Targeted Gene Knockout Research Articles

Production of Human CRISPR-Engineered CAR-T Cells

Adoptive cell therapies using chimeric antigen receptor T cells (CAR-T cells) have demonstrated remarkable clinical efficacy in patients with hematological malignancies and are currently being investigated for various solid tumors. CAR-T cells are generated by removing T cells from a patient's blood and engineering them to express a synthetic immune receptor that redirects the T-cells to recognize and eliminate target tumor cells. Gene editing of CAR-T cells has the potential to improve safety of current CAR-T cell therapies and further increase the efficacy of CAR-T cells. Here, we describe methods for the activation, expansion, and characterization of human CRISPR-engineered CD19 directed CAR-T cells. This comprises transduction of the CAR lentiviral vector and use of single guide RNA (sgRNA) and Cas9 endonuclease to target genes of interest in T cells. The methods described in this protocol can be universally applied to other CAR constructs and target genes beyond the ones used for this study. Furthermore, this protocol discusses strategies for gRNA design, lead gRNA selection and target gene knockout validation to reproducibly achieve high-efficiency, multiplex CRISPR-Cas9 engineering of clinical grade human T cells.

A novel class of sulfur-containing aminolipids widespread in marine roseobacters

Marine roseobacter group bacteria are numerically abundant and ecologically important players in ocean ecosystems. These bacteria are capable of modifying their membrane lipid composition in response to environmental change. Remarkably, a variety of lipids are produced in these bacteria, including phosphorus-containing glycerophospholipids and several amino acid-containing aminolipids such as ornithine lipids and glutamine lipids. Here, we present the identification and characterization of a novel sulfur-containing aminolipid (SAL) in roseobacters. Using high resolution accurate mass spectrometry, a SAL was found in the lipid extract of Ruegeria pomeroyi DSS-3 and Phaeobacter inhibens DSM 17395. Using comparative genomics, transposon mutagenesis and targeted gene knockout, we identified a gene encoding a putative lyso-lipid acyltransferase, designated salA, which is essential for the biosynthesis of this SAL. Multiple sequence analysis and structural modeling suggest that SalA is a novel member of the lysophosphatidic acid acyltransferase (LPAAT) family, the prototype of which is the PlsC acyltransferase responsible for the biosynthesis of the phospholipid phosphatidic acid. SAL appears to play a key role in biofilm formation in roseobacters. salA is widely distributed in Tara Oceans metagenomes and actively expressed in Tara Oceans metatranscriptomes. Our results raise the importance of sulfur-containing membrane aminolipids in marine bacteria.

Automatic reconstruction of metabolic pathways from identified biosynthetic gene clusters

BackgroundA wide range of bioactive compounds is produced by enzymes and enzymatic complexes encoded in biosynthetic gene clusters (BGCs). These BGCs can be identified and functionally annotated based on their DNA sequence. Candidates for further research and development may be prioritized based on properties such as their functional annotation, (dis)similarity to known BGCs, and bioactivity assays. Production of the target compound in the native strain is often not achievable, rendering heterologous expression in an optimized host strain as a promising alternative. Genome-scale metabolic models are frequently used to guide strain development, but large-scale incorporation and testing of heterologous production of complex natural products in this framework is hampered by the amount of manual work required to translate annotated BGCs to metabolic pathways. To this end, we have developed a pipeline for an automated reconstruction of BGC associated metabolic pathways responsible for the synthesis of non-ribosomal peptides and polyketides, two of the dominant classes of bioactive compounds.ResultsThe developed pipeline correctly predicts 72.8% of the metabolic reactions in a detailed evaluation of 8 different BGCs comprising 228 functional domains. By introducing the reconstructed pathways into a genome-scale metabolic model we demonstrate that this level of accuracy is sufficient to make reliable in silico predictions with respect to production rate and gene knockout targets. Furthermore, we apply the pipeline to a large BGC database and reconstruct 943 metabolic pathways. We identify 17 enzymatic reactions using high-throughput assessment of potential knockout targets for increasing the production of any of the associated compounds. However, the targets only provide a relative increase of up to 6% compared to wild-type production rates.ConclusionWith this pipeline we pave the way for an extended use of genome-scale metabolic models in strain design of heterologous expression hosts. In this context, we identified generic knockout targets for the increased production of heterologous compounds. However, as the predicted increase is minor for any of the single-reaction knockout targets, these results indicate that more sophisticated strain-engineering strategies are necessary for the development of efficient BGC expression hosts.

CRISPR/Cas9-mediated knockout of the NlCSAD gene results in darker cuticle pigmentation and a reduction in female fecundity in Nilaparvata lugens (Hemiptera: Delphacidae)

In insects, cuticular pigmentation genes have been exploited as potential visible markers for constructing genetic manipulation systems. Here, we cloned cysteine sulfinic acid decarboxylase (CSAD), an orthologue of melanin metabolism pathway genes, and performed RNAi experiments in the brown planthopper Nilaparvata lugens (Hemiptera: Delphacidae). The results showed that a decrease in the level of transcription of NlCSAD increased melanin deposition in the body compared to the control group, resulting in darker cuticle pigmentation. Female adults treated with dsNlCSAD and mated with wild-type males laid significantly fewer eggs than the dsGFP-treated group, and lower hatchability of the eggs was also observed. In addition, two melanic mutant N. lugens strains (NlCSAD−/+ and NlCSAD−/−) constructed by the CRISPR/Cas9 genome editing system showed darker cuticular melanisation and a reduced oviposition and hatching rate, but the homozygotes had a darker body colour, fewer eggs and lower hatchability than heterozygotes or individuals after RNAi. Thus, we have provided the first evidence that NlCSAD is required for normal body pigmentation in adults and has a role in the fecundity of females and hatchability of eggs in N. lugens via a combination of RNAi and knockout of target genes based on the CRISPR/Cas9 genome editing system. Our results suggest that NlCSAD is a candidate visual reference gene for genetic manipulation of this important crop pest.

Serine/threonine Kinases Play Important Roles in Regulating Polyunsaturated Fatty Acid Biosynthesis in Synechocystis sp. PCC6803.

Serine/threonine kinases (STKs) play important roles in prokaryotic cellular functions such as growth, differentiation, and secondary metabolism. When the external environment changes, prokaryotes rely on signal transduction systems, including STKs that quickly sense these changes and alter gene expression to induce the appropriate metabolic changes. In this study, we examined the roles of the STK genes spkD and spkG in fatty acid biosynthesis in the unicellular cyanobacterium Synechocystis sp. PCC6803, using targeted gene knockout. The linoleic acid (C18: 2), γ-linolenic acid (C18: 3n6), α-linolenic acid (C18: 3n3), and stearidonic acid (C18: 4) levels were significantly lower in spkD and spkG gene knockout mutants than in the wild type at a culture temperature of 30°C and a light intensity of 40 μmol⋅m–2⋅s–1. The expression levels of fatty acid desaturases and STK genes differed between the spkD and spkG gene knockout mutants. These observations suggest that spkD and spkG may directly or indirectly affect the fatty acid composition in Synechocystis sp. PCC6803 by regulating the expression of fatty acid desaturases genes. Therefore, the STK genes spkD and spkG play important roles in polyunsaturated fatty acid biosynthesis in Synechocystis sp. PCC6803. These findings could facilitate the development of cyanobacteria germplasm resources that yield high levels of fatty acids. In addition, they provide a theoretical basis for the genetic engineering of cyanobacteria with improved yields of secondary metabolites and increased economic benefits.

Assisted Reproductive Techniques and Genetic Manipulation in the Common Marmoset.

Genetic modification of nonhuman primate (NHP) zygotes is a useful method for the development of NHP models of human diseases. This review summarizes the recent advances in the development of assisted reproductive and genetic manipulation techniques in NHP, providing the basis for the generation of genetically modified NHP disease models. In this study, we review assisted reproductive techniques, including ovarian stimulation, in vitro maturation of oocytes, in vitro fertilization, embryo culture, embryo transfer, and intracytoplasmic sperm injection protocols in marmosets. Furthermore, we review genetic manipulation techniques, including transgenic strategies, target gene knock-out and knock-in using gene editing protocols, and newly developed gene-editing approaches that may potentially impact the production of genetically manipulated NHP models. We further discuss the progress of assisted reproductive and genetic manipulation techniques in NHP; future prospects on genetically modified NHP models for biomedical research are also highlighted.

The DEAD-box RNA helicase eIF4A1 interacts with the SWI2/SNF2-related chromatin remodelling ATPase DDM1 in the moss Physcomitrella

eIF4A is a DEAD box containing RNA helicase that plays crucial roles in regulating translation initiation, growth and abiotic stress tolerance in plants. It also functions as an ATP-dependent RNA binding protein to curb granule formation by limiting RNA-RNA interactions that promote RNA condensation and formation of ribonucleoprotein particles in vivo. Helicase activity of eIF4A is known to be dictated by its binding partners. Proteins interacting with eIF4A have been identified across land plants. In monocots a close link between eIF4A regulated processes and DNA methylation in epigenetic regulation of plant development is inferred from interaction between OseIF4A and the de novo methyltransferase OsDRM2 and loss-of-function studies of these genes in Oryza sativa and Brachypodium distachyon. In the moss Physcomitrella patens, eIF4A1 encoded by Pp3c6_1080V3.1 interacts with the heterogeneous nuclear ribonucleoprotein (hnRNP) PpLIF2L1, homolog of which in Arabidopsis regulates transcription of stress-responsive genes. In this study, using different protein-protein interaction methods, targeted gene knockout strategy and quantitative expression analysis we show genetic interaction between PpeIF4A1 and the putative nucleosome remodeler protein PpDDM1 and between PpDDM1 and PpLIF2L1 in vivo. Stress-induced co-expression of PpeIF4A1, PpDDM1 and PpLIF2L1, their roles in salt stress tolerance and differences in subnuclear distribution of PpLIF2L1 in ppeif4a1 cells in comparison to wild type suggest existence of a regulatory network comprising of RNA helicases, chromatin remodelling proteins and hnRNP active in stress-responsive biological processes in P. patens.

Two Plasmid-Based Systems for CRISPR/Cas9 Mediated Knockout of Target Genes.

CRISPR/Cas9-based gene editing is a recent advance that allows for the knockout or alteration of target genes within mammalian cells. Many variations of the technique exist, but here we describe two systems of plasmid-based CRISPR gene knockout which together allow for the selective knockout of virtually any gene target. Compared with other CRISPR-based systems, these plasmids have the advantages of delivering all the necessary components in one plasmid, choice of multiple selectable markers, and choice of route of administration into target cells. In addition, potential off-target effects from one system (dependent upon selection of target gene) can be overcome through use of the second system. Strategies for optimizing the knockout process and selection of finished cell lines are also presented.

StKU80, a component in the NHEJ repair pathway, is involved in mycelial morphogenesis, conidiation, appressorium development, and oxidative stress reactions in Exserohilum turcicum

Homologous recombination (HR) and nonhomologous end joining (NHEJ) are considered the two main double-strand break (DSB) repair approaches in eukaryotes. Inhibiting the activities of the key component in NHEJ commonly enhances the efficiency of targeted gene knockouts or affects growth and development in higher eukaryotes. However, little is known about the roles of the NHEJ pathway in foliar pathogens. Here we identified a gene designated StKU80, which encodes a putative DNA end-binding protein homologous to yeast Ku80, in the foliar pathogen Exserohilum turcicum. Conserved domain analysis showed that the typical domains VWA, Ku78 and Ku-PK-bind are usually present in Ku70/80 proteins in eukaryotes and are also present in StKu80. Phylogenetic analysis indicated that StKu80 is most closely related to Ku80 (XP_001802136.1) from Parastagonospora nodorum, followed by Ku80 (AGF90044.1) from Monascus ruber. Furthermore, the gene knockout mutants ΔStKU80-1 and ΔStKU80-2 were obtained. These mutants displayed longer septas, thinner cell walls, smaller amounts of substances on cell wall surfaces, and more mitochondria per cell than the wild-type (WT) strain but similar HT-toxin activity. The mutants did not produce conidia and mature appressoria. On the other hand, the mutants were highly sensitive to H2O2, but not to ultraviolet radiation. In summary, the StKU80 plays devious roles in regulating the development of E. turcicum.

Rab-E and its interaction with myosin XI are essential for polarised cell growth.

The fundamental process of polarised exocytosis requires the interconnected activity of molecular motors trafficking vesicular cargo within a dynamic cytoskeletal network. In plants, few mechanistic details are known about how molecular motors, such as myosin XI, associate with their secretory cargo to support the ubiquitous processes of polarised growth and cell division. Live-cell imaging coupled with targeted gene knockouts and a high-throughput RNAi assay enabled the first characterisation of the loss of Rab-E function. Yeast two-hybrid and subsequent in silico structural prediction uncovered a specific interaction between Rab-E and myosin XI that is conserved between P.patens and A.thaliana. Rab-E co-localises with myosin XI at sites of active exocytosis, and at the growing tip both proteins are spatiotemporally coupled. Rab-E is required for normal plant growth in P.patens and the rab-E and myosin XI phenotypes are rescued by A.thaliana's Rab-E1c and myosin XI-K/E, respectively. Both PpMyoXI and AtMyoXI-K interact with PpRabE14, and the interaction is specifically mediated by PpMyoXI residue V1422. This interaction is required for polarised growth. Our results suggest that the interaction of Rab-E and myosin XI is a conserved feature of polarised growth in plants.

Adenylate Kinase 2 Is a Selective Dependency in NSD2-High Multiple Myeloma

Background: Multiple myeloma (MM)-associated t (4;14) chromosomal translocation leads to overexpression of NSD2, the histone H3 lysine 36 specific methyltransferase. t(4;14) MM patients have a high risk of relapse and NSD2 overexpression drives an oncogenic epigenetic and transcriptional program promoting clonogenicity, proliferation, altered adhesion and chemoresistance in MM cells. The lack of a specific and potent NSD2 inhibitors mandates finding alternative strategies for treating NSD2-high MM. Aim: This study aims to test the hypothesis that NSD2 overexpression in MM cells generates cellular vulnerabilities that can be therapeutically exploited for treatment of t (4;14) MM. Methods: We conducted a genome wide CRISPR-based loss-of-function genetic screen using the human Brunello library in isogenic NSD2-high (NTKO) and NSD2-low (TKO) KMS-11 derived MM cells to define genes whose loss is selectively detrimental to cells with NSD2 overexpression. The cellular dependency of each identified candidate was then investigated across hundreds of human cell lines using the Cancer Dependency Map portal (www.Depmap.org). Candidate genes were validated using CRISPR-Cas9 gene knockout and shRNA knockdown of individual target genes followed by in vitro competitive growth assays and cell viability assays. Results: Our study revealed multiple candidate genes with increased dependency in NSD2-high cells including the adenine nucleotide regulator Adenylate Kinase 2 (AK2). AK2 catalyzes the reversible conversion of ADP to AMP and ATP and can thus modulates energy balance within the cell. Dependency map analysis showed that AK2 is not a commonly essential gene. The top enriched lineages with AK2 dependency included MM with notable representation of t(4;14)-positive MM cell lines. The increased dependency of NTKO and other t (4;14) MM cells on AK2 was confirmed by in vitro competition assays. Disruption of AK2 in TKO cells had a minimal effect on cellular fitness but the dependency on AK2 was restored upon engineered overexpression of NSD2 in these cells. In addition, NSD2-high cells displayed higher sensitivity to the proteasome inhibitor bortezomib than NSD2-low cells suggesting elevated levels of endoplasmic reticulum (ER) stress in cells overexpressing NSD2. Elevated ER stress necessitates increased levels of ATP to refold proteins and could underlie the increased dependency of NSD2-high cells on AK2. Notably, suppression of AK2 increased bortezomib sensitivity in t (4;14) MM cell lines. Conclusions: Our findings indicate that NSD2 high t(4;14) MM may have a vulnerability due to increased proteostatic stress. Accordingly, AK2 inhibition could be used in combination with proteasome inhibitors to treat MM patients with t (4;14) translocations by inducing the accumulation of lethal levels of unfolded proteins. Disclosures No relevant conflicts of interest to declare.

The lowest hanging fruit: Beneficial gene knockouts in past, present, and future crop evolution

It is now well-documented that gene knockout (loss-of-function) alleles have played a prominent role in crop evolution during domestication, diversification, and improvement. This includes dramatic examples from the very origins of agriculture to the monumental yield increases of the Green Revolution. Recent advances in the generation of whole genome sequence data and functional investigations into the molecular genetic basis of crop traits continue to reveal the existence of considerable beneficial loss of function in crop species. These discoveries now inspire contemporary efforts to use targeted gene knockouts powered by modern gene editing tools to accelerate crop breeding. Here we trace the history and future of loss of function as a powerful mechanism of crop evolution and review the reasons gene knockouts might have been particularly important in past crop evolution and why they continue to be a “lowest hanging fruit” for directing crop evolution through molecular breeding in pursuit of more productive, resilient, and nutritious crops.

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

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

A versatile bulk electrotransfection protocol for murine embryonic fibroblasts and iPS cells

Although electroporation has been widely accepted as the main gene transfer tool, there is still considerable scope to improve the electroporation efficiency of exogenous DNAs into primary cells. Here, we developed a square-wave pulsing protocol using OptiMEM-GlutaMAX for highly efficient transfection of murine embryonic fibroblasts (MEF) and induced pluripotency stem (iPS) cells using reporter genes as well as gRNA/Cas9-encoding plasmids. An electrotransfection efficiency of > 95% was achieved for both MEF and iPS cells using reporter-encoding plasmids. The protocol was efficient for plasmid sizes ranging from 6.2 to 13.5 kb. Inducing the error prone non-homologous end joining repair by gRNA/Cas9 plasmid transfection, a high rate of targeted gene knockouts of up to 98% was produced in transgenic cells carrying a single-copy of Venus reporter. Targeted deletions in the Venus transgene were efficiently (up to 67% deletion rate) performed by co-electroporation of two gRNA-encoding plasmids. We introduced a plasmid electrotransfection protocol which is straight-forward, cost-effective, and efficient for CRISPRing murine primary cells. This protocol is promising to make targeted genetic engineering using the CRISPR/Cas9 plasmid system.

A Site-Specific Integration Reporter System That Enables Rapid Evaluation of CRISPR/Cas9-Mediated Genome Editing Strategies in CHO Cells.

Targeted gene knockout and site-specific integration (SSI) are powerful genome editing techniques to improve the development of industrially relevant Chinese hamster ovary (CHO) cell lines. However, past efforts to perform SSI in CHO cells are characterized by low efficiencies. Moreover, numerous strategies proposed to boost SSI efficiency in mammalian cell types have yet to be evaluated head to head or in combination to appreciably boost efficiencies in CHO. To enable systematic and rapid optimization of genome editing methods, the SSIGNAL (site-specific integration and genome alteration) reporter system is developed. This tool can analyze CRISPR (clustered regularly interspaced palindromic repeats)/Cas9 (CRISPR-associated protein 9)-mediated disruption activity alone or in conjunction with SSI efficiency. The reporter system uses green and red dual-fluorescence signals to indicate genotype states within four days following transfection, facilitating rapid data acquisition via standard flow cytometry instrumentation. In addition to describing the design and development of the system, two of its applications are demonstrated by first comparing transfection conditions to maximize CRISPR/Cas9 activity and subsequently assessing the efficiency of several promising SSI strategies. Due to its sensitivity and versatility, the SSIGNAL reporter system may serve as a tool to advance genome editing technology.

Aromatic Polyketides from a Symbiotic Strain Aspergillus fumigatus D and Characterization of Their Biosynthetic Gene D8.t287.

The chemical investigation of one symbiotic strain, Aspergillus fumigatus D, from the coastal plant Edgeworthia chrysantha Lindl led to the isolation of eight compounds (1–8), which were respectively identified as rubrofusarin B (1), alternariol 9-O-methyl ether (2), fonsecinone D (3), asperpyrone A (4), asperpyrone D (5), fonsecinone B (6), fonsecinone A (7), and aurasperone A (8) by a combination of spectroscopic methods (1D NMR and ESI-MS) as well as by comparison with the literature data. An antimicrobial assay showed that these aromatic polyketides exhibited no remarkable inhibitory effect on Escherichia coli, Staphyloccocus aureus and Candida albicans. The genomic feature of strain D was analyzed, as well as its biosynthetic gene clusters, using antibiotics and Secondary Metabolite Analysis Shell 5.1.2 (antiSMASH). Plausible biosynthetic pathways for dimeric naphtho-γ-pyrones 3–8 were first proposed in this work. A non-reducing polyketide synthase (PKS) gene D8.t287 responsible for the biosynthesis of these aromatic polyketides 1–8 was identified and characterized by target gene knockout experiment and UPLC-MS analysis.

Flux Balance Analysis for Media Optimization and Genetic Targets to Improve Heterologous Siderophore Production.

SummarySiderophores are small molecule metal chelators secreted in sparse quantities by their native microbial hosts but can be engineered for enhanced production from heterologous hosts like Escherichia coli. These molecules have been proved to be capable of binding heavy metals of commercial and/or environmental interest. In this work, we incorporated, as needed, the appropriate pathways required to produce several siderophores (anguibactin, vibriobactin, bacillibactin, pyoverdine, and enterobactin) into the base E. coli K-12 MG1655 metabolic network model to computationally predict, via flux balance analysis methodologies, gene knockout targets, gene over-expression targets, and media modifications capable of improving siderophore reaction flux. E. coli metabolism proved supportive for the underlying production mechanisms of various siderophores. Within such a framework, the gene deletion and over-expression targets identified, coupled with complementary insights from medium optimization predictions, portend experimental implementation to both enable and improve heterologous siderophore production. Successful production of siderophores would then spur novel metal-binding applications.

Predictable CRISPR/Cas9-Mediated COL7A1 Reframing for Dystrophic Epidermolysis Bullosa

End-joining‒based gene editing is frequently used for efficient reframing and knockout of target genes. However, the associated random, unpredictable, and often heterogeneous repair outcomes limit its applicability for therapeutic approaches. This study revealed more precise and predictable outcomes simply on the basis of the sequence context at the CRISPR/Cas9 target site. The severe dystrophic form of the blistering skin disease epidermolysis bullosa (DEB) represents a suitable model platform to test these recent developments for the disruption and reframing of dominant and recessive alleles, respectively, both frequently seen in DEB. We delivered a CRISPR/Cas9 nuclease as ribonucleoprotein into primary wild-type and recessive DEB keratinocytes to introduce a precise predictable single adenine sense-strand insertion at the target site. We achieved type VII collagen knockout in more than 40% of ribonucleoprotein-treated primary wild-type keratinocytes and type VII collagen restoration in more than 70% of ribonucleoprotein-treated recessive DEB keratinocytes. Next-generation sequencing of the on-target site revealed the presence of the precise adenine insertion upstream of the pathogenic mutation in at least 17% of all analyzed COL7A1 alleles. This demonstrates that COL7A1 editing based on precise end-joining‒mediated DNA repair is an efficient strategy to revert the disease-associated nature of DEB regardless of the mutational inheritance.

Muscle Hyperplasia in Japanese Quail by Single Amino Acid Deletion in MSTN Propeptide.

Mutation in myostatin (MSTN), a negative regulator of muscle growth in skeletal muscle, resulted in increased muscle mass in mammals and fishes. However, MSTN mutation in avian species has not been reported. The objective of this study was to generate MSTN mutation in quail and investigate the effect of MSTN mutation in avian muscle growth. Recently, a new targeted gene knockout approach for the avian species has been developed using an adenoviral CRISPR/Cas9 system. By injecting the recombinant adenovirus containing CRISPR/Cas9 into the quail blastoderm, potential germline chimeras were generated and offspring with three base-pair deletion in the targeted region of the MSTN gene was identified. This non-frameshift mutation in MSTN resulted in deletion of cysteine 42 in the MSTN propeptide region and homozygous mutant quail showed significantly increased body weight and muscle mass with muscle hyperplasia compared to heterozygous mutant and wild-type quail. In addition, decreased fat pad weight and increased heart weight were observed in MSTN mutant quail in an age- and sex-dependent manner, respectively. Taken together, these data indicate anti-myogenic function of MSTN in the avian species and the importance of cysteine 42 in regulating MSTN function.

BAX Deletion Accelerates Progression of BCR-ABL-Induced B-ALL in Mice

To explore whether BAX plays a role in the development of Philadelphia chromosome-positive leukemia and related mechanisms. Target-gene knockout mice were used as bone marrow cell donors. Retrovirus over-expressing BCR-ABL were packaged. BCR-ABL-induced B-ALL mouse model was established through donor's B cells transfected by the retrovirus and the B cells over-expressing BCR-ABL were given to the receptor mice by tail vein injection. Western blot was used to detect the protein express and flow cytometry was used to analyze the B cell subpopulations in BAX-/- and WT mouse bone marrows. Kaplan-Meier analysis was used to estimate the survival of diseased mice. BAX deletion caused faster development of BCR-ABL-induced leukemia in vitro and in vivo. BCR-ABL increased BCL-2 expression and enhanced BCL-2/BAX heterodimer formation. The BAX deletion can accelerate the disease progression of BCR-ABL induced B-ALL.