow Transplantation

Translational and rotational diffusion of short ribonucleic acids

Inevitable precondition for ribonucleic acids to regulate gene expression and to perform gene editing is diffusion. Free three-dimensional translational diffusion velocity of RNA of up to 200 nucleotides could be predicted with high accuracy by the empirical model D = 4.58 10−10 N−0.39 m2s−1. Furthermore, the biological function of ribonucleic acids is determined by rotational diffusion. In the presented work, an empirical model is derived applying atom-level shell-modeling of electron density maps, Dr = 1.62 109 N−1.20 s−1, to predict the rotational diffusion coefficient of short ribonucleic acids based on the polymer size.

Deep learning - cancer genetics and application of deep learning to cancer oncology

Arguably the human body has been one of the most sophisticated systems we encounter but until now we are still far from understanding its complexity. We have been trying to replicate human intelligence by way of artificial intelligence but with limited success. We have discovered the molecular structure in terms of genetics, performed gene editing to change an organism’s DNA and much more, but their translatability into the field of oncology has remained limited. Conventional machine learning methods achieved some degree of success in solving problems that we do not have an explicit algorithm. However, they are basically shallow learning methods, not rich enough to discover and extract intricate features that represent patterns in the real environment. Deep learning has exceeded human performance in pattern recognition as well as strategic games and are powerful for dealing with many complex problems. High-throughput sequencing and microarray techniques have generated vast amounts of data and allowed the comprehensive study of gene expression in tumor cells. The application of deep learning with molecular data enables applications in oncology with information not available from clinical diagnosis. This paper provides fundamental concepts of deep learning, an essential knowledge of cancer genetics, and a review of applications of deep learning to cancer oncology. Importantly, it provides an insightful knowledge of deep learning and an extensive discussion on its challenges. The ultimate purpose is to germinate ideas and facilitate collaborations between cancer biologists and deep learning researchers to address challenging oncological problems using advanced deep learning technologies.

The characteristics of neutrophil extracellular traps produced by all-trans retinoic acid-induced dHL-60 under PMA stimulation

Extracellular traps released by neutrophils (neutrophil extracellular traps, NETs) are a double-edged sword, and understanding the mechanism of NET formation is of great significance for disease treatment. However, the short lifespan, the large individual differences, and the inability to perform gene editing render it difficult to decipher NET formation using neutrophils. It is necessary to find a model cell to replace neutrophils to study the mechanism of NET formation. In this study, we used different concentrations (0, 0.1, 1, and 10 μmol/L) of all-trans retinoic acid (ATRA) to differentiate HL-60 cells for different days (1, 3, 5, and 7 days). By detecting the cell viability and nuclear morphology of cells, we confirmed that HL-60 cells were differentiated to neutrophil-like cells (dHL-60) after treated with ATRA for at least 5 days. Using immunofluorescence staining to detect the formation of NETs, we demonstrated that dHL-60 cells differentiated for 5 days with 1 μmol/L ATRA could generate NETs comparable to those produced by neutrophils upon phorbol 12-myristate 13-acetate (PMA) stimulation, without histone H3 citrullination. Furthermore, the formation of NETs by dHL-60 cells were NADPH-dependent and PAD4-independent, consistent with neutrophils. Taken together, these observations suggest that dHL-60 cells differentiated with 1 μmol/L ATRA for 5 days can be used as a model cell for neutrophils to study the mechanism of NET formation.

Recent advancement in OMICS approaches to enhance abiotic stress tolerance in legumes.

The world is facing rapid climate change and a fast-growing global population. It is believed that the world population will be 9.7 billion in 2050. However, recent agriculture production is not enough to feed the current population of 7.9 billion people, which is causing a huge hunger problem. Therefore, feeding the 9.7 billion population in 2050 will be a huge target. Climate change is becoming a huge threat to global agricultural production, and it is expected to become the worst threat to it in the upcoming years. Keeping this in view, it is very important to breed climate-resilient plants. Legumes are considered an important pillar of the agriculture production system and a great source of high-quality protein, minerals, and vitamins. During the last two decades, advancements in OMICs technology revolutionized plant breeding and emerged as a crop-saving tool in wake of the climate change. Various OMICs approaches like Next-Generation sequencing (NGS), Transcriptomics, Proteomics, and Metabolomics have been used in legumes under abiotic stresses. The scientific community successfully utilized these platforms and investigated the Quantitative Trait Loci (QTL), linked markers through genome-wide association studies, and developed KASP markers that can be helpful for the marker-assisted breeding of legumes. Gene-editing techniques have been successfully proven for soybean, cowpea, chickpea, and model legumes such as Medicago truncatula and Lotus japonicus. A number of efforts have been made to perform gene editing in legumes. Moreover, the scientific community did a great job of identifying various genes involved in the metabolic pathways and utilizing the resulted information in the development of climate-resilient legume cultivars at a rapid pace. Keeping in view, this review highlights the contribution of OMICs approaches to abiotic stresses in legumes. We envisage that the presented information will be helpful for the scientific community to develop climate-resilient legume cultivars.

Transposons and CRISPR: Rewiring Gene Editing.

CRISPR-Cas is driving a gene editing revolution because of its simple reprogramming. However, off-target effects and dependence on the double-strand break repair pathways impose important limitations. Because homology-directed repair acts primarily in actively dividing cells, many of the current gene correction/replacement approaches are restricted to a minority of cell types. Furthermore, current approaches display low efficiency upon insertion of large DNA cargos (e.g., sequences containing multiple gene circuits with tunable functionalities). Recent research has revealed new links between CRISPR-Cas systems and transposons providing new scaffolds that might overcome some of these limitations. Here, we comment on two new transposon-associated RNA-guided mechanisms considering their potential as new gene editing solutions. Initially, we focus on a group of small RNA-guided endonucleases of the IS200/IS605 family of transposons, which likely evolved into class 2 CRISPR effector nucleases (Cas9s and Cas12s). We explore the diversity of these nucleases (named OMEGA, obligate mobile element-guided activity) and analyze their similarities with class 2 gene editors. OMEGA nucleases can perform gene editing in human cells and constitute promising candidates for the design of new compact RNA-guided platforms. Then, we address the co-option of the RNA-guided activity of different CRISPR effector nucleases by a specialized group of Tn7-like transposons to target transposon integration. We describe the various mechanisms used by these RNA-guided transposons for target site selection and integration. Finally, we assess the potential of these new systems to circumvent some of the current gene editing challenges.

Efficient Editing of SoCSLD2 by CRISPR/Cas9 Affects Morphogenesis of Root Hair in Spinach

CRISPR/Cas9 is a valuable tool and has been extensively employed to perform gene editing in plants. However, CRISPR/Cas9 has not been successfully used in spinach, an important leafy vegetable crop. Here, we established a CRISPR/Cas9-based gene-editing system for spinach hairy roots and edited two cellulose synthase-like D (CSLD) genes (SoCSLD2 and SoCSLD3) that were involved in root-hair formation of spinach hairy roots. Four mutation types (i.e., replacement, insertion, deletion, and combined mutations) were observed, among which the replacement accounted for the vast majority (about 64.1%). Mutation rate differed largely among different targets. Seven homozygous/bi-allelic and eight heterozygous/chimeric mutants of SoCSLD2 were obtained from 15 independent transgenic hairy root lines. All of the seven homozygous/bi-allelic mutant lines displayed bulking and short root hairs, which resembled the characteristics of Arabidopsis atcsld2 mutants. The transcriptomic analysis further revealed that multiple gene expressions for cell-wall modulation and membrane trafficking were disturbed, which might result in the inhibition of root hair growth in socsld2 mutants. This indicates that SoCSLD2 was successfully knocked out in spinach root hairs using the CRISPR/Cas9-based gene editing system.

CRISPR/Cas9 (D10A) nickase-mediated Hb CS gene editing and genetically modified fibroblast identification

ABSTRACT This study investigated whether CRISPR/Cas9 (D10A) nickase-mediated gene editing can correct the aberrant Hb Constant Spring mutation (Hb CS or HBA2: c.427 T > C) in fibroblasts. Vectors for repairing the α-globin-encoding gene, HBA2:c.427 T > C mutation, includingthe CRISPR/Cas9(D10A)-sg plasmid and donor with homology arms, were constructed and used to perform gene editing in patient-derived fibroblasts. We subsequently analyzed the genetic correction, the gene editing efficiency and off-target effect. Sequencing analysis and the BamHI assay showed that HB CS mutant cells were repaired with Hb CS point mutations, the editing efficiency was 4.18%~9.34% and no off-target effects were detected. The results indicate that the HB CS mutant gene is effectively repaired by the CRISPR/Cas9 (D10A)system, which may enable truly personalized therapy for precise repair of α-thalassemia.

Development of a Two-Dimensional Liquid Chromatography-Mass Spectrometry Platform for Simultaneous Multi-Attribute Characterization of Adeno-Associated Viruses.

Adeno-associated viruses (AAVs) are non-enveloped, single-stranded DNA viruses that have recently emerged as an attractive vector for delivering genetic materials to hosts for gene therapy applications. Due to their ability to transduce a wide range of species and tissues in vivo, low risk of immunotoxicity, and mild innate and adaptive immune responses, AAVs are currently used in research and clinical studies as a monotherapy or with other biomolecules to perform gene editing, replacement, addition, and silencing. As AAVs are a new and complex therapeutic modality with molecular weights into the megadalton range, new analytical techniques are therefore needed to support process development, product characterization, and release. In this study, an online two-dimensional liquid chromatography-mass spectrometry (2DLC-MS) method was developed for AAV characterization. Our method uses high-resolution anion-exchange chromatography (AEX) in the first dimension to separate and measure empty and full capsids in AAV samples, followed by reversed-phase liquid chromatography coupled with mass spectrometry (RPLC-MS) to separate and characterize viral proteins. In this technique, online denaturation and removal of MS-incompatible salt were performed following AEX. The viral proteins present in the peak of interest after first-dimensional AEX are subjected to intact protein separation on the second-dimensional RPLC column and then characterized by MS. The 2DLC-MS method demonstrated in this study allows for high-throughput and multi-attribute AAV characterization in a single run, with minimal sample handling required for different AAV serotypes.

SNP rs4971059 predisposes to breast carcinogenesis and chemoresistance via TRIM46-mediated HDAC1 degradation.

Identification of the driving force behind malignant transformation holds the promise to combat the relapse and therapeutic resistance of cancer. We report here that the single nucleotide polymorphism (SNP) rs4971059, one of 65 new breast cancer risk loci identified in a recent genome-wide association study (GWAS), functions as an active enhancer of TRIM46 expression. Recreating the G-to-A polymorphic switch caused by the SNP via CRISPR/Cas9-mediated homologous recombination leads to an overt upregulation of TRIM46. We find that TRIM46 is a ubiquitin ligase that targets histone deacetylase HDAC1 for ubiquitination and degradation and that the TRIM46-HDAC1 axis regulates a panel of genes, including ones critically involved in DNA replication and repair. Consequently, TRIM46 promotes breast cancer cell proliferation and chemoresistance invitro and accelerates tumor growth invivo. Moreover, TRIM46 is frequently overexpressed in breast carcinomas, and its expression is correlated with lower HDAC1 expression, higher histological grades, and worse prognosis of the patients. Together, our study links SNP rs4971059 to replication and to breast carcinogenesis and chemoresistance and support thepursuit of TRIM46 as a potential target for breast cancer intervention.

Attaining the promise of plant gene editing at scale

Crop improvement relies heavily on genetic variation that arises spontaneously through mutation. Modern breeding methods are very adept at combining this genetic variation in ways that achieve remarkable improvements in plant performance. Novel traits have also been created through mutation breeding and transgenesis. The advent of gene editing, however, marks a turning point: With gene editing, synthetic variation will increasingly supplement and, in some cases, supplant the genetic variation that occurs naturally. We are still in the very early stages of realizing the opportunity provided by plant gene editing. At present, typically only one or a few genes are targeted for mutation at a time, and most mutations result in loss of gene function. New technological developments, however, promise to make it possible to perform gene editing at scale. RNA virus vectors, for example, can deliver gene-editing reagents to the germ line through infection and create hundreds to thousands of diverse mutations in the progeny of infected plants. With developmental regulators, edited somatic cells can be induced to form meristems that yield seed-producing shoots, thereby increasing throughput and shrinking timescales for creating edited plants. As these approaches are refined and others developed, they will allow for accelerated breeding, the domestication of orphan crops and the reengineering of metabolism in a more directed manner than has ever previously been possible.

MRNA-based therapies and their clinical prospects

<p indent="0mm">The striking effectiveness of messenger ribonucleic acid (mRNA) vaccines against coronavirus disease 2019 (COVID-19) not only offers hope to end the current pandemic, but also presents mRNA-based therapeutics as a potential new treatment class that may help in the fight against other diseases. <italic>In vitro</italic>-transcribed (IVT) mRNA is engineered to structurally resemble naturally occurring mature and processed eukaryotic mRNA. Such synthetic mRNAs deliver genetic information that allows the translational machinery of the host cells to produce many copies of the encoded proteins, which can function as antigens to boost immune responses or as supplementary beneficial proteins, resulting in a therapeutic effect. With recent developments in mRNA <italic>in vivo</italic> delivery platforms and the improvement of modified regulatory systems, the stability and transfection efficiency of mRNA have been greatly improved. Modified regulatory systems utilize caps, 5′ and 3′ untranslated regions, open reading frames, Poly(A) tails, and chemically modified nucleotides. Notably, <italic>in vivo</italic> delivery platforms can either be a non-targeted or targeted delivery system. Currently, mRNA-based therapeutics, including vaccines and protein replacement therapy, are being clinically developed for cancers, infectious diseases, and rare diseases. Common mRNA cancer vaccines include dendritic cell mRNA cancer vaccine, naked mRNA injection, mRNA encoding CAR, and other combination therapies. There are two main types of mRNA vaccines against infectious diseases: Non-replicating and self-amplifying. In addition, mRNA protein replacement therapies are available; these are therapies in which body cells are transfected with mRNA, and the translated proteins are used to supplement the lack of proteins in the body or replace abnormal proteins that hinder normal cellular pathways. mRNA therapies are also used in treatment of rare genetic diseases, such as Fabry disease, methylmalonic acidemia, ornithine carbamoyltransferase deficiency, and acute intermittent porphyria. Furthermore, IVT mRNA encoding genome-editing nucleases has been used as a novel method to perform gene editing. mRNA therapies have the advantages of fast preparation, low production costs, and safety. However, there are still challenges to be overcome for the successful clinical application of mRNA-based therapies. For example, mRNA stability and target specificity still require improvement. In terms of stability, quality control needs to be improved and the negative impact of immunogenicity should be reduced. Quality control refers to improving the ability to detect residual template deoxyribonucleic acid, IVT reaction by-products, and incomplete mRNA. In addition, when comparing independent routes of administration, the variation between- and within individuals, particularly due to the mRNA dosage and protein effects has not been thoroughly studied. To understand the up-to-date development and research status of mRNA therapies, this review focuses on molecular design, delivery system, and clinical research status of mRNA therapies.

Adapting CRISPR/Cas9 System for Targeting Mitochondrial Genome.

Gene editing of the mitochondrial genome using the CRISPR-Cas9 system is highly challenging mainly due to sub-efficient delivery of guide RNA and Cas9 enzyme complexes into the mitochondria. In this study, we were able to perform gene editing in the mitochondrial DNA by appending an NADH-ubiquinone oxidoreductase chain 4 (ND4) targeting guide RNA to an RNA transport-derived stem loop element (RP-loop) and expressing the Cas9 enzyme with a preceding mitochondrial localization sequence. We observe mitochondrial colocalization of RP-loop gRNA and a marked reduction of ND4 expression in the cells carrying a 11205G variant in their ND4 sequence coincidently decreasing the mtDNA levels. This proof-of-concept study suggests that a stem-loop element added sgRNA can be transported to the mitochondria and functionally interact with Cas9 to mediate sequence-specific mtDNA cleavage. Using this novel approach to target the mtDNA, our results provide further evidence that CRISPR-Cas9-mediated gene editing might potentially be used to treat mitochondrial-related diseases.

Rational Selection of CRISPR-Cas Triggering Homology-Directed Repair in Human Cells.

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated) nucleases have been widely applied for genome engineering. Cas9 (Streptococcus pyogenes Cas9 [SpCas9] and Staphylococcus aureus Cas9 [SaCas9]) and Cpf1 (i.e., Francisella novicida U112 Cpf1 [FnCpf1], also named FnCas12a) were harnessed to perform gene editing in human cells. Precise genetic modification by homology-directed repair (HDR) is an attractive approach for in situ gene correction. However, so far, the comparative efficiencies of HDR mediated by different CRISPR orthologs remain unknown. To address this question, in this study, we developed a reporter system to investigate HDR efficiencies triggered by various CRISPR orthologs. We found that SpCas9 and SaCas9, the two most commonly used Cas9 enzymes, possessed a similar ability to induce HDR. Interestingly, with the increasing amount of coding plasmids or additional nuclear localization sequences, FnCpf1 could improve the HDR efficacy. Collectively, our study provides insights for the rational selection of appropriate tools for human genome manipulation.

CRSIPR/Cas9: Magic scissors for genome editing

The Nobel Prize in Chemistry 2020 was awarded jointly to Emmanuelle Charpentier and Jennifer A. Doudna “for the development of a method for genome editing.” Emmanuelle Charpentier and Jennifer A. Doudna have discovered one of gene technology’s sharpest tools: the CRISPR/Cas9 genetic scissors. Using this strategy, researchers can change the DNA of animals, plants and microorganisms with extremely high precision. This technology has had a revolutionary impact on the life sciences, is contributing to new cancer therapies and may make the dream of curing inherited diseases come true. The CRISPR sequence was first discovered in E. coli. by Japanese molecular biologist Yoshizumi Ishino in 1987, and the Spanish microbiologist Francisco Mojica further discovered unique non-repetitions in CRISPR in 2003. Later on, it has been realized that CRISPR is an adaptive immune system derived from bacteria. CRISPR can identify (via crRNA) viruses that invade bacteria, and use a special nuclease (Cas9 protein) to degrade the DNA sequence of the invading viruses, thereby protecting bacteria from viral invasion. Emmanuelle Charpentier discovered that there are some new small RNA molecules in Streptococcus pyogenes in the process of studying the regulatory RNA of Streptococcus pyogenes, whose genetic code partially matches the CRISPR sequence. Furthermore, they have discovered that these unknown RNA molecules (later known as trans-activated CRISPR RNA, tracrRNA) can help the long RNA molecules produced by the transcription of CRISPR sequences in the genome to be processed into mature, active RNA (crRNA). The first comprehensive analysis of the principles of CRISPR/Cas9 gene editing came from the collaboration of Emmanuelle Charpentier and Jennifer Doudna. They found that using recombinant S. pyogenes Cas9 protein and in vitro transcribed crRNA and tracrRNA, either purified DNA or E. coli gene could be efficiently edited. In addition, they proved that both crRNA and tracrRNA are necessary for Cas9 to be functional, and that the two RNAs can also function in vitro when they are fused into a single guide RNA (sgRNA). Shortly, the research group from MIT reported for the first time that CRISPR/Cas9 gene editing technology can be applied to gene editing in mammalian and human cells, thus promoting the explosive development of this technology. Using the CRISPR gene editing method, researchers can theoretically direct Cas9 nuclease to perform gene editing at any desired gene locus by designing different sgRNAs. Due to the simplicity, low-cost, and efficiency of this method, CRISPR has rapidly developed into the most mainstream gene editing technology, which is used to efficiently and accurately change, edit or replace plant, animal and even human genes. CRISPR/Cas9 gene editing technology has shown a great potential for biomedical applications, but it still associated with challenges that include biosafety, off-target effects of gene editing. For example, the human body may have an immune response to the gene-editing nuclease Cas9 derived from bacterial; it is very important to efficiently and safely introduce the CRISPR/Cas9 gene-editing system into living cells or in vivo . Viral vectors have been widely used in human gene therapy, but their loading capacity for Cas9 is greatly limited. Therefore, the development of new CRISPR/Cas9 delivery systems is particularly important for in vivo gene editing and its clinical transformation applications. To reduce off-target effects of CRISPR/Cas9 genome editing, site-directed mutation of Cas9 protein or the development of new gene editing technologies (such as base editing) are highly appreciated for promoting this technology into clinic.

Mutations in cardinal are responsible for the red-1 and peach eye color mutants of the red flour beetle Tribolium castaneum

Ommochromes are the major pigments found in the eyes, eggs, wings and epidermis of insects. Here, we report the identification and characterization of the gene responsible for red-1 locus of Tribolium, whose mutants have white eyes due to lack of ommochrome pigments in the eyes. Using a candidate gene approach, we demonstrated that red-1 and peach mutants have molecular defects in the cardinal gene, which encodes a haem peroxidase that is considered to convert 3-hydroxykynurenine into ommochromes in pigment granules. Our experiments showed that the expression pattern of cardinal correlates well with the progression of eye pigmentation during pupal stages. We performed gene editing experiments using the Receptor-Mediated Ovary Transduction of Cargo (ReMOT) Control technique to disrupt the cardinal gene by adult injection, and were able to establish a novel cardinal mutant line. Our complementation test provided definitive genetic evidence that cardinal is located at the red-1 locus. The present study will lead to a greater understanding of the function and diversity of ommochrome pathway genes in insects. Our successful use of ReMOT Control in beetles will facilitate the development of more efficient and versatile systems for insect genome editing by simple adult injection.