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Bibliography on: CRISPR-Cas

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Robert J. Robbins is a biologist, an educator, a science administrator, a publisher, an information technologist, and an IT leader and manager who specializes in advancing biomedical knowledge and supporting education through the application of information technology. More About:  RJR | OUR TEAM | OUR SERVICES | THIS WEBSITE

RJR: Recommended Bibliography 15 Jan 2021 at 01:37 Created: 

CRISPR-Cas

Clustered regularly interspaced short palindromic repeats (CRISPR, pronounced crisper) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to foreign DNA (e.g a virus or plasmid). The CRISPR/Cas system is a prokaryotic immune system that confers resistance to foreign genetic elements such as those present within plasmids and phages, and provides a form of acquired immunity. CRISPR associated proteins (Cas) use the CRISPR spacers to recognize and cut these exogenous genetic elements in a manner analogous to RNA interference in eukaryotic organisms. CRISPRs are found in approximately 40% of sequenced bacterial genomes and 90% of sequenced archaea. By delivering the Cas9 nuclease complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be cut at a desired location, allowing existing genes to be removed and/or new ones added. The Cas9-gRNA complex corresponds with the CAS III crRNA complex in the above diagram. CRISPR/Cas genome editing techniques have many potential applications, including altering the germline of humans, animals, and food crops. The use of CRISPR Cas9-gRNA complex for genome editing was the AAAS's choice for breakthrough of the year in 2015.

Created with PubMed® Query: "CRISPR.CAS" OR "crispr/cas" NOT pmcbook NOT ispreviousversion

Citations The Papers (from PubMed®)

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RevDate: 2021-01-14

Zuo F, H Marcotte (2021)

Advancing mechanistic understanding and bioengineering of probiotic lactobacilli and bifidobacteria by genome editing.

Current opinion in biotechnology, 70:75-82 pii:S0958-1669(20)30201-9 [Epub ahead of print].

Typical traditional probiotics lactobacilli and bifidobacteria are gaining great interest to be developed as living diagnostics and therapeutics for improving human health. However, the mechanistic basis underlying their inherent health beneficial property remain incompletely understood which can slow down the translational pipeline in the functional food and pharmaceutical field. Efficient genome editing will advance the understanding of the molecular mechanism of the probiotics' physiological properties and their interaction with the host and the host microbiota, thereby further promote the development of next-generation designer probiotics with improved robustness and tailored functionalities. With the expansion of genome editing strategies such as CRISPR-Cas-based tools and IPSD assisted genome engineering as well as other synthetic biology technologies, the research and application of these health-promoting bacteria for the food and pharmaceutical industry will be further enhanced.

RevDate: 2021-01-14

Li Y, J Bondy-Denomy (2020)

Anti-CRISPRs go viral: the infection biology of CRISPR-Cas inhibitors.

Cell host & microbe pii:S1931-3128(20)30669-7 [Epub ahead of print].

Bacteriophages encode diverse anti-CRISPR (Acr) proteins that inhibit CRISPR-Cas immunity during infection of their bacterial hosts. Although detailed mechanisms have been characterized for multiple Acr proteins, an understanding of their role in phage infection biology is just emerging. Here, we review recent work in this area and propose a framework of "phage autonomy" to evaluate CRISPR-immune evasion strategies. During phage infection, Acr proteins are deployed by a tightly regulated "fast on-fast off" transcriptional burst, which is necessary, but insufficient, for CRISPR-Cas inactivation. Instead of a single phage shutting down CRISPR-Cas immunity, a community of acr-carrying phages cooperate to suppress bacterial immunity, displaying low phage autonomy. Enzymatic Acr proteins with novel mechanisms have been recently revealed and are predicted to enhance phage autonomy, while phage DNA protective measures offer the highest phage autonomy observed. These varied Acr mechanisms and strengths also have unexpected impacts on the bacterial populations and competing phages.

RevDate: 2021-01-14

Kurtz S, Lucas-Hahn A, Schlegelberger B, et al (2021)

Knockout of the HMG domain of the porcine SRY gene causes sex reversal in gene-edited pigs.

Proceedings of the National Academy of Sciences of the United States of America, 118(2):.

The sex-determining region on the Y chromosome (SRY) is thought to be the central genetic element of male sex development in mammals. Pathogenic modifications within the SRY gene are associated with a male-to-female sex reversal syndrome in humans and other mammalian species, including rabbits and mice. However, the underlying mechanisms are largely unknown. To understand the biological function of the SRY gene, a site-directed mutational analysis is required to investigate associated phenotypic changes at the molecular, cellular, and morphological level. Here, we successfully generated a knockout of the porcine SRY gene by microinjection of two CRISPR-Cas ribonucleoproteins, targeting the centrally located "high mobility group" (HMG), followed by a frameshift mutation of the downstream SRY sequence. This resulted in the development of genetically male (XY) pigs with complete external and internal female genitalia, which, however, were significantly smaller than in 9-mo-old age-matched control females. Quantitative digital PCR analysis revealed a duplication of the SRY locus in Landrace pigs similar to the known palindromic duplication in Duroc breeds. Our study demonstrates the central role of the HMG domain in the SRY gene in male porcine sex determination. This proof-of-principle study could assist in solving the problem of sex preference in agriculture to improve animal welfare. Moreover, it establishes a large animal model that is more comparable to humans with regard to genetics, physiology, and anatomy, which is pivotal for longitudinal studies to unravel mammalian sex determination and relevant for the development of new interventions for human sex development disorders.

RevDate: 2021-01-14

Scott TA, KV Morris (2021)

Designer nucleases to treat malignant cancers driven by viral oncogenes.

Virology journal, 18(1):18.

Viral oncogenic transformation of healthy cells into a malignant state is a well-established phenomenon but took decades from the discovery of tumor-associated viruses to their accepted and established roles in oncogenesis. Viruses cause ~ 15% of know cancers and represents a significant global health burden. Beyond simply causing cellular transformation into a malignant form, a number of these cancers are augmented by a subset of viral factors that significantly enhance the tumor phenotype and, in some cases, are locked in a state of oncogenic addiction, and substantial research has elucidated the mechanisms in these cancers providing a rationale for targeted inactivation of the viral components as a treatment strategy. In many of these virus-associated cancers, the prognosis remains extremely poor, and novel drug approaches are urgently needed. Unlike non-specific small-molecule drug screens or the broad-acting toxic effects of chemo- and radiation therapy, the age of designer nucleases permits a rational approach to inactivating disease-causing targets, allowing for permanent inactivation of viral elements to inhibit tumorigenesis with growing evidence to support their efficacy in this role. Although many challenges remain for the clinical application of designer nucleases towards viral oncogenes; the uniqueness and clear molecular mechanism of these targets, combined with the distinct advantages of specific and permanent inactivation by nucleases, argues for their development as next-generation treatments for this aggressive group of cancers.

RevDate: 2021-01-14

Guan J, J Bondy-Denomy (2020)

Intracellular Organization by Jumbo Bacteriophages.

Journal of bacteriology, 203(2):.

Since their discovery more than 100 years ago, the viruses that infect bacteria (bacteriophages) have been widely studied as model systems. Largely overlooked, however, have been "jumbo phages," with genome sizes ranging from 200 to 500 kbp. Jumbo phages generally have large virions with complex structures and a broad host spectrum. While the majority of jumbo phage genes are poorly functionally characterized, recent work has discovered many unique biological features, including a conserved tubulin homolog that coordinates a proteinaceous nucleus-like compartment that houses and segregates phage DNA. The tubulin spindle displays dynamic instability and centers the phage nucleus within the bacterial host during phage infection for optimal reproduction. The shell provides robust physical protection for the enclosed phage genomes against attack from DNA-targeting bacterial immune systems, thereby endowing jumbo phages with broad resistance. In this review, we focus on the current knowledge of the cytoskeletal elements and the specialized nuclear compartment derived from jumbo phages, and we highlight their importance in facilitating spatial and temporal organization over the viral life cycle. Additionally, we discuss the evolutionary relationships between jumbo phages and eukaryotic viruses, as well as the therapeutic potential and drawbacks of jumbo phages as antimicrobial agents in phage therapy.

RevDate: 2021-01-14
CmpDate: 2021-01-14

Das S, Banerjee A, Kamran M, et al (2020)

A chemical inhibitor of heat shock protein 78 (HSP78) from Leishmania donovani represents a potential antileishmanial drug candidate.

The Journal of biological chemistry, 295(29):9934-9947.

The emergence of resistance to available antileishmanial drugs advocates identification of new drug targets and their inhibitors for visceral leishmaniasis. Here, we identified Leishmania donovani heat shock protein 78 (LdHSP78), a putative caseinolytic protease, as important for parasite infection of host macrophages and a potential therapeutic target. Enrichment of LdHSP78 in infected humans, hamsters, and parasite amastigotes suggested its importance for disease persistence. Heterozygous knockouts of L. donovani HSP78 (LdHSP78+/-) and Leishmania mexicana HSP78 (LmxHSP78+/-) were generated using a flanking UTR-based multifragment ligation strategy and the CRISPR-Cas9 technique, respectively to investigate the significance of HSP78 for disease manifestation. The LdHSP78+/- parasite burden was dramatically reduced in both murine bone marrow-derived macrophages and hamsters, in association with enrichment of proinflammatory cytokines and NO. This finding implies that LdHSP78+/- parasites cannot suppress immune activation and escape NO-mediated toxicity in macrophages. Furthermore, phosphorylation of the mitogen-activated protein kinase p38 was enhanced and phosphorylation of extracellular signal-regulated kinase 1/2 was decreased in cells infected with LdHSP78+/- parasites, compared with WT parasites. Virulence of the LdHSP78+/- strain was restored by episomal addition of the LdHSP78 gene. Finally, using high-throughput virtual screening, we identified P1,P5-di(adenosine-5')-pentaphosphate (Ap5A) ammonium salt as an LdHSP78 inhibitor. It selectively induced amastigote death at doses similar to amphotericin B doses, while exhibiting much less cytotoxicity to healthy macrophages than amphotericin B. In summary, using both a genetic knockout approach and pharmacological inhibition, we establish LdHSP78 as a drug target and Ap5A as a potential lead for improved antileishmanial agents.

RevDate: 2021-01-14
CmpDate: 2021-01-14

Huang T, Liu Z, Zheng Y, et al (2020)

YTHDF2 promotes spermagonial adhesion through modulating MMPs decay via m6A/mRNA pathway.

Cell death & disease, 11(1):37.

As the foundation of male fertility, spermatogenesis is a complicated and highly controlled process. YTHDF2 plays regulatory roles in biological processes through accelerating the degradation of target mRNAs. However, the function of YTHDF2 in spermatogenesis remains elusive. Here, we knocked out Ythdf2 in mouse spermatogonia via CRISPR/Cas9, and found that depletion of Ythdf2 mainly downregulated the expression of matrix metallopeptidase (MMPs), thus affecting cell adhesion and proliferation. m6A-IP-PCR and RIP-PCR analysis showed that Mmp3, Mmp13, Adamts1 and Adamts9 were modified with m6A and simultaneously interacted with YTHDF2. Moreover, inhibition of Mmp13 partially rescued the phenotypes in Ythdf2-KO cells. Taken together, YTHDF2 regulates cell-matrix adhesion and proliferation through modulating the expression of Mmps by the m6A/mRNA degradation pathway.

RevDate: 2021-01-12
CmpDate: 2021-01-12

Wang L, Zhou J, Wang Q, et al (2021)

Rapid design and development of CRISPR-Cas13a targeting SARS-CoV-2 spike protein.

Theranostics, 11(2):649-664.

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused a worldwide epidemic of the lethal respiratory coronavirus disease (COVID-19), necessitating urgent development of specific and effective therapeutic tools. Among several therapeutic targets of coronaviruses, the spike protein is of great significance due to its key role in host invasion. Here, we report a potential anti-SARS-CoV-2 strategy based on the CRISPR-Cas13a system. Methods: A comprehensive set of bioinformatics methods, including sequence alignment, structural comparison, and molecular docking, was utilized to identify a SARS-CoV-2-spike(S)-specific segment. A tiling crRNA library targeting this specific RNA segment was designed, and optimal crRNA candidates were selected using in-silico methods. The efficiencies of the crRNA candidates were tested in human HepG2 and AT2 cells. Results: The most effective crRNA sequence inducing a robust cleavage effect on S and a potent collateral cleavage effect were identified. Conclusions: This study provides a rapid design pipeline for a CRISPR-Cas13a-based antiviral tool against SARS-CoV-2. Moreover, it offers a novel approach for anti-virus study even if the precise structures of viral proteins are indeterminate.

RevDate: 2021-01-12
CmpDate: 2021-01-12

Dronina J, Bubniene US, A Ramanavicius (2021)

The application of DNA polymerases and Cas9 as representative of DNA-modifying enzymes group in DNA sensor design (review).

Biosensors & bioelectronics, 175:112867.

Rapid detection of nucleic acids (DNA or RNA) by inexpensive, selective, accurate, and highly sensitive methods is very important for biosensors. DNA-sensors based on DNA-modifying enzymes for fast determination and monitoring of pathogenic (Zika, Dengue, SARS-Cov-2 (inducer of COVID-19), human papillomavirus, HIV, etc.) viruses and diagnosis of virus-induced diseases is a key factor of this overview. Recently, DNA-modifying enzymes (Taq DNA polymerase, Phi29 DNA polymerase) have been widely used for the diagnosis of virus or pathogenic disease by gold standard (PCR, qPCR, RT-qPCR) methods, therefore, alternative methods have been reviewed. The main mechanisms of DNA metabolism (replication cycle, amplification) and the genomeediting tool CRISPR-Cas9 are purposefully discussed in order to address strategic possibility to design DNA-sensors based on immobilized DNA-enzymes. However, the immobilization of biologically active proteins on a gold carrier technique with the ability to detect viral or bacterial nucleic acids is individual for each DNA-modifying enzyme group, due to a different number of active sites, C and N terminal locations and arrangement, therefore, individual protocols based on the 'masking' of active sites should be elaborated for each enzyme.

RevDate: 2021-01-13
CmpDate: 2021-01-13

Sgro A, P Blancafort (2020)

Epigenome engineering: new technologies for precision medicine.

Nucleic acids research, 48(22):12453-12482.

Chromatin adopts different configurations that are regulated by reversible covalent modifications, referred to as epigenetic marks. Epigenetic inhibitors have been approved for clinical use to restore epigenetic aberrations that result in silencing of tumor-suppressor genes, oncogene addictions, and enhancement of immune responses. However, these drugs suffer from major limitations, such as a lack of locus selectivity and potential toxicities. Technological advances have opened a new era of precision molecular medicine to reprogram cellular physiology. The locus-specificity of CRISPR/dCas9/12a to manipulate the epigenome is rapidly becoming a highly promising strategy for personalized medicine. This review focuses on new state-of-the-art epigenome editing approaches to modify the epigenome of neoplasms and other disease models towards a more 'normal-like state', having characteristics of normal tissue counterparts. We highlight biomolecular engineering methodologies to assemble, regulate, and deliver multiple epigenetic effectors that maximize the longevity of the therapeutic effect, and we discuss limitations of the platforms such as targeting efficiency and intracellular delivery for future clinical applications.

RevDate: 2021-01-13
CmpDate: 2021-01-13

Mehta HM, SJ Corey (2020)

Getting Back to Normal: Correcting SCN by Universal or Precision Strikes.

Molecular therapy : the journal of the American Society of Gene Therapy, 28(12):2525-2526.

RevDate: 2021-01-13
CmpDate: 2021-01-13

Cooper SE, Schwartzentruber J, Bello E, et al (2020)

Screening for functional transcriptional and splicing regulatory variants with GenIE.

Nucleic acids research, 48(22):e131.

Genome-wide association studies (GWAS) have identified numerous genetic loci underlying human diseases, but a fundamental challenge remains to accurately identify the underlying causal genes and variants. Here, we describe an arrayed CRISPR screening method, Genome engineering-based Interrogation of Enhancers (GenIE), which assesses the effects of defined alleles on transcription or splicing when introduced in their endogenous genomic locations. We use this sensitive assay to validate the activity of transcriptional enhancers and splice regulatory elements in human induced pluripotent stem cells (hiPSCs), and develop a software package (rgenie) to analyse the data. We screen the 99% credible set of Alzheimer's disease (AD) GWAS variants identified at the clusterin (CLU) locus to identify a subset of likely causal variants, and employ GenIE to understand the impact of specific mutations on splicing efficiency. We thus establish GenIE as an efficient tool to rapidly screen for the role of transcribed variants on gene expression.

RevDate: 2021-01-13
CmpDate: 2021-01-13

Xu H, Wang J, Liang Y, et al (2020)

TriTag: an integrative tool to correlate chromatin dynamics and gene expression in living cells.

Nucleic acids research, 48(22):e127.

A wealth of single-cell imaging studies have contributed novel insights into chromatin organization and gene regulation. However, a comprehensive understanding of spatiotemporal gene regulation requires developing tools to combine multiple monitoring systems in a single study. Here, we report a versatile tag, termed TriTag, which integrates the functional capabilities of CRISPR-Tag (DNA labeling), MS2 aptamer (RNA imaging) and fluorescent protein (protein tracking). Using this tag, we correlate changes in chromatin dynamics with the progression of endogenous gene expression, by recording both transcriptional bursting and protein production. This strategy allows precise measurements of gene expression at single-allele resolution across the cell cycle or in response to stress. TriTag enables capturing an integrated picture of gene expression, thus providing a powerful tool to study transcriptional heterogeneity and regulation.

RevDate: 2021-01-12
CmpDate: 2021-01-12

Li G, Zhang X, Wang H, et al (2020)

Increasing CRISPR/Cas9-mediated homology-directed DNA repair by histone deacetylase inhibitors.

The international journal of biochemistry & cell biology, 125:105790.

Histone deacetylase inhibitors (HDACis) affect DNA repair pathways by modulating multiple cellular machineries, including chromatin state, DNA repair factor modification, and the cell cycle. These machineries can differentially affect DNA repair outcomes. With the aim to investigate the impacts of HDACis on DNA repair following CRISPR/Cas9 cleavage from the mixed actions, we used two pan-HDACis, trichostatin A (TSA) and PCI-24781, to treat animal immortalized and primary cells, and studied CRISPR/Cas9-mediated genome editing results by nonhomologous end joining (NHEJ) and homology-directed repair (HDR) pathways. We first found that TSA and PCI-24781 increased NHEJ efficiency. However, further analysis of the total NHEJ events demonstrated that alternative end joining (alt-EJ) mainly contributed to the enhanced total NHEJ by HDACis. We then analyzed HDR efficiency with HDACi treatment and found that multiple HDR pathways, including homologous recombination, single strand annealing and single-stranded oligonucleotide (ssODN)-mediated HDR, were all increased with HDACi treatment. TSA also increased CRISPR-induced ssODN-mediated HDR rate in pig parthenogenetic embryos. Analyzing acetylation status of DNA repair factors showed that acetylation levels of classical NHEJ (c-NHEJ) factors KU70 and KU80 and alt-EJ factor PARP1 were significantly enhanced, but alt-EJ factor LIG3 and HDR factors Rad51 and Rad52 were not affected greatly, implying a differential impact on these repair pathways by HDACis. In addition, TSA and PCI-24781 can enrich cells in G2/M phase of the cell cycle which is beneficial for occurrence of HDR. These findings show that HDACis can effectively promote CRISPR-mediated homology-involved DNA repair, including HDR and alt-EJ pathways, through concerted action of multiple cellular machineries.

RevDate: 2021-01-12
CmpDate: 2021-01-12

Strezoska Ž, Dickerson SM, Maksimova E, et al (2020)

CRISPR-mediated transcriptional activation with synthetic guide RNA.

Journal of biotechnology, 319:25-35.

The CRISPR-Cas9 system has been adapted for transcriptional activation (CRISPRa) and several second-generation CRISPRa systems (including VPR, SunTag, and SAM) have been developed to recruit different transcriptional activators to a deactivated Cas9, which is guided to a transcriptional start site via base complementarity with a target guide RNA. Multiple studies have shown the benefit of CRISPRa using plasmid or lentiviral expressed guide RNA, but the use of synthetic guide RNA has not been reported. Here we demonstrate the effective use of synthetic guide RNA for gene activation via CRISPRa. CRISPRa crRNA may be used with a canonical tracrRNA using the VPR or SunTag activation systems or with an extended tracrRNA containing an aptamer sequence for the SAM system. Transcriptional activation with synthetic crRNA:tracrRNA is comparable to activation achieved with expression vectors and combining several crRNA sequences targeting the same gene can enhance transcriptional activation. The use of synthetic crRNA is also ideal for simultaneous activation of multiple genes or use with dCas9-VPR mRNA when viral transduction is not feasible. Here, we perform a proof-of-principle arrayed screen using a CRISPRa crRNA library consisting of 153 cytokine receptor targets to identify regulators of IL-6 cytokine secretion. Together, these results demonstrate the suitability of synthetic CRISPRa guide RNA for high throughput, arrayed screening applications which allow for more complex phenotypic readouts to complement viability and drug resistance assays typically used in a pooled screening format.

RevDate: 2021-01-13
CmpDate: 2021-01-13

Zhang Q, Fu Y, Thakur C, et al (2020)

CRISPR-Cas9 gene editing causes alternative splicing of the targeting mRNA.

Biochemical and biophysical research communications, 528(1):54-61.

The technique of CRISPR-Cas9 gene editing has been widely used to specifically delete the selected target genes through generating double strand breaks (DSBs) and inducing insertion and/or deletion (indel) of the genomic DNAs in the cells. We recently applied this technique to disrupt mineral dust-induced gene (mdig), a potential oncogene as previously reported, by single guide RNA (sgRNA) targeting the third exon of mdig gene in several cell types, including human bronchial epithelial cell line BEAS-2B, lung cancer cell line A549, and human triple negative breast cancer cell line MDA-MB-231 cells. In addition to the successful knockout of mdig gene in these cells, we unexpectedly noted generation of several alternatively spliced mdig mRNAs. Amplification of the mdig mRNAs during the screening of knockout clones by reverse transcription-polymerase chain reaction (RT-PCR) and the subsequent sanger sequencing of DNA revealed deletion and alternative splicing of mdig mRNAs induced by CRISPR-Cas9 gene editing. The most common deletions include nine and twenty-four nucleotides deletion around the DSBs. In addition, interestingly, some mdig mRNAs showed skipping of the entire exon 3, or alternative splicing between exon 2 and exon 8 using the new donor and accept splicing sites, leading to deletion of exons 3, 4, 5, 6, and 7. Accordingly, cautions should be taken when using CRISPR-Cas9 strategy to edit human genes due to the unintended alterative splicing of the target mRNAs. It is very likely that new proteins, some of which may be highly oncogenic, may be generated from CRISPR-Cas9 gene editing.

RevDate: 2021-01-12
CmpDate: 2021-01-12

Tyagi S, Kumar R, Das A, et al (2020)

CRISPR-Cas9 system: A genome-editing tool with endless possibilities.

Journal of biotechnology, 319:36-53.

The discovery of CRISPR: Cas9 and its application as a powerful gene-editing tool has transformed the world of basic and applied science, especially the molecular biology dome. Also, the smooth, quick, flexible, and very efficient nature of this technology has enabled the biologists to alter the genome of prokaryotes to complex eukaryotic systems, including plants and animals. Using CRISPR and associated tools, investigation, control, and modification of significant biological events have been more accessible than before. These biological scissors are now being used to accelerate breeding programs of crop and livestock, engineer new antimicrobials, and control disease-carrying pathogens. However, like other techniques, these cutters emerged as a double-edged sword and put several challenges to the scientific society. Here in this review article, we summarized the beneficial application of the CRISPR: Cas9 system and unsafe perception to the society if handled carelessly. We also discussed the limitations and ethical issues related to CRISPR: Cas9 technology.

RevDate: 2021-01-12
CmpDate: 2021-01-12

Thom CS, Jobaliya CD, Lorenz K, et al (2020)

Tropomyosin 1 genetically constrains in vitro hematopoiesis.

BMC biology, 18(1):52.

BACKGROUND: Identifying causal variants and genes from human genetic studies of hematopoietic traits is important to enumerate basic regulatory mechanisms underlying these traits, and could ultimately augment translational efforts to generate platelets and/or red blood cells in vitro. To identify putative causal genes from these data, we performed computational modeling using available genome-wide association datasets for platelet and red blood cell traits.

RESULTS: Our model identified a joint collection of genomic features enriched at established trait associations and plausible candidate variants. Additional studies associating variation at these loci with change in gene expression highlighted Tropomyosin 1 (TPM1) among our top-ranked candidate genes. CRISPR/Cas9-mediated TPM1 knockout in human induced pluripotent stem cells (iPSCs) enhanced hematopoietic progenitor development, increasing total megakaryocyte and erythroid cell yields.

CONCLUSIONS: Our findings may help explain human genetic associations and identify a novel genetic strategy to enhance in vitro hematopoiesis. A similar trait-specific gene prioritization strategy could be employed to help streamline functional validation experiments for virtually any human trait.

RevDate: 2021-01-13
CmpDate: 2021-01-13

Chen X, Gao YQ, Zheng YY, et al (2020)

The intragenic microRNA miR199A1 in the dynamin 2 gene contributes to the pathology of X-linked centronuclear myopathy.

The Journal of biological chemistry, 295(26):8656-8667.

Mutations in the myotubularin 1 (MTM1) gene can cause the fatal disease X-linked centronuclear myopathy (XLCNM), but the underlying mechanism is incompletely understood. In this report, using an Mtm1-/y disease model, we found that expression of the intragenic microRNA miR-199a-1 is up-regulated along with that of its host gene, dynamin 2 (Dnm2), in XLCNM skeletal muscle. To assess the role of miR-199a-1 in XLCNM, we crossed miR-199a-1-/- with Mtm1-/y mice and found that the resultant miR-199a-1-Mtm1 double-knockout mice display markers of improved health, as evidenced by lifespans prolonged by 30% and improved muscle strength and histology. Mechanistic analyses showed that miR-199a-1 directly targets nonmuscle myosin IIA (NM IIA) expression and, hence, inhibits muscle postnatal development as well as muscle maturation. Further analysis revealed that increased expression and phosphorylation of signal transducer and activator of transcription 3 (STAT3) up-regulates Dnm2/miR-199a-1 expression in XLCNM muscle. Our results suggest that miR-199a-1 has a critical role in XLCNM pathology and imply that this microRNA could be targeted in therapies to manage XLCNM.

RevDate: 2021-01-13
CmpDate: 2021-01-13

Vidyanti AN, Hsieh JY, Lin KJ, et al (2020)

Role of HMGB1 in an Animal Model of Vascular Cognitive Impairment Induced by Chronic Cerebral Hypoperfusion.

International journal of molecular sciences, 21(6):.

The pathophysiology of vascular cognitive impairment (VCI) is associated with chronic cerebral hypoperfusion (CCH). Increased high-mobility group box protein 1 (HMGB1), a nonhistone protein involved in injury and inflammation, has been established in the acute phase of CCH. However, the role of HMGB1 in the chronic phase of CCH remains unclear. We developed a novel animal model of CCH with a modified bilateral common carotid artery occlusion (BCCAO) in C57BL/6 mice. Cerebral blood flow (CBF) reduction, the expression of HMGB1 and its proinflammatory cytokines (tumor necrosis factor-alpha [TNF-α], interleukin [IL]-1β, and IL-6), and brain pathology were assessed. Furthermore, we evaluated the effect of HMGB1 suppression through bilateral intrahippocampus injection with the CRISPR/Cas9 knockout plasmid. Three months after CCH induction, CBF decreased to 30-50% with significant cognitive decline in BCCAO mice. The 7T-aMRI showed hippocampal atrophy, but amyloid positron imaging tomography showed nonsignificant amyloid-beta accumulation. Increased levels of HMGB1, TNF-α, IL-1β, and IL-6 were observed 3 months after BCCAO. HMGB1 suppression with CRISPR/Cas9 knockout plasmid restored TNF-α, IL-1β, and IL-6 and attenuated hippocampal atrophy and cognitive decline. We believe that HMGB1 plays a pivotal role in CCH-induced VCI pathophysiology and can be a potential therapeutic target of VCI.

RevDate: 2021-01-12
CmpDate: 2021-01-12

Martin J, T Free (2020)

A look back at 2019 in BioTechniques.

BioTechniques, 68(1):2-3.

RevDate: 2021-01-11

Safari F, Afarid M, Rastegari B, et al (2021)

CRISPR systems: Novel approaches for detection and combating COVID-19.

Virus research pii:S0168-1702(20)31189-8 [Epub ahead of print].

Type V and VI CRISPR enzymes are RNA-guided, DNA and RNA-targeting effectors that allow specific gene knockdown. Cas12 and Cas13 are CRISPR proteins that are efficient agents for diagnosis and combating single-stranded RNA (ssRNA) viruses. The programmability of these proteins paves the way for the detection and degradation of RNA viruses by targeting RNAs complementary to its CRISPR RNA (crRNA). Approximately two-thirds of viruses causing diseases contain ssRNA genomes. The Severe Acute Respiratory Syndrome coronavirus 2 (SARS-CoV-2) has caused the outbreak of the coronavirus disease 2019 (COVID-19), which has infected more than fifty million people worldwide with near 1.3 million deaths since December 2019. Thus, accurate and rapid diagnostic and therapeutic tools are essential for early detection and treatment of this widespread infectious disease. For us, the CRISPR based platforms seem to be a plausible new approach for an accurate detection and treatment of SARS-CoV-2. In this review, we talk about Cas12 and Cas13 CRISPR systems and their applications in diagnosis and treatment of RNA virus mediated diseases. In continue, the SARS-CoV-2 pathogenicity, and its conventional diagnostics and antivirals will be discussed. Moreover, we highlight novel CRISPR based diagnostic platforms and therapies for COVID-19. We also discuss the challenges of diagnostic CRISPR based platforms as well as clarifying the proposed solution for high efficient selective in vivo delivery of CRISPR components into SARS-CoV-2-infected cells.

RevDate: 2021-01-11

Palaz F, Kerem Kalkan A, Tozluyurt A, et al (2021)

CRISPR-based tools: alternative methods for the diagnosis of COVID-19.

Clinical biochemistry pii:S0009-9120(21)00001-1 [Epub ahead of print].

The recently emerged severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) spread all over the world rapidly and caused a global pandemic. To prevent the virus from spreading to more individuals, it is of great importance to identify and isolate infected individuals through testing. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the gold standard method for the diagnosis of coronavirus disease (COVID-19) worldwide. However, performing RT-qPCR is limited to centralized laboratories because of the need for sophisticated laboratory equipment and skilled personnel. Further, it can sometimes give false negative or uncertain results. Recently, new methods have been developed for nucleic acid detection and pathogen diagnosis using CRISPR-Cas systems. These methods present rapid and cost-effective diagnostic platforms that provide high sensitivity and specificity without the need for complex instrumentation. Using the CRISPR-based SARS-CoV-2 detection methods, it is possible to increase the number of daily tests in existing laboratories, reduce false negative or uncertain result rates obtained with RT-qPCR, and perform testing in resource-limited settings or at points of need where performing RT-qPCR is not feasible. Here, we briefly describe the RT-qPCR method, and discuss its limitations in meeting the current diagnostic needs. We explain how the unique properties of various CRISPR-associated enzymes are utilized for nucleic acid detection and pathogen diagnosis. Then, we highlight the important features of CRISPR-based diagnostic methods developed for SARS-CoV-2 detection. Finally, we examine the advantages and limitations of these methods, and discuss how they can contribute to improving the efficiency of the current testing systems for combating SARS-CoV-2.

RevDate: 2021-01-11

Srivastava S, Upadhyay DJ, A Srivastava (2020)

Next-Generation Molecular Diagnostics Development by CRISPR/Cas Tool: Rapid Detection and Surveillance of Viral Disease Outbreaks.

Frontiers in molecular biosciences, 7:582499.

Virus disease spreads effortlessly mechanically or through minute insect vectors that are extremely challenging to avoid. Emergence and reemergence of new viruses such as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), H1N1 influenza virus, avian influenza virus, dengue virus, Citrus tristeza virus, and Tomato yellow leaf curl virus have paralyzed the economy of many countries. The cure for major viral diseases is not feasible; however, early detection and surveillance of the disease can obstruct their spread. Therefore, advances in the field of virus diagnosis and the development of new point-of-care testing kits become necessary globally. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) is an emerging technology for gene editing and diagnostics development. Several rapid nucleic acid diagnostic kits have been developed and validated using Cas9, Cas12, and Cas13 proteins. This review summarizes the CRISPR/Cas-based next-generation molecular diagnostic techniques and portability of devices for field-based utilization.

RevDate: 2021-01-11
CmpDate: 2021-01-11

House NCM, Parasuram R, Layer JV, et al (2020)

Site-specific targeting of a light activated dCas9-KillerRed fusion protein generates transient, localized regions of oxidative DNA damage.

PloS one, 15(12):e0237759.

DNA repair requires reorganization of the local chromatin structure to facilitate access to and repair of the DNA. Studying DNA double-strand break (DSB) repair in specific chromatin domains has been aided by the use of sequence-specific endonucleases to generate targeted breaks. Here, we describe a new approach that combines KillerRed, a photosensitizer that generates reactive oxygen species (ROS) when exposed to light, and the genome-targeting properties of the CRISPR/Cas9 system. Fusing KillerRed to catalytically inactive Cas9 (dCas9) generates dCas9-KR, which can then be targeted to any desired genomic region with an appropriate guide RNA. Activation of dCas9-KR with green light generates a local increase in reactive oxygen species, resulting in "clustered" oxidative damage, including both DNA breaks and base damage. Activation of dCas9-KR rapidly (within minutes) increases both γH2AX and recruitment of the KU70/80 complex. Importantly, this damage is repaired within 10 minutes of termination of light exposure, indicating that the DNA damage generated by dCas9-KR is both rapid and transient. Further, repair is carried out exclusively through NHEJ, with no detectable contribution from HR-based mechanisms. Surprisingly, sequencing of repaired DNA damage regions did not reveal any increase in either mutations or INDELs in the targeted region, implying that NHEJ has high fidelity under the conditions of low level, limited damage. The dCas9-KR approach for creating targeted damage has significant advantages over the use of endonucleases, since the duration and intensity of DNA damage can be controlled in "real time" by controlling light exposure. In addition, unlike endonucleases that carry out multiple cut-repair cycles, dCas9-KR produces a single burst of damage, more closely resembling the type of damage experienced during acute exposure to reactive oxygen species or environmental toxins. dCas9-KR is a promising system to induce DNA damage and measure site-specific repair kinetics at clustered DNA lesions.

RevDate: 2021-01-11
CmpDate: 2021-01-11

Kang D, Shin W, Yoo H, et al (2020)

Cep215 is essential for morphological differentiation of astrocytes.

Scientific reports, 10(1):17000.

Cep215 (also known as Cdk5rap2) is a centrosome protein which is involved in microtubule organization. Cep215 is also placed at specific subcellular locations and organizes microtubules outside the centrosome. Here, we report that Cep215 is involved in morphological differentiation of astrocytes. Cep215 was specifically localized at the glial processes as well as centrosomes in developing astrocytes. Morphological differentiation of astrocytes was suppressed in the Cep215-deleted P19 cells and in the Cep215-depleted embryonic hippocampal culture. We confirm that the microtubule organizing function of Cep215 is critical for the glial process formation. However, Cep215 is not involved in the regulation of cell proliferation nor cell specification. Based on the results, we propose that Cep215 organizes microtubules for glial process formation during astrocyte differentiation.

RevDate: 2021-01-11
CmpDate: 2021-01-11

Zhao N, Li L, Luo G, et al (2020)

Multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1-RecE/T system in Corynebacterium glutamicum.

Journal of industrial microbiology & biotechnology, 47(8):599-608.

Corynebacterium glutamicum is an essential industrial strain that has been widely harnessed for the production of all kinds of value-added products. Efficient multiplex gene editing and large DNA fragment deletion are essential strategies for industrial biotechnological research. Cpf1 is a robust and simple genome editing tool for simultaneous editing of multiplex genes. However, no studies on effective multiplex gene editing and large DNA fragment deletion by the CRISPR/Cpf1 system in C. glutamicum have been reported. Here, we developed a multiplex gene editing method by optimizing the CRISPR/Cpf1-RecT system and a large chromosomal fragment deletion strategy using the CRISPR/Cpf1-RecET system in C. glutamicum ATCC 14067. The CRISPR/Cpf1-RecT system exhibited a precise editing efficiency of more than 91.6% with the PAM sequences TTTC, TTTG, GTTG or CTTC. The sites that could be edited were limited due to the PAM region and the 1-7 nt at the 5' end of the protospacer region. Mutations in the PAM region increased the editing efficiency of the - 6 nt region from 0 to 96.7%. Using a crRNA array, two and three genes could be simultaneously edited in one step via the CRISPR/Cpf1-RecT system, and the efficiency of simultaneously editing two genes was 91.6%, but the efficiency of simultaneously editing three genes was below 10%. The editing efficiency for a deletion of 1 kb was 79.6%, and the editing efficiencies for 5- and 20 kb length DNA fragment deletions reached 91.3% and 36.4%, respectively, via the CRISPR/Cpf1-RecET system. This research provides an efficient and simple tool for C. glutamicum genome editing that can further accelerate metabolic engineering efforts and genome evolution.

RevDate: 2021-01-11
CmpDate: 2021-01-11

Akkaya M, Bansal A, Sheehan PW, et al (2020)

Testing the impact of a single nucleotide polymorphism in a Plasmodium berghei ApiAP2 transcription factor on experimental cerebral malaria in mice.

Scientific reports, 10(1):13630.

Cerebral malaria (CM) is the deadliest form of severe Plasmodium infections. Currently, we have limited understanding of the mechanisms by which Plasmodium parasites induce CM. The mouse model of CM, experimental CM (ECM), induced by infection with the rodent parasite, Plasmodium berghei ANKA (PbANKA) has been extensively used to study the pathophysiology of CM. Recent genomic analyses revealed that the coding regions of PbANKA and the closely related Plasmodium berghei NK65 (PbNK65), that does not cause ECM, differ in only 21 single nucleotide polymorphysims (SNPs). Thus, the SNP-containing genes might contribute to the pathogenesis of ECM. Although the majority of these SNPs are located in genes of unknown function, one SNP is located in the DNA binding site of a member of the Plasmodium ApiAP2 transcription factor family, that we recently showed functions as a virulence factor alternating the host's immune response to the parasite. Here, we investigated the impact of this SNP on the development of ECM. Our results using CRISPR-Cas9 engineered parasites indicate that despite its immune modulatory function, the SNP is neither necessary nor sufficient to induce ECM and thus cannot account for parasite strain-specific differences in ECM phenotypes.

RevDate: 2021-01-11
CmpDate: 2021-01-11

Gans I, Hartig EI, Zhu S, et al (2020)

Klf9 is a key feedforward regulator of the transcriptomic response to glucocorticoid receptor activity.

Scientific reports, 10(1):11415.

The zebrafish has recently emerged as a model system for investigating the developmental roles of glucocorticoid signaling and the mechanisms underlying glucocorticoid-induced developmental programming. To assess the role of the Glucocorticoid Receptor (GR) in such programming, we used CRISPR-Cas9 to produce a new frameshift mutation, GR369-, which eliminates all potential in-frame initiation codons upstream of the DNA binding domain. Using RNA-seq to ask how this mutation affects the larval transcriptome under both normal conditions and with chronic cortisol treatment, we find that GR mediates most of the effects of the treatment, and paradoxically, that the transcriptome of cortisol-treated larvae is more like that of larvae lacking a GR than that of larvae with a GR, suggesting that the cortisol-treated larvae develop GR resistance. The one transcriptional regulator that was both underexpressed in GR369- larvae and consistently overexpressed in cortisol-treated larvae was klf9. We therefore used CRISPR-Cas9-mediated mutation of klf9 and RNA-seq to assess Klf9-dependent gene expression in both normal and cortisol-treated larvae. Our results indicate that Klf9 contributes significantly to the transcriptomic response to chronic cortisol exposure, mediating the upregulation of proinflammatory genes that we reported previously.

RevDate: 2021-01-11
CmpDate: 2021-01-11

Modell AE, Siriwardena SU, A Choudhary (2020)

A Jumbo Phage Forms a Nucleus-like Compartment to Evade Bacterial Defense Systems.

Biochemistry, 59(20):1869-1870.

RevDate: 2021-01-11
CmpDate: 2021-01-11

Lu M, TA Tokuyasu (2020)

CRISPR-Cas13-Based RNA-Interacting Protein Detection in Living Cells.

Biochemistry, 59(19):1791-1792.

RevDate: 2021-01-11
CmpDate: 2021-01-11

Wu H, Qian C, Wu C, et al (2020)

End-point dual specific detection of nucleic acids using CRISPR/Cas12a based portable biosensor.

Biosensors & bioelectronics, 157:112153.

A CRISPR/Cas12a based portable biosensor (Cas12a-PB) was developed to simultaneously visually detect CaMV35S promoter and Lectin gene from genetically modified (GM) soybean powders (Roundup Ready@). The Cas12a-PB, mainly made of polymethylmethacrylate (PMMA) and PMMA tape, has a connection structure, three channels and three detection chambers. The CRISPR/Cas12a detection reagents were preloaded in detection chambers and the reaction tube was connected to the connection structure by screw threads. After amplification, the amplicons were gone into three detection chambers by swinging the Cas12a-PB to conduct dual detection. Positive samples would produce green fluorescence while negative samples were black under the irradiation of 490 nm LED light. In this study, the Cas12a-PB successively combined with ordinary PCR, rapid PCR and loop-mediated isothermal amplification (LAMP) to achieve dual detection, which made detection process more convenient and portable. As low as 0.1% transgenic ingredients in soybean powders could be detected and the specificity of Cas12a-PB was confirmed with GM maize powders (MON810, GA21), GM soybean powders (DP305423), non-GM peanut and rice as targets. In the end, an amplification chamber combining with Cas12a-PB on a PMMA chip was further designed to eliminate the use of reaction tube and mineral oil, which made operation simpler. The established Cas12a-PB would provide a new reliable solution for multiple targets detection in clinic diagnostics, food safety, etc.

RevDate: 2021-01-11
CmpDate: 2021-01-11

Ramesh A, Ong T, Garcia JA, et al (2020)

Guide RNA Engineering Enables Dual Purpose CRISPR-Cpf1 for Simultaneous Gene Editing and Gene Regulation in Yarrowia lipolytica.

ACS synthetic biology, 9(4):967-971.

Yarrowia lipolytica has fast become a biotechnologically significant yeast for its ability to accumulate lipids to high levels. While there exists a suite of synthetic biology tools for genetic engineering in this yeast, there is a need for multipurposed tools for rapid strain generation. Here, we describe a dual purpose CRISPR-Cpf1 system that is capable of simultaneous gene disruption and gene regulation. Truncating guide RNA spacer length to 16 nt inhibited nuclease activity but not binding to the target loci, enabling gene activation and repression with Cpf1-fused transcriptional regulators. Gene repression was demonstrated using a Cpf1-Mxi1 fusion achieving a 7-fold reduction in mRNA, while CRISPR-activation with Cpf1-VPR increased hrGFP expression by 10-fold. High efficiency disruptions were achieved with gRNAs 23-25 bp in length, and efficiency and repression levels were maintained with multiplexed expression of truncated and full-length gRNAs. The developed CRISPR-Cpf1 system should prove useful in metabolic engineering, genome wide screening, and functional genomics studies.

RevDate: 2021-01-11
CmpDate: 2021-01-11

Liow LT, Go MK, Chang MW, et al (2020)

Toolkit Development for Cyanogenic and Gold Biorecovery Chassis Chromobacterium violaceum.

ACS synthetic biology, 9(4):953-961.

Chromobacterium violaceum has been of interest recently due to its cyanogenic ability and its potential role in environmental sustainability via the biorecovery of gold from electronic waste. However, as with many nonmodel bacteria, there are limited genetic tools to implement the use of this Gram-negative chassis in synthetic biology. We propose a system that involves assaying spontaneous antibiotic resistances and using broad host range vectors to develop episomal vectors for nonmodel Gram-negative bacteria. These developed vectors can subsequently be used to characterize inducible promoters for gene expressions and implementing CRISPRi to inhibit endogenous gene expression for further studies. Here, we developed the first episomal genetic toolkit for C. violaceum consisting of two origins of replication, three antibiotic resistance genes, and four inducible promoter systems. We examined the occurrences of spontaneous resistances of the bacterium to the chosen selection markers to prevent incidences of false positives. We also tested broad host range vectors from four different incompatibility groups and characterized four inducible promoter systems, which potentially can be applied in other Gram-negative nonmodel bacteria. CRISPRi was also implemented to inhibit violacein pigment production in C. violaceum. This systematic toolkit will aid future genetic circuitry building in this chassis and other nonmodel bacteria for synthetic biology and biotechnological applications.

RevDate: 2021-01-11
CmpDate: 2021-01-11

Wrist A, Sun W, RM Summers (2020)

The Theophylline Aptamer: 25 Years as an Important Tool in Cellular Engineering Research.

ACS synthetic biology, 9(4):682-697.

The theophylline aptamer was isolated from an oligonucleotide library in 1994. Since that time, the aptamer has found wide utility, particularly in synthetic biology, cellular engineering, and diagnostic applications. The primary application of the theophylline aptamer is in the construction and characterization of synthetic riboswitches for regulation of gene expression. These riboswitches have been used to control cellular motility, regulate carbon metabolism, construct logic gates, screen for mutant enzymes, and control apoptosis. Other applications of the theophylline aptamer in cellular engineering include regulation of RNA interference and genome editing through CRISPR systems. Here we describe the uses of the theophylline aptamer for cellular engineering over the past 25 years. In so doing, we also highlight important synthetic biology applications to control gene expression in a ligand-dependent manner.

RevDate: 2021-01-11
CmpDate: 2021-01-11

Zhang L, He A, Chen B, et al (2020)

A HOTAIR regulatory element modulates glioma cell sensitivity to temozolomide through long-range regulation of multiple target genes.

Genome research, 30(2):155-163.

Temozolomide (TMZ) is a frequently used chemotherapy for glioma; however, chemoresistance is a major problem limiting its effectiveness. Thus, knowledge of mechanisms underlying this outcome could improve patient prognosis. Here, we report that deletion of a regulatory element in the HOTAIR locus increases glioma cell sensitivity to TMZ and alters transcription of multiple genes. Analysis of a combination of RNA-seq, Capture Hi-C, and patient survival data suggests that CALCOCO1 and ZC3H10 are target genes repressed by the HOTAIR regulatory element and that both function in regulating glioma cell sensitivity to TMZ. Rescue experiments and 3C data confirmed this hypothesis. We propose a new regulatory mechanism governing glioma cell TMZ sensitivity.

RevDate: 2021-01-11
CmpDate: 2021-01-11

Ou B, Jiang B, Jin D, et al (2020)

Engineered Recombinant Escherichia coli Probiotic Strains Integrated with F4 and F18 Fimbriae Cluster Genes in the Chromosome and Their Assessment of Immunogenic Efficacy in Vivo.

ACS synthetic biology, 9(2):412-426.

F4 (K88) and F18 fimbriaed enterotoxigenic Escherichia coli (ETEC) are the predominant causes of porcine postweaning diarrhea (PWD), and vaccines are considered the most effective preventive approach against PWD. Since heterologous DNA integrated into bacterial chromosomes could be effectively expressed with stable inheritance, we chose probiotic EcNc (E. coli Nissle 1917 prototype cured of cryptic plasmids) as a delivery vector to express the heterologous F4 or both F4 and F18 fimbriae and sequentially assessed their immune efficacy of anti-F4 and F18 fimbriae in both murine and piglet models. Employing the CRISPR-cas9 technology, yjcS, pcadA, lacZ, yieN/trkD, maeB, and nth/tppB sites in the chromosome of an EcNc strain were targeted as integration sites to integrate F4 or F18 fimbriae cluster genes under the Ptet promotor to construct two recombinant integration probiotic strains (RIPSs), i.e., nth integration strain (EcNcΔnth/tppB::PtetF4) and multiple integration strain (EcNc::PtetF18x4::PtetF4x2). Expression of F4, both F4 and F18 fimbriae on the surfaces of two RIPSs, was verified with combined methods of agglutination assay, Western blot, and immunofluorescence microscopy. The recombinant strains have improved adherence to porcine intestinal epithelial cell lines. Mice and piglets immunized with the nth integration strain and multiple integration strain through gavage developed anti-F4 and both anti-F4 and anti-F18 IgG immune responses. Moreover, the serum antibodies from the immunized mice and piglets significantly inhibited the adherence of F4+ or both F4+ and F18+ ETEC wild-type strains to porcine intestinal cell lines in vitro, indicating the potential of RIPSs as promising probiotic strains plus vaccine candidates against F4+/F18+ ETEC infection.

RevDate: 2021-01-11
CmpDate: 2021-01-11

Anonymous (2020)

Mucopolysaccharidosis Type I Phenotypically Corrected with Edited Hematopoietic Stem Cells: Instead of altering the IDUA gene, a protein was inserted in a repurposable place in the genome known as a "safe harbor locus".

American journal of medical genetics. Part A, 182(2):275-276.

RevDate: 2021-01-11
CmpDate: 2021-01-11

Fan J, Liu Y, Liu L, et al (2020)

A Multifunction Lipid-Based CRISPR-Cas13a Genetic Circuit Delivery System for Bladder Cancer Gene Therapy.

ACS synthetic biology, 9(2):343-355.

The treatment of bladder cancer has recently shown minimal progress. Gene therapy mediated by CRISPR provides a new option for bladder cancer treatment. In this study, we developed a versatile liposome system to deliver the CRISPR-Cas13a gene circuits into bladder cancer cells. After in vitro studies and intravesical perfusion studies in mice, this system showed five advantages: (1) CRISPR-Cas13a, a transcriptional targeting and cleavage tool for gene expression editing, did not affect the stability of the cell genome; (2) the prepared liposome systems were targeted to hVEGFR2, which is always highly expressed in bladder cancer cells; (3) the CRISPR-Cas13a sequence was driven by an artificial tumor specific promoter to achieve further targeting; (4) a near-infrared photosensitizer released using near-infrared light was introduced to control the delivery system; and (5) the plasmids were constructed with three crRNA tandem sequences to achieve multiple targeting and wider therapeutic results. This tumor cell targeting lipid delivery system with near-infrared laser-controlled ability provided a versatile strategy for CRISPR-Cas13a based gene therapy of bladder cancer.

RevDate: 2021-01-11
CmpDate: 2021-01-11

Katayama S, Sato K, T Nakazawa (2020)

In vivo and in vitro knockout system labelled using fluorescent protein via microhomology-mediated end joining.

Life science alliance, 3(1):.

Gene knockout is important for understanding gene function and genetic disorders. The CRISPR/Cas9 system has great potential to achieve this purpose. However, we cannot distinguish visually whether a gene is knocked out and in how many cells it is knocked out among a population of cells. Here, we developed a new system that enables the labelling of knockout cells with fluorescent protein through microhomology-mediated end joining-based knock-in. Using a combination with recombinant adeno-associated virus, we delivered our system into the retina, where the expression of Staphylococcus aureus Cas9 was driven by a retina ganglion cell (RGC)-specific promoter, and knocked out carnitine acetyltransferase (CAT). We evaluated RGCs and revealed that CAT is required for RGC survival. Furthermore, we applied our system to Keap1 and confirmed that Keap1 is not expressed in fluorescently labelled cells. Our system provides a promising framework for cell type-specific genome editing and fluorescent labelling of gene knockout based on knock-in.

RevDate: 2021-01-11
CmpDate: 2021-01-11

Jacot D, D Soldati-Favre (2020)

CRISPR/Cas9-Mediated Generation of Tetracycline Repressor-Based Inducible Knockdown in Toxoplasma gondii.

Methods in molecular biology (Clifton, N.J.), 2071:125-141.

The phylum Apicomplexa groups numerous pathogenic protozoan parasites including Plasmodium, the causative agent of malaria, Cryptosporidium which can cause severe gastrointestinal infections, as well as Babesia, Eimeria, and Theileria that account for considerable economic burdens to poultry and cattle industry. Toxoplasma gondii is the most ubiquitous and opportunistic member of this phylum able to infect all warm-blooded animals and responsible for severe disease in immunocompromised individuals and unborn fetuses.Due to its ease of cultivation and genetic tractability T. gondii has served as recipient for the transfer and adaptation of multiple genetic tools developed to control gene expression. In these parasites, a collection of tight conditional systems exists to control gene expression at the levels of transcription, RNA degradation or protein stability. The recent implementation of the CRISPR/Cas9 technology considerably reduces time and effort to generate transgenic parasites and at the same time increases to an ultimate level of precision the editing of the parasite genome. Here, we provide a step-by-step protocol for CRISPR/Cas9-mediated generation of tetracycline repressor-based inducible knockdown in T. gondii.

RevDate: 2021-01-10

Behbahani RG, Danyaei A, Teimoori A, et al (2021)

Breast cancer radioresistance may be overcome by osteopontin gene knocking out with CRISPR/Cas9 technique.

Cancer radiotherapie : journal de la Societe francaise de radiotherapie oncologique pii:S1278-3218(20)30336-X [Epub ahead of print].

PURPOSE: Osteopontin (OPN) is a phosphoglycoprotein, with a wide range of physiological and pathological roles. High expression of OPN promotes aggressive behavior, causes poor prognosis in tumor cells, and reduces the survival of patients. Since overexpression of OPN gives rise to radioresistance, the effects of the gene knock out using the CRISPR/Cas9 system in combination with radiation are emphasized.

MATERIAL AND METHODS: We used the CRISPR/Cas9 technique to knock out the OPN gene in the MDA-MB-231 cell line. After transfection, the cells were irradiated. The changes of the OPN mRNA levels, the apoptosis, and the differences in cell viability were assessed.

RESULTS: A significant reduction in the OPN expression was observed alone or along with irradiation. The knocked out gene alone increased apoptosis rate. The cell viability decreased to after knocking out of the OPN gene. The gene knocking-out combined with irradiation led to more decline of cell viability.

CONCLUSION: Our results demonstrated that after knocking out the OPN gene, the MDA-MB-231 cells showed a significant radiosensitivity. Therefore, the OPN knock out in combination with conventional radiotherapy, may become an efficient therapeutic target in the future.

RevDate: 2021-01-09

Oh SH, Lee HJ, Ahn MK, et al (2021)

Multiplex gene targeting in the mouse embryo using a Cas9-Cpf1 hybrid guide RNA.

Biochemical and biophysical research communications, 539:48-55 pii:S0006-291X(20)32249-X [Epub ahead of print].

CRISPR-Cas systems, including Cas9 and Cpf1 (Cas12a), are promising tools for generating gene knockout mouse models. Unlike Cas9, Cpf1 can generate multiple crRNAs from a single concatemeric crRNA precursor, which is favorable for multiplex gene editing. Recently, a hybrid guide RNA (hgRNA) system employing both Cas9 and Cpf1 was developed for multiplex gene editing. As the crRNA of Cpf1 was linked to the 3' end of the sgRNA for Cas9, it can be split into separate guide RNAs by Cpf1. To examine whether this Cas9-Cpf1 hybrid system is suitable for multiplex gene knockouts in the mouse embryo, we generated an hgRNA that simultaneously targets the mouse Il10ra gene by Cas9 and mouse Dr3 (or Tnfrsf25, death receptor3) gene by Cpf1. The expression of hgRNA from a single promoter induced significant indels at each gene in cultured mouse cells upon the co-expression of both Cas9 and Cpf1. Interestingly, the hgRNA exhibited comparable Cas9-mediated indel activity without Cpf1 expression. Similarly, when the hgRNA was co-microinjected with both Cas9 and Cpf1 mRNAs into mouse zygotes at the pronuclear stage, founder mice were generated harboring mutations in both the Il10ra and Dr3 genes. However, when Cas9 mRNA was used alone without Cpf1 mRNA, the mouse Il10ra gene targeting was significantly decreased. These results indicate that the hgRNA system is a possible tool for multiplex gene targeting in the mouse embryo.

RevDate: 2021-01-09

Ansari I, Chaturvedi A, Chitkara D, et al (2021)

CRISPR/Cas mediated epigenome editing for cancer therapy.

Seminars in cancer biology pii:S1044-579X(20)30278-9 [Epub ahead of print].

The understanding of the relationship between epigenetic alterations, their effects on gene expression and the knowledge that these epigenetic alterations are reversible, have opened up new therapeutic pathways for treating various diseases, including cancer. This has led the research for a better understanding of the mechanism and pathways of carcinogenesis and provided the opportunity to develop the therapeutic approaches by targeting such pathways. Epi-drugs, DNA methyl transferase (DNMT) inhibitors and histone deacetylase (HDAC) inhibitors are the best examples of epigenetic therapies with clinical applicability. Moreover, precise genome editing technologies such as CRISPR/Cas has proven their efficacy in epigenome editing, including the alteration of epigenetic markers, such as DNA methylation or histone modification. The main disadvantage with DNA gene editing technologies is off-target DNA sequence alteration, which is not an issue with epigenetic editing. It is known that cancer is linked with epigenetic alteration, and thus CRISPR/Cas system shows potential for cancer therapy via epigenome editing. This review outlines the epigenetic therapeutic approach for cancer therapy using CRISPR/Cas, from the basic understanding of cancer epigenetics to potential applications of CRISPR/Cas in treating cancer.

RevDate: 2021-01-09

Workman RE, Pammi T, Nguyen BTK, et al (2021)

A natural single-guide RNA repurposes Cas9 to autoregulate CRISPR-Cas expression.

Cell pii:S0092-8674(20)31687-1 [Epub ahead of print].

CRISPR-Cas systems provide prokaryotes with acquired immunity against viruses and plasmids, but how these systems are regulated to prevent autoimmunity is poorly understood. Here, we show that in the S. pyogenes CRISPR-Cas system, a long-form transactivating CRISPR RNA (tracr-L) folds into a natural single guide that directs Cas9 to transcriptionally repress its own promoter (Pcas). Further, we demonstrate that Pcas serves as a critical regulatory node. De-repression causes a dramatic 3,000-fold increase in immunization rates against viruses; however, heightened immunity comes at the cost of increased autoimmune toxicity. Using bioinformatic analyses, we provide evidence that tracrRNA-mediated autoregulation is widespread in type II-A CRISPR-Cas systems. Collectively, we unveil a new paradigm for the intrinsic regulation of CRISPR-Cas systems by natural single guides, which may facilitate the frequent horizontal transfer of these systems into new hosts that have not yet evolved their own regulatory strategies.

RevDate: 2021-01-09

Yuza K, Nagahashi M, Ichikawa H, et al (2021)

Activin a Receptor Type 2A Mutation Affects the Tumor Biology of Microsatellite Instability-High Gastric Cancer.

Journal of gastrointestinal surgery : official journal of the Society for Surgery of the Alimentary Tract [Epub ahead of print].

BACKGROUND: Activin A receptor type 2A (ACVR2A) is one of the most frequently mutated genes in microsatellite instability-high (MSI-H) gastric cancer. However, the clinical relevance of the ACVR2A mutation in MSI-H gastric cancer patients remains unclear. The aims of this study were to explore the effect of ACVR2A mutation on the tumor behavior and to identify the clinicopathological characteristics of gastric cancer patients with ACVR2A mutations.

METHODS: An in vitro study was performed to investigate the biological role of ACVR2A via CRISPR/Cas9-mediated ACVR2A knockout MKN74 human gastric cancer cells. One hundred twenty-four patients with gastric cancer were retrospectively analyzed, and relations between MSI status, ACVR2A mutations, and clinicopathological factors were evaluated.

RESULTS: ACVR2A knockout cells showed less aggressive tumor biology than mock-transfected cells, displaying reduced proliferation, migration, and invasion (P < 0.05). MSI mutations were found in 10% (13/124) of gastric cancer patients, and ACVR2A mutations were found in 8.1% (10/124) of patients. All ACVR2A mutations were accompanied by MSI. The 5-year overall survival rates of ACVR2A wild-type patients and ACVR2A-mutated patients were 57% and 90%, respectively (P = 0.048). Multivariate analysis revealed that older age (P = 0.015), distant metastasis (P < 0.001), and ACVR2A wild-type status (P = 0.040) were independent prognostic factors for overall survival.

CONCLUSIONS: Our study demonstrated that gastric cancer patients with ACVR2A mutation have a significantly better prognosis than those without. Dysfunction of ACVR2A in MKN74 human gastric cancer cells caused less aggressive tumor biology, indicating the importance of ACVR2A in the progression of MSI-H tumors.

RevDate: 2021-01-08

Das Mukhopadhyay C, Sharma P, Sinha K, et al (2020)

Recent trends in analytical and digital techniques for the detection of the SARS-Cov-2.

Biophysical chemistry, 270:106538 pii:S0301-4622(20)30246-5 [Epub ahead of print].

The current global outbreak of COVID-19 due to SARS-CoV-2 is an unprecedented humanitarian crisis. Considering the gravity of its impact there is an immediate need to develop a detection technique that is sensitive, specific, fast, and affordable for the clinical diagnosis of the disease. Real time Polymerase Chain Reaction (RT-PCR)-based detection platforms are contemplated to be the gold standard to detect viral RNA. However, that may be susceptible to errors, and there is a risk of obtaining false results, which ultimately compromises the strategy of efficient disease management. Several modern techniques exhibiting assured results with enhanced sensitivity and specificity against the SARS-CoV-2 associated viral components or immune response against it have been developed and may be implemented. The review deals with the conventional RT-PCR detection techniques and compares them to other detection platforms viz., biosensor based detection of antigens, fluorescent or colorimetric detection systems including CRISPR-Cas 13 based SHERLOCK kit, CRISPR Cas-9 based FELUDA test kit, CRISPR DETECTR kit, Next Generation Sequencing or microarray-based kits. These modern techniques are great as a point of care detection methods but should be followed by RT PCR based detection for the confirmation of COVID-19 status.

RevDate: 2021-01-08

Park YK, Yoon BH, Park SJ, et al (2020)

BaSDAS: a web-based pooled CRISPR-Cas9 knockout screening data analysis system.

Genomics & informatics, 18(4):e46.

We developed the BaSDAS (Barcode-Seq Data Analysis System), a GUI-based pooled knockout screening data analysis system, to facilitate the analysis of pooled knockout screen data easily and effectively by researchers with limited bioinformatics skills. The BaSDAS supports the analysis of various pooled screening libraries, including yeast, human, and mouse libraries, and provides many useful statistical and visualization functions with a user-friendly web interface for convenience. We expect that BaSDAS will be a useful tool for the analysis of genome-wide screening data and will support the development of novel drugs based on functional genomics information.

RevDate: 2021-01-08
CmpDate: 2021-01-08

Das S, Chandrasekaran AP, Jo KS, et al (2020)

HAUSP stabilizes Cdc25A and protects cervical cancer cells from DNA damage response.

Biochimica et biophysica acta. Molecular cell research, 1867(12):118835.

Resistance to DNA-damaging agents is one of the main reasons for the low survival of cervical cancer patients. Previous reports have suggested that the Cdc25A oncoprotein significantly affects the level of susceptibility to DNA-damaging agents, but the molecular mechanism remains unclear. In this study, we used Western blot and flow cytometry analyses to demonstrate that the deubiquitinating enzyme HAUSP stabilizes Cdc25A protein level. Furthermore, in a co-immunoprecipitation assay, we found that HAUSP interacts with and deubiquitinates Cdc25A both exogenously and endogenously. HAUSP extends the half-life of the Cdc25A protein by circumventing turnover. HAUSP knockout in HeLa cells using the CRISPR/Cas9 system caused a significant delay in Cdc25A-mediated cell cycle progression, cell migration, and colony formation and attenuated tumor progression in a mouse xenograft model. Furthermore, HAUSP-mediated stabilization of the Cdc25A protein produced enhanced resistance to DNA-damaging agents. Overall, our study suggests that targeting Cdc25A and HAUSP could be a promising combinatorial approach to halt progression and minimize antineoplastic resistance in cervical cancer.

RevDate: 2021-01-08
CmpDate: 2021-01-08

Zhang W, Kataoka M, Yen Doan H, et al (2020)

Isolation and characterization of mammalian orthoreoviruses using a cell line resistant to sapelovirus infection.

Transboundary and emerging diseases, 67(6):2849-2859.

Porcine sapelovirus (PSV) is a causative agent of acute diarrhoea, pneumonia and reproductive disorders in swine. Since PSV infection interrupts the growth of other viruses due to its high replication capability in cell culture, the prevention of PSV replication is a keystone to the isolation of non-PSV agents from PSV-contaminated samples. In the present study, we established the PSV infection-resistant cell line N1380 and isolated three mammalian orthoreoviruses (MRV) strains, sR1521, sR1677 and sR1590, from swine in Taiwan. These Taiwanese isolates induced an extensive cytopathic effect in N1380 cells upon infection. The complete and empty virus particles were purified from the cell culture supernatants. Next-generation sequencing analyses revealed that the complete virus particles contained 10 segments, including 3 large (L1, L2 and L3), 3 medium (M1, M2 and M3) and 4 small (S1, S2, S3 and S4) segments. In contrast, the empty virus particles without genome were non-infectious. Phylogenetic analyses revealed that the Taiwanese strains belong to serotype 2 MRV (MRV2). We established an ELISA for the detection of IgG antibody against MRV2 by using the empty virus particles as the antigen. A total of 540 swine and 95 wild boar serum samples were collected in Japan, and the positive rates were 100% and 52.6%, respectively. These results demonstrated that MRV infection occurred frequently in both swine and wild boar in Japan. We established a cell line that is efficient for the isolation of MRV, and the ELISA based on the naturally occurring empty particles would be of great value for the surveillance of MRV-related diseases.

RevDate: 2021-01-08
CmpDate: 2021-01-08

He XY, Ren XH, Peng Y, et al (2020)

Aptamer/Peptide-Functionalized Genome-Editing System for Effective Immune Restoration through Reversal of PD-L1-Mediated Cancer Immunosuppression.

Advanced materials (Deerfield Beach, Fla.), 32(17):e2000208.

Effective reversal of tumor immunosuppression is of critical importance in cancer therapy. A multifunctional delivery vector that can effectively deliver CRISPR-Cas9 plasmid for β-catenin knockout to reverse tumor immunosuppression is constructed. The multi-functionalized delivery vector is decorated with aptamer-conjugated hyaluronic acid and peptide-conjugated hyaluronic acid to combine the tumor cell/nuclear targeting function of AS1411 with the cell penetrating/nuclear translocation function of TAT-NLS. Due to the significantly enhanced plasmid enrichment in malignant cell nuclei, the genome editing system can induce effective β-catenin knockout and suppress Wnt/β-catenin pathway, resulting in notably downregulated proteins involved in tumor progression and immunosuppression. Programmed death-ligand 1 (PD-L1) downregulation in edited tumor cells not only releases the PD-1/PD-L1 brake to improve the cancer killing capability of CD8+ T cells, but also enhances antitumor immune responses of immune cells. This provides a facile strategy to reverse tumor immunosuppression and to restore immunosurveillance and activate anti-tumor immunity.

RevDate: 2021-01-08
CmpDate: 2021-01-08

Chen L, Cai Y, Qu M, et al (2020)

Soybean adaption to high-latitude regions is associated with natural variations of GmFT2b, an ortholog of FLOWERING LOCUS T.

Plant, cell & environment, 43(4):934-944.

Day length has an important influence on flowering and growth habit in many plant species. In crops such as soybean, photoperiod sensitivity determines the geographical range over which a given cultivar can grow and flower. The soybean genome contains ~10 genes homologous to FT, a central regulator of flowering from Arabidopsis thaliana. However, the precise roles of these soybean FTs are not clearly. Here we show that one such gene, GmFT2b, promotes flowering under long-days (LDs). Overexpression of GmFT2b upregulates expression of flowering-related genes which are important in regulating flowering time. We propose a 'weight' model for soybean flowering under short-day (SD) and LD conditions. Furthermore, we examine GmFT2b sequences in 195 soybean cultivars, as well as flowering phenotypes, geographical distributions and maturity groups. We found that Hap3, a major GmFT2b haplotype, is associated with significantly earlier flowering at higher latitudes. We anticipate our assay to provide important resources for the genetic improvement of soybean, including new germplasm for soybean breeding, and also increase our understanding of functional diversity in the soybean FT gene family.

RevDate: 2021-01-08
CmpDate: 2021-01-08

Kandul NP, Liu J, Buchman A, et al (2020)

Assessment of a Split Homing Based Gene Drive for Efficient Knockout of Multiple Genes.

G3 (Bethesda, Md.), 10(2):827-837.

Homing based gene drives (HGD) possess the potential to spread linked cargo genes into natural populations and are poised to revolutionize population control of animals. Given that host encoded genes have been identified that are important for pathogen transmission, targeting these genes using guide RNAs as cargo genes linked to drives may provide a robust method to prevent disease transmission. However, effectiveness of the inclusion of additional guide RNAs that target separate genes has not been thoroughly explored. To test this approach, we generated a split-HGD in Drosophila melanogaster that encoded a drive linked effector consisting of a second gRNA engineered to target a separate host-encoded gene, which we term a gRNA-mediated effector (GME). This design enabled us to assess homing and knockout efficiencies of two target genes simultaneously, and also explore the timing and tissue specificity of Cas9 expression on cleavage/homing rates. We demonstrate that inclusion of a GME can result in high efficiency of disruption of both genes during super-Mendelian propagation of split-HGD. Furthermore, both genes were knocked out one generation earlier than expected indicating the robust somatic expression of Cas9 driven by Drosophila germline-limited promoters. We also assess the efficiency of 'shadow drive' generated by maternally deposited Cas9 protein and accumulation of drive-induced resistance alleles along multiple generations, and discuss design principles of HGD that could mitigate the accumulation of resistance alleles while incorporating a GME.

RevDate: 2021-01-08
CmpDate: 2021-01-08

Dekkers JF, Whittle JR, Vaillant F, et al (2020)

Modeling Breast Cancer Using CRISPR-Cas9-Mediated Engineering of Human Breast Organoids.

Journal of the National Cancer Institute, 112(5):540-544.

Breast cancer is characterized by histological and functional heterogeneity, posing a clinical challenge for patient treatment. Emerging evidence suggests that the distinct subtypes reflect the repertoire of genetic alterations and the target cell. However, the precise initiating events that predispose normal epithelium to neoplasia are poorly understood. Here, we demonstrate that breast epithelial organoids can be generated from human reduction mammoplasties (12 out of 12 donors), thus creating a tool to study the clonal evolution of breast cancer. To recapitulate de novo oncogenesis, we exploited clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 for targeted knockout of four breast cancer-associated tumor suppressor genes (P53, PTEN, RB1, NF1) in mammary progenitor cells from six donors. Mutant organoids gained long-term culturing capacity and formed estrogen-receptor positive luminal tumors on transplantation into mice for one out of six P53/PTEN/RB1-mutated and three out of six P53/PTEN/RB1/NF1-mutated lines. These organoids responded to endocrine therapy or chemotherapy, supporting the potential utility of this model to enhance our understanding of the molecular events that culminate in specific subtypes of breast cancer.

RevDate: 2021-01-07

Liu PF, Zhao KR, Liu ZJ, et al (2021)

Cas12a-based electrochemiluminescence biosensor for target amplification-free DNA detection.

Biosensors & bioelectronics, 176:112954 pii:S0956-5663(20)30939-8 [Epub ahead of print].

CRISPR/Cas system have drawn increasing attention in accurate and sensitive nucleic acids detection. Herein, we reported a novel Cas12a-based electrochemiluminescence biosensor for target amplification-free human papilloma virus subtype (HPV-16) DNA detection. During this detection process, Cas12a employed its two-part recognition mechanism to improve the specificity and trans-cleavage capability to achieve signal amplification, while L-Methionine stabilized gold nanoclusters (Met-AuNCs) were served as high-efficiency ECL emitters to achieve ECL signal transition. Given the unique combination of Cas12a with ECL technique, the detection limit was determined as 0.48 pM and the whole detection could be completed within 70 min. We also validated the practical application of the proposed biosensor by using undiluted human blood samples, which gives impetus to the design of new generations of CRISPR/Cas detection system beyond the traditional ones with ultimate applications in sensing analysis and diagnostic technologies.

RevDate: 2021-01-07

Jubair L, Lam AK, Fallaha S, et al (2021)

CRISPR/Cas9-loaded stealth liposomes effectively cleared established HPV16-driven tumours in syngeneic mice.

PloS one, 16(1):e0223288 pii:PONE-D-19-24737.

Gene-editing has raised the possibility of being able to treat or cure cancers, but key challenges remain, including efficient delivery, in vivo efficacy, and its safety profile. Ideal targets for cancer therapy are oncogenes, that when edited, cause cell death. Here, we show, using the human papillomavirus (HPV) type 16 cancer cell line TC1, that CRISPR/Cas9 targeting the E7 oncogene and packaged in PEGylated liposomes cleared established tumours in immunocompetent mice. Treatment caused no significant toxicity in the spleen or liver. An ideal therapeutic outcome would be the induction of an immunogenic cell death (ICD), such that recurrent tumours would be eliminated by the host immune system. We show here for the first time that CRISPR/Cas9-mediated cell death via targeting E7 did not result in ICD. Overall, our data show that in vivo CRISPR/Cas targeting of oncogenes is an effective treatment approach for cancer.

RevDate: 2021-01-07

Zhou T, Huang M, Lin J, et al (2021)

High-Fidelity CRISPR/Cas13a trans-Cleavage-Triggered Rolling Circle Amplified DNAzyme for Visual Profiling of MicroRNA.

Analytical chemistry [Epub ahead of print].

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) (CRISPR/Cas) system innovates a next-generation biosensor due to its high-fidelity, programmability, and efficient signal amplification ability. Developing a CRISPR/Cas-based visual detection system could contribute to point-of-care biomarker diagnosis. Existing CRISPR/Cas9-mediated visual detection methods are limited by the inherent properties of Cas9. Herein, we explored the trans-cleavage ability of Cas13a on ribonucleotide-bearing DNA oligo, eliminated the unavailability of the trans-cleavage substrate for subsequent polymerization reaction, and developed a homogeneous CRISPR/Cas13a-based visual detection system (termed vCas) for specific and sensitive detection of miRNA. The results indicated that vCas can provide a detection limit of 1 fM for miR-10b with single-base specificity and can be used to analyze miRNA in serum and cell extracts. Conclusively, vCas holds a great application prospective for clinical molecular diagnosis.

RevDate: 2021-01-07

Sreepadmanabh M, Sahu AK, A Chande (2020)

COVID-19: Advances in diagnostic tools, treatment strategies, and vaccine development.

Journal of biosciences, 45:.

An unprecedented worldwide spread of the SARS-CoV-2 has imposed severe challenges on healthcare facilities and medical infrastructure. The global research community faces urgent calls for the development of rapid diagnostic tools, effective treatment protocols, and most importantly, vaccines against the pathogen. Pooling together expertise across broad domains to innovate effective solutions is the need of the hour. With these requirements in mind, in this review, we provide detailed critical accounts on the leading efforts at developing diagnostics tools, therapeutic agents, and vaccine candidates. Importantly, we furnish the reader with a multidisciplinary perspective on how conventional methods like serology and RT-PCR, as well as cutting-edge technologies like CRISPR/Cas and artificial intelligence/machine learning, are being employed to inform and guide such investigations. We expect this narrative to serve a broad audience of both active and aspiring researchers in the field of biomedical sciences and engineering and help inspire radical new approaches towards effective detection, treatment, and prevention of this global pandemic.

RevDate: 2021-01-06

Konishi CT, C Long (2020)

Progress and challenges in CRISPR-mediated therapeutic genome editing for monogenic diseases.

Journal of biomedical research [Epub ahead of print].

There are an estimated 10 000 monogenic diseases affecting tens of millions of individuals worldwide. The application of CRISPR/Cas genome editing tools to treat monogenic diseases is an emerging strategy with the potential to generate personalized treatment approaches for these patients. CRISPR/Cas-based systems are programmable and sequence-specific genome editing tools with the capacity to generate base pair resolution manipulations to DNA or RNA. The complexity of genomic insults resulting in heritable disease requires patient-specific genome editing strategies with consideration of DNA repair pathways, and CRISPR/Cas systems of different types, species, and those with additional enzymatic capacity and/or delivery methods. In this review we aim to discuss broad and multifaceted therapeutic applications of CRISPR/Cas gene editing systems including in harnessing of homology directed repair, non-homologous end joining, microhomology-mediated end joining, and base editing to permanently correct diverse monogenic diseases.

RevDate: 2021-01-06

Zhang Y, Xi H, M Juhas (2020)

Biosensing Detection of the SARS-CoV-2 D614G Mutation.

Trends in genetics : TIG pii:S0168-9525(20)30334-6 [Epub ahead of print].

The emergence of a mutant strain of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with an amino acid change from aspartate to a glycine residue at position 614 (D614G) has been reported and this mutant appears to be now dominant in the pandemic. Efficient detection of the SARS-CoV-2 D614G mutant by biosensing technologies is therefore crucial for the control of the pandemic.

RevDate: 2021-01-06

Taliansky M, Samarskaya V, Zavriev SK, et al (2021)

RNA-Based Technologies for Engineering Plant Virus Resistance.

Plants (Basel, Switzerland), 10(1): pii:plants10010082.

In recent years, non-coding RNAs (ncRNAs) have gained unprecedented attention as new and crucial players in the regulation of numerous cellular processes and disease responses. In this review, we describe how diverse ncRNAs, including both small RNAs and long ncRNAs, may be used to engineer resistance against plant viruses. We discuss how double-stranded RNAs and small RNAs, such as artificial microRNAs and trans-acting small interfering RNAs, either produced in transgenic plants or delivered exogenously to non-transgenic plants, may constitute powerful RNA interference (RNAi)-based technology that can be exploited to control plant viruses. Additionally, we describe how RNA guided CRISPR-CAS gene-editing systems have been deployed to inhibit plant virus infections, and we provide a comparative analysis of RNAi approaches and CRISPR-Cas technology. The two main strategies for engineering virus resistance are also discussed, including direct targeting of viral DNA or RNA, or inactivation of plant host susceptibility genes. We also elaborate on the challenges that need to be overcome before such technologies can be broadly exploited for crop protection against viruses.

RevDate: 2021-01-07
CmpDate: 2021-01-07

Carlson-Stevermer J, Das A, Abdeen AA, et al (2020)

Design of efficacious somatic cell genome editing strategies for recessive and polygenic diseases.

Nature communications, 11(1):6277.

Compound heterozygous recessive or polygenic diseases could be addressed through gene correction of multiple alleles. However, targeting of multiple alleles using genome editors could lead to mixed genotypes and adverse events that amplify during tissue morphogenesis. Here we demonstrate that Cas9-ribonucleoprotein-based genome editors can correct two distinct mutant alleles within a single human cell precisely. Gene-corrected cells in an induced pluripotent stem cell model of Pompe disease expressed the corrected transcript from both corrected alleles, leading to enzymatic cross-correction of diseased cells. Using a quantitative in silico model for the in vivo delivery of genome editors into the developing human infant liver, we identify progenitor targeting, delivery efficiencies, and suppression of imprecise editing outcomes at the on-target site as key design parameters that control the efficacy of various therapeutic strategies. This work establishes that precise gene editing to correct multiple distinct gene variants could be highly efficacious if designed appropriately.

RevDate: 2021-01-06
CmpDate: 2021-01-06

Lin MT, Lai LL, Zhao M, et al (2020)

[Construction of a striatum-specific Slc20a2 gene knockout mice model by CRISPR/Cas9 AAV system].

Yi chuan = Hereditas, 42(10):1017-1027.

Primary familial brain calcification (PFBC) is a chronic progressive neurogenetic disorder. Its clinical symptoms mainly include dyskinesia, cognitive disorder and mental impairment; and the pathogenesis remains unclear. Studies have shown that SLC20A2 is the most common pathogenic gene of the disease. Since the Slc20a2 gene knockout mouse model could result in fetal growth restriction, in order to better understand the pathogenesis of PFBC, the present study used the CRISPR/Cas9 technology to construct a conditional knockout model of Slc20a2 gene in the striatum of mice. First, three sgRNAs (single guide RNAs) were designed to target the exon3 of Slc20a2 gene. The activity of the respective sgRNA was verified by constructing expression plasmids, transfecting cells and Surveyor assay. Second, the SgRNA with the highest activity was selected to generate the recombinant AAV-Cre virus, which was injected into the striatum of mice by stereotactic method. In vitro experiments showed that the three sgRNAs could effectively mediate Cas9 cleavage of the respective target DNA. The activity of Cre recombinase of the AAV-Cre was confirmed by immunofluorescence assay. Immunohistochemistry, TA clone, high-throughput sequencing and Western blot were used to detect and evaluate the efficiency of Slc20a2 gene knockout. The results showed that the Slc20a2 expression in the striatum of mice in the experimental group decreased significantly. In this study, three sgRNAs capable of knockout of Slc20a2 were successfully designed, and the conditional knockout of the Slc20a2 gene in the striatum of mouse was successfully established by the CRISPR/Cas9 technology, thereby providing an effective animal model for studying the pathogenesis of PFBC.

RevDate: 2021-01-06
CmpDate: 2021-01-06

Bao LW, Zhou YY, FY Zeng (2020)

[Advances in gene therapy for β-thalassemia and hemophilia based on the CRISPR/Cas9 technology].

Yi chuan = Hereditas, 42(10):949-964.

Thalassemia and hemophilia are common inherited blood disorders caused by genetic abnormalities. These diseases are difficult to cure and can be inherited to the next generation, causing severe family and social burden. The emergence of gene therapy provides a new treatment for genetic diseases. However, since its first clinical trial in 1990, the development of gene therapy has not been as optimistic in the past three decades as one could hope. The development of gene-editing technology, particularly the third generation gene-editing technology CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9), has given hope in such therapeutic approach for having advantages in high editing efficiency, simple operation, and low cost. Gene editing-mediated gene therapy has thus received increasing attention from the biomedical community. It has shown promises for the treatment of inherited blood disorders, such as thalassemia and hemophilia. This paper reviews the fundamental research progress of gene therapy for β-thalassemia and hemophilia based on CRISPR/Cas9 technology in the past six years. It also summarizes the CRISPR/Cas9-based clinical trials of gene therapy. The problems and possible solutions to this technology for gene therapy are also discussed, thereby providing a reference for the research on gene therapy of inherited blood disorders based on CRISPR/Cas9 technology.

RevDate: 2021-01-06
CmpDate: 2021-01-06

Alghamdi A, Aldossary W, Albahkali S, et al (2020)

The loss of microglia activities facilitates glaucoma progression in association with CYP1B1 gene mutation (p.Gly61Glu).

PloS one, 15(11):e0241902.

BACKGROUND: Glaucoma represents the second main cause of irreversible loss of eyesight worldwide. Progression of the disease is due to changes around the optic nerve, eye structure and optic nerve environment. Focusing on primary congenital glaucoma, which is not completely understood, we report an evaluation of an untested mutation (c.182G>A, p.Gly61Glu) within the CYP1B1 gene in the context of microglia, astrocytes and mesenchymal stem cells. We investigated the behaviours of these cells, which are needed to maintain eye homeostasis, in response to the CYP1B1 mutation.

METHODS AND RESULTS: CRISPR technology was used to edit normal CYP1B1 genes within normal astrocytes, microglia and stem cells in vitro. Increased metabolic activities were found in microglia and astrocytes 24 hours after CYP1B1 manipulation. However, these activities dropped by 40% after 72 hrs. In addition, the nicotinamide adenine dinucleotide phosphate (NADP)/NADPH reducing equivalent process decreased by 50% on average after 72 hrs of manipulation. The cytokines measured in mutated microglia showed progressive activation leading to apoptosis, which was confirmed with annexin-V. The cytokines evaluated in mutant astrocytes were abnormal in comparison to those in the control.

CONCLUSIONS: The results suggest a progressive inflammation that was induced by mutations (p.Gly61Glu) on CYP1B1. Furthermore, the mutations enhanced the microglia's loss of activity. We are the first to show the direct impact of the mutation on microglia. This progressive inflammation might be responsible for primary congenital glaucoma complications, which could be avoided via an anti-inflammatory regimen. This finding also reveals that progressive inflammation affects recovery failure after surgeries to relieve glaucoma. Moreover, microglia are important for the survival of ganglion cells, along with the clearing of pathogens and inflammation. The reduction of their activities may jeopardise homeostasis within the optic nerve environment and complicate the protection of optic nerve components (such as retinal ganglion and glial cells).

RevDate: 2021-01-06
CmpDate: 2021-01-06

Liu H, Robinson DS, Wu ZY, et al (2020)

Bacterial genome editing by coupling Cre-lox and CRISPR-Cas9 systems.

PloS one, 15(11):e0241867.

The past decade has been a golden age for microbiology, marked by the discovery of an unprecedented increase in the number of novel bacterial species. Yet gaining biological knowledge of those organisms has not kept pace with sequencing efforts. To unlock this genetic potential there is an urgent need for generic (i.e. non-species specific) genetic toolboxes. Recently, we developed a method, termed chassis-independent recombinase-assisted genome engineering (CRAGE), enabling the integration and expression of large complex gene clusters directly into the chromosomes of diverse bacteria. Here we expand upon this technology by incorporating CRISPR-Cas9 allowing precise genome editing across multiple bacterial species. To do that we have developed a landing pad that carries one wild-type and two mutant lox sites to allow integration of foreign DNA at two locations through Cre-lox recombinase-mediated cassette exchange (RMCE). The first RMCE event is to integrate the Cas9 and the DNA repair protein genes RecET, and the second RMCE event enables the integration of customized sgRNA and a repair template. Following this workflow, we achieved precise genome editing in four different gammaproteobacterial species. We also show that the inserted landing pad and the entire editing machinery can be removed scarlessly after editing. We report here the construction of a single landing pad transposon and demonstrate its functionality across multiple species. The modular design of the landing pad and accessory vectors allows design and assembly of genome editing platforms for other organisms in a similar way. We believe this approach will greatly expand the list of bacteria amenable to genetic manipulation and provides the means to advance our understanding of the microbial world.

RevDate: 2021-01-07
CmpDate: 2021-01-07

Schleicher EM, Dhoonmoon A, Jackson LM, et al (2020)

Dual genome-wide CRISPR knockout and CRISPR activation screens identify mechanisms that regulate the resistance to multiple ATR inhibitors.

PLoS genetics, 16(11):e1009176.

The ataxia telangiectasia and Rad3-related (ATR) protein kinase is a key regulator of the cellular response to DNA damage. Due to increased amount of replication stress, cancer cells heavily rely on ATR to complete DNA replication and cell cycle progression. Thus, ATR inhibition is an emerging target in cancer therapy, with multiple ATR inhibitors currently undergoing clinical trials. Here, we describe dual genome-wide CRISPR knockout and CRISPR activation screens employed to comprehensively identify genes that regulate the cellular resistance to ATR inhibitors. Specifically, we investigated two different ATR inhibitors, namely VE822 and AZD6738, in both HeLa and MCF10A cells. We identified and validated multiple genes that alter the resistance to ATR inhibitors. Importantly, we show that the mechanisms of resistance employed by these genes are varied, and include restoring DNA replication fork progression, and prevention of ATR inhibitor-induced apoptosis. In particular, we describe a role for MED12-mediated inhibition of the TGFβ signaling pathway in regulating replication fork stability and cellular survival upon ATR inhibition. Our dual genome-wide screen findings pave the way for personalized medicine by identifying potential biomarkers for ATR inhibitor resistance.

RevDate: 2021-01-07
CmpDate: 2021-01-07

Chen K, Yu Y, Yang D, et al (2020)

Gtsf1 is essential for proper female sex determination and transposon silencing in the silkworm, Bombyx mori.

PLoS genetics, 16(11):e1009194.

Sex determination pathways are astoundingly diverse in insects. For instance, the silk moth Bombyx mori uniquely use various components of the piRNA pathway to produce the Fem signal for specification of the female fate. In this study, we identified BmGTSF1 as a novel piRNA factor which participates in B. mori sex determination. We found that BmGtsf1 has a distinct expression pattern compared to Drosophila and mouse. CRISPR/Cas9 induced mutation in BmGtsf1 resulted in partial sex reversal in genotypically female animals by shifting expression of the downstream targets BmMasc and Bmdsx to the male pattern. As levels of Fem piRNAs were substantially reduced in female mutants, we concluded that BmGtsf1 plays a critical role in the biogenesis of the feminizing signal. We also demonstrated that BmGTSF1 physically interacted with BmSIWI, a protein previously reported to be involved in female sex determination, indicating BmGTSF1 function as the cofactor of BmSIWI. BmGtsf1 mutation resulted in piRNA pathway dysregulation, including piRNA biogenesis defects and transposon derepression, suggesting BmGtsf1 is also a piRNA factor in the silkworm. Furthermore, we found that BmGtsf1 mutation leads to gametogenesis defects in both male and female. Our data suggested that BmGtsf1 is a new component involved in the sex determination pathway in B. mori.

RevDate: 2021-01-07
CmpDate: 2021-01-07

Lin RJ, Kuo MW, Yang BC, et al (2020)

B3GALT5 knockout alters gycosphingolipid profile and facilitates transition to human naïve pluripotency.

Proceedings of the National Academy of Sciences of the United States of America, 117(44):27435-27444.

Conversion of human pluripotent stem cells from primed to naïve state is accompanied by altered transcriptome and methylome, but glycosphingolipid (GSL) profiles in naïve human embryonic stem cells (hESCs) have not been systematically characterized. Here we showed a switch from globo-(SSEA-3, SSEA-4, and Globo H) and lacto-series (fucosyl-Lc4Cer) to neolacto-series GSLs (SSEA-1 and H type 2 antigen), along with marked down-regulation of β-1,3-galactosyltransferase (B3GALT5) upon conversion to naïve state. CRISPR/Cas9-generated B3GALT5-knockout (KO) hESCs displayed an altered GSL profile, increased cloning efficiency and intracellular Ca2+, reminiscent of the naïve state, while retaining differentiation ability. The altered GSLs could be rescued through overexpression of B3GALT5. B3GALT5-KO cells cultured with 2iLAF exhibited naïve-like transcriptome, global DNA hypomethylation, and X-chromosome reactivation. In addition, B3GALT5-KO rendered hESCs more resistant to calcium chelator in blocking entry into naïve state. Thus, loss of B3GALT5 induces a distinctive state of hESCs displaying unique GSL profiling with expression of neolacto-glycans, increased Ca2+, and conducive for transition to naïve pluripotency.

RevDate: 2021-01-07
CmpDate: 2021-01-07

Müller M, Schneider M, Salathé M, et al (2020)

Assessing Public Opinion on CRISPR-Cas9: Combining Crowdsourcing and Deep Learning.

Journal of medical Internet research, 22(8):e17830.

BACKGROUND: The discovery of the CRISPR-Cas9-based gene editing method has opened unprecedented new potential for biological and medical engineering, sparking a growing public debate on both the potential and dangers of CRISPR applications. Given the speed of technology development and the almost instantaneous global spread of news, it is important to follow evolving debates without much delay and in sufficient detail, as certain events may have a major long-term impact on public opinion and later influence policy decisions.

OBJECTIVE: Social media networks such as Twitter have shown to be major drivers of news dissemination and public discourse. They provide a vast amount of semistructured data in almost real-time and give direct access to the content of the conversations. We can now mine and analyze such data quickly because of recent developments in machine learning and natural language processing.

METHODS: Here, we used Bidirectional Encoder Representations from Transformers (BERT), an attention-based transformer model, in combination with statistical methods to analyze the entirety of all tweets ever published on CRISPR since the publication of the first gene editing application in 2013.

RESULTS: We show that the mean sentiment of tweets was initially very positive, but began to decrease over time, and that this decline was driven by rare peaks of strong negative sentiments. Due to the high temporal resolution of the data, we were able to associate these peaks with specific events and to observe how trending topics changed over time.

CONCLUSIONS: Overall, this type of analysis can provide valuable and complementary insights into ongoing public debates, extending the traditional empirical bioethics toolset.

RevDate: 2021-01-07
CmpDate: 2021-01-07

Yokouchi Y, Suzuki S, Ohtsuki N, et al (2020)

Rapid repair of human disease-specific single-nucleotide variants by One-SHOT genome editing.

Scientific reports, 10(1):13927.

Many human diseases ranging from cancer to hereditary disorders are caused by single-nucleotide mutations in critical genes. Repairing these mutations would significantly improve the quality of life for patients with hereditary diseases. However, current procedures for repairing deleterious single-nucleotide mutations are not straightforward, requiring multiple steps and taking several months to complete. In the current study, we aimed to repair pathogenic allele-specific single-nucleotide mutations using a single round of genome editing. Using high-fidelity, site-specific nuclease AsCas12a/Cpf1, we attempted to repair pathogenic single-nucleotide variants (SNVs) in disease-specific induced pluripotent stem cells. As a result, we achieved repair of the Met918Thr SNV in human oncogene RET with the inclusion of a single-nucleotide marker, followed by absolute markerless, scarless repair of the RET SNV with no detected off-target effects. The markerless method was then confirmed in human type VII collagen-encoding gene COL7A1. Thus, using this One-SHOT method, we successfully reduced the number of genetic manipulations required for genome repair from two consecutive events to one, resulting in allele-specific repair that can be completed within 3 weeks, with or without a single-nucleotide marker. Our findings suggest that One-SHOT can be used to repair other types of mutations, with potential beyond human medicine.

RevDate: 2021-01-07
CmpDate: 2021-01-07

D'Amore C, Moro E, Borgo C, et al (2020)

"Janus" efficacy of CX-5011: CK2 inhibition and methuosis induction by independent mechanisms.

Biochimica et biophysica acta. Molecular cell research, 1867(11):118807.

Methuosis has been described as a distinctive form of cell death characterized by the displacement of large fluid-filled vacuoles derived from uncontrolled macropinocytosis. Its induction has been proposed as a new strategy against cancer cells. Small molecules, such as indole-based calchones, have been identified as methuosis inducers and, recently, the CK2 inhibitor CX-4945 has been shown to have a similar effect on different cell types. However, the contribution of protein kinase CK2 to methuosis signalling is still controversial. Here we show that methuosis is not related to CK2 activity since it is not affected by structurally unrelated CK2 inhibitors and genetic reduction/ablation of CK2 subunits. Interestingly, CX-5011, a CK2 inhibitor related to CX-4945, behaves as a CK2-independent methuosis inducer, four times more powerful than its parental compound and capable to promote the formation on enlarged cytosolic vacuoles at low micromolar concentrations. We show that pharmacological inhibition of the small GTPase Rac-1, its downregulation by siRNA treatment, or the over-expression of the dominant-negative mutated form of Rac-1 (Rac-1 T17N), impairs CX-5011 ability to induce methuosis. Furthermore, cell treatment with CX-5011 induces a durable activation of Rac-1 that persists for at least 24 h. Worthy of note, CX-5011 is able to promote macropinocytosis not only in mammalian cells, but also in an in-vivo zebrafish model. Based on these evidences, CX-5011 is, therefore, proposed as a potential promising compound for cancer therapies for its dual efficacy as an inhibitor of the pro-survival kinase CK2 and inducer of methuosis.

RevDate: 2021-01-07
CmpDate: 2021-01-07

Domènech EB, Andrés R, López-Iniesta MJ, et al (2020)

A New Cerkl Mouse Model Generated by CRISPR-Cas9 Shows Progressive Retinal Degeneration and Altered Morphological and Electrophysiological Phenotype.

Investigative ophthalmology & visual science, 61(8):14.

Purpose: Close to 100 genes cause retinitis pigmentosa, a Mendelian rare disease that affects 1 out of 4000 people worldwide. Mutations in the ceramide kinase-like gene (CERKL) are a prevalent cause of autosomal recessive cause retinitis pigmentosa and cone-rod dystrophy, but the functional role of this gene in the retina has yet to be fully determined. We aimed to generate a mouse model that resembles the phenotypic traits of patients carrying CERKL mutations to undertake functional studies and assay therapeutic approaches.

Methods: The Cerkl locus has been deleted (around 97 kb of genomic DNA) by gene editing using the CRISPR-Cas9 D10A nickase. Because the deletion of the Cerkl locus is lethal in mice in homozygosis, a double heterozygote mouse model with less than 10% residual Cerkl expression has been generated. The phenotypic alterations of the retina of this new model have been characterized at the morphological and electrophysiological levels.

Results: This CerklKD/KO model shows retinal degeneration, with a decreased number of cones and progressive photoreceptor loss, poorly stacked photoreceptor outer segment membranes, defective retinal pigment epithelium phagocytosis, and altered electrophysiological recordings in aged retinas.

Conclusions: To our knowledge, this is the first Cerkl mouse model to mimic many of the phenotypic traits, including the slow but progressive retinal degeneration, shown by human patients carrying CERKL mutations. This useful model will provide unprecedented insights into the retinal molecular pathways altered in these patients and will contribute to the design of effective treatments.

RevDate: 2021-01-06
CmpDate: 2021-01-06

Przewrocka J, Rowan A, Rosenthal R, et al (2020)

Unintended on-target chromosomal instability following CRISPR/Cas9 single gene targeting.

Annals of oncology : official journal of the European Society for Medical Oncology, 31(9):1270-1273.

RevDate: 2021-01-06
CmpDate: 2021-01-06

Xie T, Chen X, Guo T, et al (2020)

Targeted Knockout of BnTT2 Homologues for Yellow-Seeded Brassica napus with Reduced Flavonoids and Improved Fatty Acid Composition.

Journal of agricultural and food chemistry, 68(20):5676-5690.

Brassica napus is one of the important oil crops grown worldwide, and oil quality improvement is a major goal in rapeseed breeding. Yellow seed is an excellent trait, which has great potential in improving seed quality and economic value. In this study, we created stable yellow seed mutants using a CRISPR/Cas9 system and obtained the yellow seed phenotype only when the four alleles of two BnTT2 homologues were knocked out, indicating that the two BnTT2 homologues had conserved but redundant functions in regulating seed color. Histochemical staining and flavonoid metabolic analysis proved that the BnTT2 mutation hindered the synthesis and accumulation of proanthocyanidins. Transcriptome analysis also showed that the BnTT2 mutation inhibited the expression of genes in the phenylpropanoid and flavonoid biosynthetic pathway, which might be regulated by the complex of BnTT2, BnTT8 and BnTTG1. In addition, the homozygous mutants of BnTT2 homologues increased oil content and improved fatty acid composition with higher linoleic acid (C18:2) and linolenic acid (C18:3), which could be used for the genetic improvement of rapeseed. Overall, this research showed that the BnTT2 mutation can be used for yellow seed breeding and oil improvement, which is of great significance in improving the economic value of rapeseeds.

RevDate: 2021-01-06
CmpDate: 2021-01-06

Shu P, Li Z, Min D, et al (2020)

CRISPR/Cas9-Mediated SlMYC2 Mutagenesis Adverse to Tomato Plant Growth and MeJA-Induced Fruit Resistance to Botrytis cinerea.

Journal of agricultural and food chemistry, 68(20):5529-5538.

Methyl jasmonate (MeJA), a natural phytohormone, played a critical role not only in plant growth but also in plant defense response to biotic and abiotic stresses. MYC2, a basic helix-loop-helix transcription factor, is a master regulator in MeJA signaling pathway. In the present work, slmyc2 mutants were generated by the clustered regularly interspaced short palindromic repeats and associated Cas9 protein (CRISPR/Cas9) system to investigate the role of SlMYC2 in tomato plant growth and fruit disease resistance induced by exogenous MeJA. The results showed that slmyc2 mutants possessed a higher number of flowers and a lower fruit setting rate in comparison with wild-type plants. In addition, the fruit shape of slmyc2 mutant was prolate, while the control fruits were oblate. Knockout of SlMYC2 significantly decreased the activities of disease defensive and antioxidant enzymes, as well as the expression levels of pathogen-related (PR) genes (SlPR-1 and SlPR-STH2) and the key genes related to jasmonic acid (JA) biosynthesis and signaling pathway including allene oxide cyclase (SlAOC), lipoxygenase D (SlLOXD), SlMYC2, and coronatine insensitive 1 (SlCOI1), and consequently aggravated the disease symptoms. By contrast, the disease symptoms were largely reduced in MeJA-treated fruit that possessed higher activities of these enzymes and expression levels of genes. However, the induction effects of MeJA on fruit disease resistance and these enzymes' activities and genes' expressions were significantly attenuated by knockout of SlMYC2. Therefore, the results indicated that SlMYC2 played positive regulatory roles not only in the growth of tomato plants but also in MeJA-induced disease resistance and the antioxidant process in tomato fruits.

RevDate: 2021-01-06
CmpDate: 2021-01-06

Pinzon-Arteaga C, Snyder MD, Lazzarotto CR, et al (2020)

Efficient correction of a deleterious point mutation in primary horse fibroblasts with CRISPR-Cas9.

Scientific reports, 10(1):7411.

Phenotypic selection during animal domestication has resulted in unwanted incorporation of deleterious mutations. In horses, the autosomal recessive condition known as Glycogen Branching Enzyme Deficiency (GBED) is the result of one of these deleterious mutations (102C > A), in the first exon of the GBE1 gene (GBE1102C>A). With recent advances in genome editing, this type of genetic mutation can be precisely repaired. In this study, we used the RNA-guided nuclease CRISPR-Cas9 (clustered regularly-interspaced short palindromic repeats/CRISPR-associated protein 9) to correct the GBE1102C>A mutation in a primary fibroblast cell line derived from a high genetic merit heterozygous stallion. To correct this mutation by homologous recombination (HR), we designed a series of single guide RNAs (sgRNAs) flanking the mutation and provided different single-stranded donor DNA templates. The distance between the Cas9-mediated double-stranded break (DSB) to the mutation site, rather than DSB efficiency, was the primary determinant for successful HR. This framework can be used for targeting other harmful diseases in animal populations.

RevDate: 2021-01-06
CmpDate: 2021-01-06

Audebert C, Bonardi F, Caboche S, et al (2020)

Genetic basis for virulence differences of various Cryptosporidium parvum carcinogenic isolates.

Scientific reports, 10(1):7316.

Cryptosporidium parvum is known to cause life-threatening diarrhea in immunocompromised hosts and was also reported to be capable of inducing digestive adenocarcinoma in a rodent model. Interestingly, three carcinogenic isolates of C. parvum, called DID, TUM1 and CHR, obtained from fecal samples of naturally infected animals or humans, showed higher virulence than the commercially available C. parvum IOWA isolate in our animal model in terms of clinical manifestations, mortality rate and time of onset of neoplastic lesions. In order to discover the potential genetic basis of the differential virulence observed between C. parvum isolates and to contribute to the understanding of Cryptosporidium virulence, entire genomes of the isolates DID, TUM1 and CHR were sequenced then compared to the C. parvum IOWA reference genome. 125 common SNVs corresponding to 90 CDSs were found in the C. parvum genome that could explain this differential virulence. In particular variants in several membrane and secreted proteins were identified. Besides the genes already known to be involved in parasite virulence, this study identified potential new virulence factors whose functional characterization can be achieved through CRISPR/Cas9 technology applied to this parasite.

RevDate: 2021-01-06
CmpDate: 2021-01-06

Soliman SHA, Stark AE, Gardner ML, et al (2020)

Tagging enhances histochemical and biochemical detection of Ran Binding Protein 9 in vivo and reveals its interaction with Nucleolin.

Scientific reports, 10(1):7138.

The lack of tools to reliably detect RanBP9 in vivo has significantly hampered progress in understanding the biological functions of this scaffold protein. We report here the generation of a novel mouse strain, RanBP9-TT, in which the endogenous protein is fused with a double (V5-HA) epitope tag at the C-terminus. We show that the double tag does not interfere with the essential functions of RanBP9. In contrast to RanBP9 constitutive knock-out animals, RanBP9-TT mice are viable, fertile and do not show any obvious phenotype. The V5-HA tag allows unequivocal detection of RanBP9 both by IHC and WB. Importantly, immunoprecipitation and mass spectrometry analyses reveal that the tagged protein pulls down known interactors of wild type RanBP9. Thanks to the increased detection power, we are also unveiling a previously unknown interaction with Nucleolin, a protein proposed as an ideal target for cancer treatment. In summary, we report the generation of a new mouse line in which RanBP9 expression and interactions can be reliably studied by the use of commercially available αtag antibodies. The use of this line will help to overcome some of the existing limitations in the study of RanBP9 and potentially unveil unknown functions of this protein in vivo such as those linked to Nucleolin.

RevDate: 2021-01-06
CmpDate: 2021-01-06

Wu C, Chen Y, Qiu Y, et al (2020)

A simple approach to mediate genome editing in the filamentous fungus Trichoderma reesei by CRISPR/Cas9-coupled in vivo gRNA transcription.

Biotechnology letters, 42(7):1203-1210.

OBJECTIVE: To simplify CRISPR/Cas9 genome editing in the industrial filamentous fungus Trichoderma reesei based on in vivo guide RNA (gRNA) transcription.

RESULTS: Two putative RNA polymerase III U6 snRNA genes were identified in the genome of T. reesei QM6a by BLASTN using Myceliophthora. thermophila U6 snRNA gene as the template. The regions approximately 500 bp upstream of two U6 genes were efficient promoters for the in vivo expression of gRNA. The CRISPR system consisting of Cas9 and in vivo synthesized gRNA under control of the T. reesei U6 snRNA promoters was sufficient to cause a frameshift mutation in the ura5 gene via non-homologous end-joining-mediated events.

CONCLUSIONS: We report a simple gene editing method using a CRISPR/Cas9-coupled in vivo gRNA transcription system in T. reesei.

RevDate: 2021-01-07
CmpDate: 2021-01-07

Anwar A, JK Kim (2020)

Transgenic Breeding Approaches for Improving Abiotic Stress Tolerance: Recent Progress and Future Perspectives.

International journal of molecular sciences, 21(8):.

The recent rapid climate changes and increasing global population have led to an increased incidence of abiotic stress and decreased crop productivity. Environmental stresses, such as temperature, drought, nutrient deficiency, salinity, and heavy metal stresses, are major challenges for agriculture, and they lead to a significant reduction in crop growth and productivity. Abiotic stress is a very complex phenomenon, involving a variety of physiological and biochemical changes in plant cells. Plants exposed to abiotic stress exhibit enhanced levels of ROS (reactive oxygen species), which are highly reactive and toxic and affect the biosynthesis of chlorophyll, photosynthetic capacity, and carbohydrate, protein, lipid, and antioxidant enzyme activities. Transgenic breeding offers a suitable alternative to conventional breeding to achieve plant genetic improvements. Over the last two decades, genetic engineering/transgenic breeding techniques demonstrated remarkable developments in manipulations of the genes for the induction of desired characteristics into transgenic plants. Transgenic approaches provide us with access to identify the candidate genes, miRNAs, and transcription factors (TFs) that are involved in specific plant processes, thus enabling an integrated knowledge of the molecular and physiological mechanisms influencing the plant tolerance and productivity. The accuracy and precision of this phenomenon assures great success in the future of plant improvements. Hence, transgenic breeding has proven to be a promising tool for abiotic stress improvement in crops. This review focuses on the potential and successful applications, recent progress, and future perspectives of transgenic breeding for improving abiotic stress tolerance and productivity in plants.

RevDate: 2021-01-06
CmpDate: 2021-01-06

Hanzawa N, Hashimoto K, Yuan X, et al (2020)

Targeted DNA demethylation of the Fgf21 promoter by CRISPR/dCas9-mediated epigenome editing.

Scientific reports, 10(1):5181.

Recently, we reported PPARα-dependent DNA demethylation of the Fgf21 promoter in the postnatal mouse liver, where reduced DNA methylation is associated with enhanced gene expression after PPARα activation. However, there is no direct evidence for the effect of site-specific DNA methylation on gene expression. We employed the dCas9-SunTag and single-chain variable fragment (scFv)-TET1 catalytic domain (TET1CD) system to induce targeted DNA methylation of the Fgf21 promoter both in vitro and in vivo. We succeeded in targeted DNA demethylation of the Fgf 21 promoter both in Hepa1-6 cells and PPARα-deficient mice, with increased gene expression response to PPARα synthetic ligand administration and fasting, respectively. This study provides direct evidence that the DNA methylation status of a particular gene may determine the magnitude of the gene expression response to activation cues.

RevDate: 2021-01-06
CmpDate: 2021-01-06

Tu K, Deng H, Kong L, et al (2020)

Reshaping Tumor Immune Microenvironment through Acidity-Responsive Nanoparticles Featured with CRISPR/Cas9-Mediated Programmed Death-Ligand 1 Attenuation and Chemotherapeutics-Induced Immunogenic Cell Death.

ACS applied materials & interfaces, 12(14):16018-16030.

Blocking immune checkpoints with monoclonal antibody has been verified to achieve potential clinical successes for cancer immunotherapy. However, its application has been impeded by the "cold" tumor microenvironment. Here, weak acidity-responsive nanoparticles co-loaded with CRISPR/Cas9 and paclitaxel (PTX) with the ability to convert "cold" tumor into "hot" tumor are reported. The nanoparticles exhibited high cargo packaging capacity, superior transfection efficiency, well biocompatibility, and effective tumor accumulation. The CRISPR/Cas9 encapsulated in nanoparticles could specifically knock out cyclin-dependent kinase 5 gene to significantly attenuate the expression of programmed death-ligand 1 on tumor cells. More importantly, PTX co-delivered in nanoparticles could significantly induce immunogenic cell death, reduce regulatory T lymphocytes, repolarize tumor-associated macrophages, and enhance antitumor immunity. Therefore, the nanoparticles could effectively convert cold tumor into hot tumor, achieve effective tumor growth inhibition, and prolong overall survival from 16 to 36 days. This research provided a referable strategy for the development of combinatorial immunotherapy and chemotherapy.

RevDate: 2021-01-07
CmpDate: 2021-01-07

Zhang XK, Wang DN, Chen J, et al (2020)

Metabolic engineering of β-carotene biosynthesis in Yarrowia lipolytica.

Biotechnology letters, 42(6):945-956.

OBJECTIVE: Carotenoids, as potent antioxidant compounds, have gained extensive attention, especially in human health. In this study, the combination of CRISPR/Cas9 integration strategy and fermenter cultivation was utilized to obtain efficient β-carotene-producing Yarrowia lipolytica cell factories for potential industrial application.

RESULTS: The introduction of the genes of Mucor circinelloides, encoding phytoene dehydrogenase (carB) and bifunctional phytoene synthase/lycopene cyclase (carRP), contributed to the heterologous production of β-carotene in Y. lipolytica XK2. Furthermore, β-carotene production was efficiently enhanced by increasing the copy numbers of the carB and carRP genes and overexpressing of GGS1, ERG13, and HMG, the genes related to the mevalonate (MVA) pathway. Thus, the optimized strain overexpressed a total of eight genes, including three copies of carRP, two copies of carB, and single copies of GGS1, HMG, and ERG13. As a consequence, strain Y. lipolytica XK19 accumulated approximately 408 mg/L β-carotene in shake flask cultures, a twenty-four-fold increase compared to the parental strain Y. lipolytica XK2.

CONCLUSIONS: 4.5 g/L β-carotene was obtained in a 5-L fermenter through a combination of genetic engineering and culture optimization, suggesting a great capacity and flexibility of Y. lipolytica in the production of carotenoids.

RevDate: 2021-01-07
CmpDate: 2021-01-07

Rojas-Sánchez U, López-Calleja AC, Millán-Chiu BE, et al (2020)

Enhancing the yield of human erythropoietin in Aspergillus niger by introns and CRISPR-Cas9.

Protein expression and purification, 168:105570.

Aspergillus niger has been employed to produce heterologous proteins due to its high capacity for expression and secretion; nevertheless, expression levels of human proteins have been modest. We were interested in investigating whether A. niger can express and secret human erythropoietin (HuEPO) at high yields. Our strategy was to combine the presence of introns with CRISPR-Cas9 to increase the yield of the recombinant protein. The epo gene was codon-optimized and its expression driven by the PmbfA promoter. Another version of epo contained introns from the fructose-1,6-bisphosphatase (fbp) gene. Two recombinant clones, uME12 (no introns) and uME23 (with introns), were selected based on the resistance to the antibiotic and because they showed a protein profile different from that of the parental strain, as shown by SDS-PAGE. Expression of epo was confirmed by RT-PCR in both colonies but the recombinant EPO protein (rHUEPO) was detected by Western blot only in uME23. The rHuEPO yield from uME23 was estimated at about 1.8 mg L-1 by ELISA, demonstrating that the presence of introns resulted in higher yield, possibly by conferring more stability to mRNA. On the other hand, as part of our strategy we decided to inactivate in the strain uME23 the following genes vps, prtT, algC and och1 which are involved in protein secretion, regulating of protease expression and protein glycosylation in A. niger, with CRISPR-Cas9, yielding the muPS20 transformant. muPS20 is a protease-free strain and its rHuEPO production level was increased 41.1-fold. Moreover, its molecular weight was ≈27 kDa showing that mutations in the above mentioned genes improved secretion, prevented proteolytic degradation and hyperglycosylation of heterologous protein.

RevDate: 2021-01-06
CmpDate: 2021-01-06

Kondo S, Takahashi T, Yamagata N, et al (2020)

Neurochemical Organization of the Drosophila Brain Visualized by Endogenously Tagged Neurotransmitter Receptors.

Cell reports, 30(1):284-297.e5.

Neurotransmitters often have multiple receptors that induce distinct responses in receiving cells. Expression and localization of neurotransmitter receptors in individual neurons are therefore critical for understanding the operation of neural circuits. Here we describe a comprehensive library of reporter strains in which a convertible T2A-GAL4 cassette is inserted into endogenous neurotransmitter receptor genes of Drosophila. Using this library, we profile the expression of 75 neurotransmitter receptors in the brain. Cluster analysis reveals neurochemical segmentation of the brain, distinguishing higher brain centers from the rest. By recombinase-mediated cassette exchange, we convert T2A-GAL4 into split-GFP and Tango to visualize subcellular localization and activation of dopamine receptors in specific cell types. This reveals striking differences in their subcellular localization, which may underlie the distinct cellular responses to dopamine in different behavioral contexts. Our resources thus provide a versatile toolkit for dissecting the cellular organization and function of neurotransmitter systems in the fly brain.

RevDate: 2021-01-06
CmpDate: 2021-01-06

Schuh RS, Gonzalez EA, Tavares AMV, et al (2020)

Neonatal nonviral gene editing with the CRISPR/Cas9 system improves some cardiovascular, respiratory, and bone disease features of the mucopolysaccharidosis I phenotype in mice.

Gene therapy, 27(1-2):74-84.

Mucopolysaccharidosis type I (MPS I) is caused by deficiency of alpha-L-iduronidase (IDUA), leading to multisystemic accumulation of glycosaminoglycans (GAG). Untreated MPS I patients may die in the first decades of life, mostly due to cardiovascular and respiratory complications. We previously reported that the treatment of newborn MPS I mice with intravenous administration of lipossomal CRISPR/Cas9 complexes carrying the murine Idua gene aiming at the ROSA26 locus resulted in long-lasting IDUA activity and GAG reduction in various tissues. Following this, the present study reports the effects of gene editing in cardiovascular, respiratory, bone, and neurologic functions in MPS I mice. Bone morphology, specifically the width of zygomatic and femoral bones, showed partial improvement. Although heart valves were still thickened, cardiac mass and aortic elastin breaks were reduced, with normalization of aortic diameter. Pulmonary resistance was normalized, suggesting improvement in respiratory function. In contrast, behavioral abnormalities and neuroinflammation still persisted, suggesting deterioration of the neurological functions. The set of results shows that gene editing performed in newborn animals improved some manifestations of the MPS I disorder in bone, respiratory, and cardiovascular systems. However, further studies will be imperative to find better delivery strategies to reach "hard-to-treat" tissues to ensure better systemic and neurological effects.

RevDate: 2021-01-06
CmpDate: 2021-01-06

Zhang J, Späth SS, SG Katz (2020)

Genome-Wide CRISPRi/a Screening in an In Vitro Coculture Assay of Human Immune Cells with Tumor Cells.

Methods in molecular biology (Clifton, N.J.), 2097:231-252.

Cell-based immunotherapy has achieved preclinical success in certain types of cancer patients, with a few approved cell-based products for clinical use. These achievements revitalized the field of cell engineering/ immunotherapy and brought attention to the opportunities that cell-based immunotherapeutics can offer to patients. On the other hand, obvious indications emphasize the need for a better understanding of the biological mechanisms involved in the immune response. This knowledge may not only ameliorate safety and efficacy, but also determine the possibilities and limitations in use of immune cell engineering for cancer treatment, and facilitate developing novel immunotherapeutic strategies. Recently developed technology based on CRISPR-dCas9 has an immense potential to systematically uncover genetic mechanisms by identifying subsets of essential genes involved in interactions of cancer cells with the immune system. This chapter will present a reliable and reproducible general protocol for the application of genome-wide sgRNA gene-editing tools in the recently established two-cell type co-culture, consisting of immune cells as effectors and cancer cells as targets, utilizing CRISPRi/a-dCas9-based technology.

RevDate: 2021-01-06
CmpDate: 2021-01-06

Bailis W (2020)

CRISPR/Cas9 Gene Targeting in Primary Mouse Bone Marrow-Derived Macrophages.

Methods in molecular biology (Clifton, N.J.), 2097:223-230.

CRISPR-Cas9 technology allows for rapid, targeted genome editing at nearly any loci with limited off-target effects. Here, we describe a method for using retroviral transduction to deliver single-guide RNA to primary bone marrow-derived macrophages. This protocol allows for high-throughput reverse genetics assays in primary immune cells and is also compatible with retroviral systems for transgene expression.

RevDate: 2021-01-06
CmpDate: 2021-01-06

Niu Q, Wu S, Li Y, et al (2020)

Expanding the scope of CRISPR/Cas9-mediated genome editing in plants using an xCas9 and Cas9-NG hybrid.

Journal of integrative plant biology, 62(4):398-402.

The widely used Streptococcus pyogenes Cas9 (SpCas9) requires NGG as a protospacer adjacent motif (PAM) for genome editing. Although SpCas9 is a powerful genome-editing tool, its use has been limited on the targetable genomic locus lacking NGG PAM. The SpCas9 variants xCas9 and Cas9-NG have been developed to recognize NG, GAA, and GAT PAMs in human cells. Here, we show that xCas9 cannot recognize NG PAMs in tomato, and Cas9-NG can recognize some of our tested NG PAMs in the tomato and Arabidopsis genomes. In addition, we engineered SpCas9 (XNG-Cas9) based on mutations from both xCas9 and Cas9-NG, and found that XNG-Cas9 can efficiently mutagenize endogenous target sites with NG, GAG, GAA, and GAT PAMs in the tomato or Arabidopsis genomes. The PAM compatibility of XNG-Cas9 is the broadest reported to date among Cas9s (SpCas9 and Cas9-NG) active in plant.

RevDate: 2021-01-07
CmpDate: 2021-01-07

Nicolai S, Mahen R, Raschellà G, et al (2020)

ZNF281 is recruited on DNA breaks to facilitate DNA repair by non-homologous end joining.

Oncogene, 39(4):754-766.

Efficient repair of DNA double-strand breaks (DSBs) is of critical importance for cell survival. Although non-homologous end joining (NHEJ) is the most used DSBs repair pathway in the cells, how NHEJ factors are sequentially recruited to damaged chromatin remains unclear. Here, we identify a novel role for the zinc-finger protein ZNF281 in participating in the ordered recruitment of the NHEJ repair factor XRCC4 at damage sites. ZNF281 is recruited to DNA lesions within seconds after DNA damage through a mechanism dependent on its DNA binding domain and, at least in part, on poly-ADP ribose polymerase (PARP) activity. ZNF281 binds XRCC4 through its zinc-finger domain and facilitates its recruitment to damaged sites. Consequently, depletion of ZNF281 impairs the efficiency of the NHEJ repair pathway and decreases cell viability upon DNA damage. Survival analyses from datasets of commonly occurring human cancers show that higher levels of ZNF281 correlate with poor prognosis of patients treated with DNA-damaging therapies. Thus, our results define a late ZNF281-dependent regulatory step of NHEJ complex assembly at DNA lesions and suggest additional possibilities for cancer patients' stratification and for the development of personalised therapeutic strategies.

RevDate: 2021-01-07
CmpDate: 2021-01-07

Ko T, Sharma R, S Li (2020)

Genome-wide screening identifies novel genes implicated in cellular sensitivity to BRAFV600E expression.

Oncogene, 39(4):723-738.

The V600E mutation of BRAF (BRAFV600E), which constitutively activates the ERK/MAPK signaling pathway, is frequently found in melanoma and other cancers. Like most other oncogenes, BRAFV600E causes oncogenic stress to normal cells, leading to growth arrest (senescence) or apoptosis. Through genome-wide screening, we identified genes implicated in sensitivity of human skin melanocytes and fibroblasts to BRAFV600E overexpression. Among the identified genes shared by the two cell types are proto-oncogenes ERK2, a component of the ERK/MAPK pathway, and VAV1, a guanine nucleotide exchange factor for Rho family GTPases that also activates the ERK/MAPK pathway. CDKN1A, which has been known to promote senescence of fibroblasts but not melanocytes, is implicated in sensitivity of the fibroblasts but not the melanocytes to BRAFV600E overexpression. Disruptions of GPR4, a pH-sensing G-protein coupled receptor, and DBT, a subunit of the branched chain α-keto acid dehydrogenase that is required for the second and rate-limiting step of branched amino acid catabolism and implicated in maple syrup urine disease, are the most highly selected in the melanocytes upon BRAFV600E overexpression. Disruption of DBT severely attenuates ERK/MAPK signaling, p53 activation, and apoptosis in melanocytes, at least in part due to accumulation of branched chain α-keto acids. The expression level of BRAF positively correlates with that of DBT in all cancer types and with that of GPR4 in most cancer types. Overexpression of DBT kills all four melanoma cell lines tested regardless of the presence of BRAFV600E mutation. Our findings shed new lights on regulations of oncogenic stress signaling and may be informative for development of novel cancer treatment strategies.

RevDate: 2021-01-05

Pan C, Sretenovic S, Y Qi (2021)

CRISPR/dCas-mediated transcriptional and epigenetic regulation in plants.

Current opinion in plant biology, 60:101980 pii:S1369-5266(20)30137-0 [Epub ahead of print].

The CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR Associated) system-mediated precise genome editing has revolutionized genome engineering due to ease of use and versatility of multiplexing. Catalytically inactivated Cas variants (dCas) further expand the usefulness of the CRISPR/Cas system for genetics studies and translational research without inducing DNA double-strand breaks. Fusion of diverse effector domains to dCas proteins empowers the CRISPR/dCas system as a multifunctional platform for gene expression regulation, epigenetic regulation and sequence-specific imaging. In this short review, we summarize the recent advances of CRISPR/dCas-mediated transcriptional activation and repression, and epigenetic modifications. We also highlight the future directions and broader applications of the CRISPR/dCas systems in plants.

RevDate: 2021-01-05

Qin Z, Yang Y, Yu S, et al (2021)

Repurposing the Endogenous Type I-E CRISPR/Cas System for Gene Repression in Gluconobacter oxydans WSH-003.

ACS synthetic biology [Epub ahead of print].

Gluconobacter oxydans is well-known for its incomplete oxidizing capacity and has been widely applied in industrial production. However, genetic tools in G. oxydans are still scarce compared with model microorganisms, limiting its metabolic engineering. This study aimed to develop a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system based on the typical type I-E endogenous CRISPR/CRISPR-associated proteins (Cas) system in G. oxydans WSH-003. The nuclease Cas3 in this system was inactivated naturally and hence did not need to be knocked out. Subsequently, the CRISPRi effect was verified by repressing the expression of fluorescent proteins, revealing effective multiplex gene repression. Finally, the endogenous CRISPRi system was used to study the role of the central carbon metabolism pathway, including the pentose phosphate pathway (PPP) and Entner-Doudoroff pathway (EDP), in G. oxydans WSH-003. This was done to demonstrate a metabolic engineering application. The PPP was found to be important for cell growth and the substrate conversion rate. The development of the CRISPRi system enriched the gene regulation tools in G. oxydans and promoted the metabolic engineering modification of G. oxydans to improve its performance. In addition, it might have implications for metabolic engineering modification of other genetically recalcitrant strains.

RevDate: 2021-01-05

Smith LM, Jackson SA, Malone LM, et al (2021)

The Rcs stress response inversely controls surface and CRISPR-Cas adaptive immunity to discriminate plasmids and phages.

Nature microbiology [Epub ahead of print].

Bacteria harbour multiple innate defences and adaptive CRISPR-Cas systems that provide immunity against bacteriophages and mobile genetic elements. Although some bacteria modulate defences in response to population density, stress and metabolic state, a lack of high-throughput methods to systematically reveal regulators has hampered efforts to understand when and how immune strategies are deployed. We developed a robust approach called SorTn-seq, which combines saturation transposon mutagenesis, fluorescence-activated cell sorting and deep sequencing to characterize regulatory networks controlling CRISPR-Cas immunity in Serratia sp. ATCC 39006. We applied our technology to assess csm gene expression for ~300,000 mutants and uncovered multiple pathways regulating type III-A CRISPR-Cas expression. Mutation of igaA or mdoG activated the Rcs outer-membrane stress response, eliciting cell-surface-based innate immunity against diverse phages via the transcriptional regulators RcsB and RcsA. Activation of this Rcs phosphorelay concomitantly attenuated adaptive immunity by three distinct type I and III CRISPR-Cas systems. Rcs-mediated repression of CRISPR-Cas defence enabled increased acquisition and retention of plasmids. Dual downregulation of cell-surface receptors and adaptive immunity in response to stress by the Rcs pathway enables protection from phage infection without preventing the uptake of plasmids that may harbour beneficial traits.

RevDate: 2021-01-05

Goh YJ, R Barrangou (2021)

A portable CRISPR-Cas9N system for flexible genome engineering in Lactobacillus acidophilus, Lactobacillus gasseri and Lactobacillus paracasei.

Applied and environmental microbiology pii:AEM.02669-20 [Epub ahead of print].

Diverse Lactobacillus strains are widely used as probiotic cultures in the dairy and dietary supplements industries, and specific strains such as Lactobacillus acidophilus NCFM have been engineered for the development of biotherapeutics. To expand the Lactobacillus manipulation toolbox with enhanced efficiency and ease, we present here a CRISPR-SpyCas9D10A nickase (Cas9N)-based system for programmable engineering of L. acidophilus NCFM, a model probiotic bacterium. Successful single-plasmid delivery system was achieved with the engineered pLbCas9N vector harboring cas9N under the regulation of a Lactobacillus promoter and a cloning region for customized sgRNA and editing template. The functionality of the pLbCas9N system was validated in NCFM with targeted chromosomal deletions ranging between 300 bp and 1.9 kb at various loci (rafE, lacS and ltaS), yielding 35-100% mutant recovery rates. Genome analysis of the mutants confirmed precision and specificity of the pLbCas9N system. To showcase the versatility of this system, we also inserted a mCherry fluorescent protein gene downstream of the pgm gene to create a polycistronic transcript. The pLbCas9N system was further deployed in other species to generate concurrent single base substitution and gene deletion in Lactobacillus gasseri ATCC 33323, and an in-frame gene deletion in Lactobacillus paracasei Lpc-37, highlighting the portability of the system in phylogenetically distant Lactobacillus species, where its targeting activity was not interfered by endogenous CRISPR-Cas systems. Collectively, these editing outcomes illustrate the robustness and versatility of the pLbCas9N system for genome manipulations in diverse lactobacilli, and open new avenues for the engineering of health-promoting lactic acid bacteria.Importance This work describes the development of a broad-host range CRISPR-based editing system for genome manipulations in three Lactobacillus species, which belong to lactic acid bacteria (LAB) commonly known for their long history of use in food fermentations and as indigenous members of healthy microbiota, and their emerging roles in human and animal commercial health-promoting applications. We exploited the established CRISPR-SpyCas9 nickase for flexible and precise genome editing applications in Lactobacillus acidophilus, and further demonstrated the efficacy of this universal system in two distantly related Lactobacillus species. This versatile Cas9-based system facilitates genome engineering compared to conventional gene replacement systems, and represents a valuable gene editing modality in species that do not possess native CRISPR-Cas systems. Overall, this portable tool contributes to expanding the genome editing toolbox of LAB for studying their health-promoting mechanisms and engineering of these beneficial microbes as next-generation vaccines and designer probiotics.

RevDate: 2021-01-05

Synefiaridou D, JW Veening (2021)

Harnessing CRISPR-Cas9 for genome editing in Streptococcus pneumoniae D39V.

Applied and environmental microbiology pii:AEM.02762-20 [Epub ahead of print].

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by detection and cleavage of invading foreign DNA. Modified versions of this system can be exploited as a biotechnological tool for precise genome editing at a targeted locus. Here, we developed a replicative plasmid that carries the CRISPR-Cas9 system for RNA-programmable, genome editing by counterselection in the opportunistic human pathogen Streptococcus pneumoniae. Specifically, we demonstrate an approach for making targeted, marker-less gene knockouts and large genome deletions. After a precise double-stranded break (DSB) is introduced, the cells' DNA repair mechanism of homology-directed repair (HDR) pathway is being exploited to select successful transformants. This is achieved through the transformation of a template DNA fragment that will recombine in the genome and eliminate recognition of the target of the Cas9 endonuclease. Next, the newly engineered strain can be easily cured from the plasmid that is temperature-sensitive for replication, by growing it at the non-permissive temperature. This allows for consecutive rounds of genome editing. Using this system, we engineered a strain with three major virulence factors deleted. The here developed approaches could be potentially transported to other Gram-positive bacteria.ImportanceStreptococcus pneumoniae (the pneumococcus) is an important opportunistic human pathogen killing over a million people each year. Having the availability of a system capable of easy genome editing would significantly facilitate drug discovery and efforts in identifying new vaccine candidates. Here, we introduced an easy to use system to perform multiple rounds of genome editing in the pneumococcus by putting the CRISPR-Cas9 system on a temperature-sensitive replicative plasmid. The here used approaches will advance genome editing projects in this important human pathogen.

RevDate: 2021-01-05

Rai KK, Pandey N, Meena RP, et al (2021)

Biotechnological strategies for enhancing heavy metal tolerance in neglected and underutilized legume crops: A comprehensive review.

Ecotoxicology and environmental safety, 208:111750.

Contamination of agricultural land and water by heavy metals due to rapid industrialization and urbanization including various natural processes have become one of the major constraints to crop growth and productivity. Several studies have reported that to counteract heavy metal stress, plants should be able to maneuver various physiological, biochemical and molecular processes to improve their growth and development under heavy metal stress. With the advent of modern biotechnological tools and techniques it is now possible to tailor legume and other plants overexpressing stress-induced genes, transcription factors, proteins, and metabolites that are directly involved in heavy metal stress tolerance. This review provides an in-depth overview of various biotechnological approaches and/or strategies that can be used for enhancing detoxification of the heavy metals by stimulating phytoremediation processes. Synthetic biology tools involved in the engineering of legume and other crop plants against heavy metal stress tolerance are also discussed herewith some pioneering examples where synthetic biology tools that have been used to modify plants for specific traits. Also, CRISPR based genetic engineering of plants, including their role in modulating the expression of several genes/ transcription factors in the improvement of abiotic stress tolerance and phytoremediation ability using knockdown and knockout strategies has also been critically discussed.

RevDate: 2021-01-05
CmpDate: 2021-01-05

Otte K, Kühne NM, Furrer AD, et al (2020)

A CRISPR-Cas9 tool to explore the genetics of Bacillus subtilis phages.

Letters in applied microbiology, 71(6):588-595.

Here, we present pRH030, a new CRISPR-Cas9 tool for the genetic engineering of Bacillus phages and beyond. It is based on the Streptococcus pyogenes cas9 with its native constitutive promoter, tracrRNA, and a gRNA precursor. The constitutive expression of Cas9 was conducive to the inactivation of viral attackers and enhanced phage mutagenesis efficiency up to 100%. The gRNA precursor can be built up to an artificial CRISPR array with up to 5 spacers (target sequences) assembled from ordinary oligonucleotides and directly cloned into pRH030. Required time and resources remain comparable to a single gRNA cloning. These properties make pRH030 an attractive new system for the modification of Bacillus phages and qualify it for research beyond genetic construction.

RevDate: 2021-01-05
CmpDate: 2021-01-05

Schwinn MK, Steffen LS, Zimmerman K, et al (2020)

A Simple and Scalable Strategy for Analysis of Endogenous Protein Dynamics.

Scientific reports, 10(1):8953.

The ability to analyze protein function in a native context is central to understanding cellular physiology. This study explores whether tagging endogenous proteins with a reporter is a scalable strategy for generating cell models that accurately quantitate protein dynamics. Specifically, it investigates whether CRISPR-mediated integration of the HiBiT luminescent peptide tag can easily be accomplished on a large-scale and whether integrated reporter faithfully represents target biology. For this purpose, a large set of proteins representing diverse structures and functions, some of which are known or potential drug targets, were targeted for tagging with HiBiT in multiple cell lines. Successful insertion was detected for 86% of the targets, as determined by luminescence-based plate assays, blotting, and imaging. In order to determine whether endogenously tagged proteins yield more representative models, cells expressing HiBiT protein fusions either from endogenous loci or plasmids were directly compared in functional assays. In the tested cases, only the edited lines were capable of accurately reproducing the anticipated biology. This study provides evidence that cell lines expressing HiBiT fusions from endogenous loci can be rapidly generated for many different proteins and that these cellular models provide insight into protein function that may be unobtainable using overexpression-based approaches.

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RJR Experience and Expertise

Researcher

Robbins holds BS, MS, and PhD degrees in the life sciences. He served as a tenured faculty member in the Zoology and Biological Science departments at Michigan State University. He is currently exploring the intersection between genomics, microbial ecology, and biodiversity — an area that promises to transform our understanding of the biosphere.

Educator

Robbins has extensive experience in college-level education: At MSU he taught introductory biology, genetics, and population genetics. At JHU, he was an instructor for a special course on biological database design. At FHCRC, he team-taught a graduate-level course on the history of genetics. At Bellevue College he taught medical informatics.

Administrator

Robbins has been involved in science administration at both the federal and the institutional levels. At NSF he was a program officer for database activities in the life sciences, at DOE he was a program officer for information infrastructure in the human genome project. At the Fred Hutchinson Cancer Research Center, he served as a vice president for fifteen years.

Technologist

Robbins has been involved with information technology since writing his first Fortran program as a college student. At NSF he was the first program officer for database activities in the life sciences. At JHU he held an appointment in the CS department and served as director of the informatics core for the Genome Data Base. At the FHCRC he was VP for Information Technology.

Publisher

While still at Michigan State, Robbins started his first publishing venture, founding a small company that addressed the short-run publishing needs of instructors in very large undergraduate classes. For more than 20 years, Robbins has been operating The Electronic Scholarly Publishing Project, a web site dedicated to the digital publishing of critical works in science, especially classical genetics.

Speaker

Robbins is well-known for his speaking abilities and is often called upon to provide keynote or plenary addresses at international meetings. For example, in July, 2012, he gave a well-received keynote address at the Global Biodiversity Informatics Congress, sponsored by GBIF and held in Copenhagen. The slides from that talk can be seen HERE.

Facilitator

Robbins is a skilled meeting facilitator. He prefers a participatory approach, with part of the meeting involving dynamic breakout groups, created by the participants in real time: (1) individuals propose breakout groups; (2) everyone signs up for one (or more) groups; (3) the groups with the most interested parties then meet, with reports from each group presented and discussed in a subsequent plenary session.

Designer

Robbins has been engaged with photography and design since the 1960s, when he worked for a professional photography laboratory. He now prefers digital photography and tools for their precision and reproducibility. He designed his first web site more than 20 years ago and he personally designed and implemented this web site. He engages in graphic design as a hobby.

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CRISPR-Cas

By delivering the Cas9 nuclease, complexed with a synthetic guide RNA (gRNA) into a cell, the cell's genome can be precisely cut at any desired location, allowing existing genes to be removed and/or new ones added. That is, the CRISPR-Cas system provides a tool for the cut-and-paste editing of genomes. Welcome to the brave new world of genome editing. R. Robbins

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Collection of publications by R J Robbins

Reprints and preprints of publications, slide presentations, instructional materials, and data compilations written or prepared by Robert Robbins. Most papers deal with computational biology, genome informatics, using information technology to support biomedical research, and related matters.

Research Gate page for R J Robbins

ResearchGate is a social networking site for scientists and researchers to share papers, ask and answer questions, and find collaborators. According to a study by Nature and an article in Times Higher Education , it is the largest academic social network in terms of active users.

Curriculum Vitae for R J Robbins

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Curriculum Vitae for R J Robbins

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