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25 Sep 2020 at 01:41
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Bibliography on: CRISPR-Cas


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RJR: Recommended Bibliography 25 Sep 2020 at 01:41 Created: 


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®)


RevDate: 2020-09-24

Değirmenci L, Geiger D, Rogé Ferreira FL, et al (2020)

CRISPR/Cas 9 mediated mutations as a new tool for studying taste in honeybees.

Chemical senses pii:5910794 [Epub ahead of print].

Honeybees rely on nectar as their main source of carbohydrates. Sucrose, glucose and fructose are the main components of plant nectars. Intriguingly, honeybees express only three putative sugar receptors (AmGr1, AmGr2 and AmGr3), which is in stark contrast to many other insects and vertebrates. The sugar receptors are only partially characterized. AmGr1 detects different sugars including sucrose and glucose. AmGr2 is assumed to act as a co-receptor only, while AmGr3 is assumedly a fructose receptor. We show that honeybee gustatory receptor AmGr3 is highly specialized for fructose perception when expressed in Xenopus oocytes. When we introduced nonsense mutations to the respective AmGr3 gene using CRISPR/Cas9 in eggs of female workers, the resulting mutants displayed almost a complete loss of responsiveness to fructose. In contrast, responses to sucrose were normal. Nonsense mutations introduced by CRISPR/Cas9 in honeybees can thus induce a measurable behavioural change and serve to characterize the function of taste receptors in vivo. CRISPR/Cas9 is an excellent novel tool for characterizing honeybee taste receptors in vivo. Biophysical receptor characterisation in Xenopus oocytes and nonsense mutation of AmGr3 in honeybees unequivocally demonstrate that this receptor is highly specific for fructose.

RevDate: 2020-09-24
CmpDate: 2020-09-24

Shinoda K, Maman Y, Canela A, et al (2019)

Intra-Vκ Cluster Recombination Shapes the Ig Kappa Locus Repertoire.

Cell reports, 29(13):4471-4481.e6.

During V(D)J recombination, RAG proteins introduce DNA double-strand breaks (DSBs) at recombination signal sequences (RSSs) that contain either 12- or 23-nt spacer regions. Coordinated 12/23 cleavage predicts that DSBs at variable (V) gene segments should equal the level of breakage at joining (J) segments. Contrary to this, here we report abundant RAG-dependent DSBs at multiple Vκ gene segments independent of V-J rearrangement. We find that a large fraction of Vκ gene segments are flanked not only by a bone-fide 12 spacer but also an overlapping, 23-spacer flipped RSS. These compatible pairs of RSSs mediate recombination and deletion inside the Vκ cluster even in the complete absence of Jκ gene segments and support a V(D)J recombination center (RC) independent of the conventional Jκ-centered RC. We propose an improved model of Vκ-Jκ repertoire formation by incorporating these surprisingly frequent, evolutionarily conserved intra-Vκ cluster recombination events.

RevDate: 2020-09-24
CmpDate: 2020-09-24

Liu XS, R Jaenisch (2019)

Editing the Epigenome to Tackle Brain Disorders.

Trends in neurosciences, 42(12):861-870.

Genetic studies of epigenetic modifiers such as DNA methyltransferases and histone acetyltransferases have revealed a critical role for epigenetic regulation during brain development and function. Alteration of epigenetic modifications have been documented in a variety of brain disorders, including neurodevelopmental, psychiatric, and neurodegenerative diseases. Development of epigenome editing tools enables a functional dissection of the link between altered epigenetic changes and disease outcomes. Here, we review the development of epigenome editing tools, summarize proof of concept applications focusing on brain disease-associated genes, and discuss the promising application and challenges of epigenome editing to tackle brain disorders.

RevDate: 2020-09-24
CmpDate: 2020-09-24

Li Y, De la Paz JA, Jiang X, et al (2019)

Coevolutionary Couplings Unravel PAM-Proximal Constraints of CRISPR-SpCas9.

Biophysical journal, 117(9):1684-1691.

The clustered regularly interspaced short palindromic repeats (CRISPR) system, an immune system analog found in prokaryotes, allows a single-guide RNA to direct a CRISPR-associated protein (Cas) with combined helicase and nuclease activity to DNA. The presence of a specific protospacer adjacent motif (PAM) next to the DNA target site plays a crucial role in determining both efficacy and specificity of gene editing. Herein, we introduce a coevolutionary framework to computationally unveil nonobvious molecular interactions in CRISPR systems and experimentally probe their functional role. Specifically, we use direct coupling analysis, a statistical inference framework used to infer direct coevolutionary couplings, in the context of protein/nucleic acid interactions. Applied to Streptococcus pyogenes Cas9, a Hamiltonian metric obtained from coevolutionary relationships reveals, to our knowledge, novel PAM-proximal nucleotide preferences at the seventh position of S. pyogenes Cas9 PAM (5'-NGRNNNT-3'), which was experimentally confirmed by in vitro and functional assays in human cells. We show that coevolved and conserved interactions point to specific clues toward rationally engineering new generations of Cas9 systems and may eventually help decipher the diversity of this family of proteins.

RevDate: 2020-09-24
CmpDate: 2020-09-24

Kang W, Sun Z, Zhao X, et al (2019)

Gene editing based hearing impairment research and therapeutics.

Neuroscience letters, 709:134326.

Hearing impairment affects 1 in 500 newborns worldwide and nearly one out of three people over the age of 65 (WHO, 2019). Hereditary hearing loss is the most common type of congenital deafness; genetic factors also affect deafness susceptibility. Gene therapies may preserve or restore natural sound perception, and have rescued deafness in multiple hereditary murine models. CRISPR-Cas9 and base editors (BEs) are newly developed gene editing technologies that can facilitate gene studies in the inner ear and provide therapeutic approaches for hearing impairment. Here, we present recent applications of gene editing in the inner ear.

RevDate: 2020-09-23

Montaña S, Vilacoba E, Fernandez JS, et al (2020)

Genomic analysis of two Acinetobacter baumannii strains belonging to two different STs (ST172 and ST25).

Journal of global antimicrobial resistance pii:S2213-7165(20)30237-X [Epub ahead of print].

OBJECTIVE: Acinetobacter baumannii is an opportunistic nosocomial pathogen that is the main focus of attention in clinical settings, due to its intrinsic ability to persist in hospital environment and its capacity to acquire determinants of resistance and virulence. Here, we present the genomic sequencing, the molecular characterization, and genomic comparison of two A. baumannii strains, that belong to two different STs, one sporadic and one widely distributed in our region.

METHODS: The whole genome sequence (WGS) of Ab42 and Ab376 were obtained using Illumina MiSeq- I and assembled with SPAdes. ARG-ANNOT, CARD-RGI, ISfinder, PHAST, Plasmid Finder, PlasmidSpades and Islandviewer were used to analyze both genomes.

RESULTS: Genome analysis revealed that Ab42 belongs to ST172, an uncommon ST, while Ab376 belongs to ST25 a widely distributed ST. The molecular characterization showed the presence of two antibiotic resistance genes in Ab42 and nine in Ab376. No insertion sequences were detected in the Ab42, however, 22 were detected in Ab376. Moreover, two prophages were found in Ab42 and three in Ab376. In addition, a CRISPR-cas type I-Fb and two plasmids, one of which harbored an AbGRI1-like island, were found in Ab376.

CONCLUSION: We present the WGS analysis of twoA. baumannii strains that belong to two different STs. These findings allowed us to characterize a not previously described ST (ST172) and provide new insights to the widely studied ST25.

RevDate: 2020-09-23

Rafia C, Harly C, E Scotet (2020)

Beyond CAR T cells: Engineered Vγ9Vδ2 T cells to fight solid tumors.

Immunological reviews [Epub ahead of print].

Despite recent significant progress in cancer immunotherapies based on adoptive cell transfer(s)(ACT), the eradication of cancers still represents a major clinical challenge. In particular, the efficacy of current ACT-based therapies against solid tumors is dramatically reduced by physical barriers that prevent tumor infiltration of adoptively transferred effectors, and the tumor environment that suppress their anti-tumor functions. Novel immunotherapeutic strategies are thus needed to circumvent these issues. Human peripheral blood Vγ9Vδ2 T cells, a non-alloreactive innate-like T lymphocyte subset, recently proved to be a promising anti-tumor effector subset for ACT-based immunotherapies. Furthermore, new cell engineering tools that leverage the potential of CRISPR/Cas technology open astounding opportunities to optimize their anti-tumor effector functions. In this review, we present the current ACT strategies based on engineered T cells and their limitations. We then discuss the potential of engineered Vγ9Vδ2 T cell to overcome these limitations and improve ACT-based cancer immunotherapies.

RevDate: 2020-09-23
CmpDate: 2020-09-23

Li Y, Weng Y, Bai D, et al (2020)

Precise allele-specific genome editing by spatiotemporal control of CRISPR-Cas9 via pronuclear transplantation.

Nature communications, 11(1):4593 pii:10.1038/s41467-020-18391-y.

Gene-targeted animal models that are generated by injecting Cas9 and sgRNAs into zygotes are often accompanied by undesired double-strand break (DSB)-induced byproducts and random biallelic targeting due to uncontrollable Cas9 targeting activity. Here, we establish a parental allele-specific gene-targeting (Past-CRISPR) method, based on the detailed observation that pronuclear transfer-mediated cytoplasmic dilution can effectively terminate Cas9 activity. We apply this method in embryos to efficiently target the given parental alleles of a gene of interest and observed little genomic mosaicism because of the spatiotemporal control of Cas9 activity. This method allows us to rapidly explore the function of individual parent-of-origin effects and to construct animal models with a single genomic change. More importantly, Past-CRISPR could also be used for therapeutic applications or disease model construction.

RevDate: 2020-09-23

Nemudryi A, Nemudraia A, Wiegand T, et al (2020)

Temporal Detection and Phylogenetic Assessment of SARS-CoV-2 in Municipal Wastewater.

Cell reports. Medicine, 1(6):100098.

SARS-CoV-2 has recently been detected in feces, which indicates that wastewater may be used to monitor viral prevalence in the community. Here, we use RT-qPCR to monitor wastewater for SARS-CoV-2 RNA over a 74-day time course. We show that changes in SARS-CoV-2 RNA concentrations follow symptom onset gathered by retrospective interview of patients but precedes clinical test results. In addition, we determine a nearly complete (98.5%) SARS-CoV-2 genome sequence from wastewater and use phylogenetic analysis to infer viral ancestry. Collectively, this work demonstrates how wastewater can be used as a proxy to monitor viral prevalence in the community and how genome sequencing can be used for genotyping viral strains circulating in a community.

RevDate: 2020-09-23
CmpDate: 2020-09-23

Crone MA, Priestman M, Ciechonska M, et al (2020)

A role for Biofoundries in rapid development and validation of automated SARS-CoV-2 clinical diagnostics.

Nature communications, 11(1):4464 pii:10.1038/s41467-020-18130-3.

The SARS-CoV-2 pandemic has shown how a rapid rise in demand for patient and community sample testing can quickly overwhelm testing capability globally. With most diagnostic infrastructure dependent on specialized instruments, their exclusive reagent supplies quickly become bottlenecks, creating an urgent need for approaches to boost testing capacity. We address this challenge by refocusing the London Biofoundry onto the development of alternative testing pipelines. Here, we present a reagent-agnostic automated SARS-CoV-2 testing platform that can be quickly deployed and scaled. Using an in-house-generated, open-source, MS2-virus-like particle (VLP) SARS-CoV-2 standard, we validate RNA extraction and RT-qPCR workflows as well as two detection assays based on CRISPR-Cas13a and RT-loop-mediated isothermal amplification (RT-LAMP). In collaboration with an NHS diagnostic testing lab, we report the performance of the overall workflow and detection of SARS-CoV-2 in patient samples using RT-qPCR, CRISPR-Cas13a, and RT-LAMP. The validated RNA extraction and RT-qPCR platform has been installed in NHS diagnostic labs, increasing testing capacity by 1000 samples per day.

RevDate: 2020-09-23
CmpDate: 2020-09-23

Benslimane Y, Bertomeu T, Coulombe-Huntington J, et al (2020)

Genome-Wide Screens Reveal that Resveratrol Induces Replicative Stress in Human Cells.

Molecular cell, 79(5):846-856.e8.

Resveratrol is a natural product associated with wide-ranging effects in animal and cellular models, including lifespan extension. To identify the genetic target of resveratrol in human cells, we conducted genome-wide CRISPR-Cas9 screens to pinpoint genes that confer sensitivity or resistance to resveratrol. An extensive network of DNA damage response and replicative stress genes exhibited genetic interactions with resveratrol and its analog pterostilbene. These genetic profiles showed similarity to the response to hydroxyurea, an inhibitor of ribonucleotide reductase that causes replicative stress. Resveratrol, pterostilbene, and hydroxyurea caused similar depletion of nucleotide pools, inhibition of replication fork progression, and induction of replicative stress. The ability of resveratrol to inhibit cell proliferation and S phase transit was independent of the histone deacetylase sirtuin 1, which has been implicated in lifespan extension by resveratrol. These results establish that a primary impact of resveratrol on human cell proliferation is the induction of low-level replicative stress.

RevDate: 2020-09-23
CmpDate: 2020-09-23

van de Weijer ML, Krshnan L, Liberatori S, et al (2020)

Quality Control of ER Membrane Proteins by the RNF185/Membralin Ubiquitin Ligase Complex.

Molecular cell, 79(5):768-781.e7.

Misfolded proteins in the endoplasmic reticulum (ER) are degraded by ER-associated degradation (ERAD). Although ERAD components involved in degradation of luminal substrates are well characterized, much less is known about quality control of membrane proteins. Here, we analyzed the degradation pathways of two short-lived ER membrane model proteins in mammalian cells. Using a CRISPR-Cas9 genome-wide library screen, we identified an ERAD branch required for quality control of a subset of membrane proteins. Using biochemical and mass spectrometry approaches, we showed that this ERAD branch is defined by an ER membrane complex consisting of the ubiquitin ligase RNF185, the ubiquitin-like domain containing proteins TMUB1/2 and TMEM259/Membralin, a poorly characterized protein. This complex cooperates with cytosolic ubiquitin ligase UBE3C and p97 ATPase in degrading their membrane substrates. Our data reveal that ERAD branches have remarkable specificity for their membrane substrates, suggesting that multiple, perhaps combinatorial, determinants are involved in substrate selection.

RevDate: 2020-09-23
CmpDate: 2020-09-23

Becker M, Noll-Puchta H, Amend D, et al (2020)

CLUE: a bioinformatic and wet-lab pipeline for multiplexed cloning of custom sgRNA libraries.

Nucleic acids research, 48(13):e78.

The systematic perturbation of genomes using CRISPR/Cas9 deciphers gene function at an unprecedented rate, depth and ease. Commercially available sgRNA libraries typically contain tens of thousands of pre-defined constructs, resulting in a complexity challenging to handle. In contrast, custom sgRNA libraries comprise gene sets of self-defined content and size, facilitating experiments under complex conditions such as in vivo systems. To streamline and upscale cloning of custom libraries, we present CLUE, a bioinformatic and wet-lab pipeline for the multiplexed generation of pooled sgRNA libraries. CLUE starts from lists of genes or pasted sequences provided by the user and designs a single synthetic oligonucleotide pool containing various libraries. At the core of the approach, a barcoding strategy for unique primer binding sites allows amplifying different user-defined libraries from one single oligonucleotide pool. We prove the approach to be straightforward, versatile and specific, yielding uniform sgRNA distributions in all resulting libraries, virtually devoid of cross-contaminations. For in silico library multiplexing and design, we established an easy-to-use online platform at www.crispr-clue.de. All in all, CLUE represents a resource-saving approach to produce numerous high quality custom sgRNA libraries in parallel, which will foster their broad use across molecular biosciences.

RevDate: 2020-09-23
CmpDate: 2020-09-23

Nishizono H, Darwish M, Uosaki H, et al (2020)

Use of Freeze-thawed Embryos for High-efficiency Production of Genetically Modified Mice.

Journal of visualized experiments : JoVE.

The use of genetically modified (GM) mice has become crucial for understanding gene function and deciphering the underlying mechanisms of human diseases. The CRISPR/Cas9 system allows researchers to modify the genome with unprecedented efficiency, fidelity, and simplicity. Harnessing this technology, researchers are seeking a rapid, efficient, and easy protocol for generating GM mice. Here we introduce an improved method for cryopreservation of one-cell embryos that leads to a higher developmental rate of the freeze-thawed embryos. By combining it with optimized electroporation conditions, this protocol allows for the generation of knockout and knock-in mice with high efficiency and low mosaic rates within a short time. Furthermore, we show a step-by-step explanation of our optimized protocol, covering CRISPR reagent preparation, in vitro fertilization, cryopreservation and thawing of one-cell embryos, electroporation of CRISPR reagents, mouse generation, and genotyping of the founders. Using this protocol, researchers should be able to prepare GM mice with unparalleled ease, speed, and efficiency.

RevDate: 2020-09-23
CmpDate: 2020-09-23

Kim DH, Kim EJ, Kim DH, et al (2020)

Dact2 is involved in the regulation of epithelial-mesenchymal transition.

Biochemical and biophysical research communications, 524(1):190-197.

Dishevelled-associated antagonist of beta-catenin 2 (Dact2) is involved in the regulation of intracellular signaling pathways during development. It negatively regulates the Nodal signaling pathway, possibly by promoting lysosomal degradation of Nodal receptors such as TGFBR1, and plays an inhibitory role during the re-epithelialization of skin wounds by attenuating transforming growth factor-β signaling. Dact2 is known to act as a functional tumor suppressor in colon cancer; reduced Dact2 can promote liver cancer progression and suppress gastric cancer proliferation, invasion, and metastasis by inhibiting Wnt signaling. Zebrafish is used as a model of cancer biology because it shows similar tumorigenesis and morphogenesis as in humans and gene manipulation in this organism is possible. This study was performed to explore phenotypic changes in Dact2 knockout zebrafish and investigate the function of Dact2. A 10-base pair deletion Dact2 knockout zebrafish was prepared using the CRISPR-Cas9 genome editing system. Dact2 knockout enhanced the expression of the MMP2 and MMP9 genes, which are related to tumor invasion and migration, and the Snail, VEGF, and ZEB genes, which are related to epithelial-mesenchymal transition (EMT). The absence of Dact2 also resulted in hyperplasia of the gastrointestinal epithelium, fibrosis in the pancreas and liver, increased proliferation of the pancreatic and hepatic bile ducts, and invasive proliferation into the pancreas. A wound healing assay confirmed that the absence of Dact2 enhanced EMT, thus accelerating wound healing. This study suggests that a loss of function of Dact2 impacts EMT-related gene regulation and tumor generation in a zebrafish knockout model, which is a useful model for exploring the mechanisms of these processes.

RevDate: 2020-09-23
CmpDate: 2020-09-23

Zhang L, Wang L, Xie Y, et al (2019)

Triple-Targeting Delivery of CRISPR/Cas9 To Reduce the Risk of Cardiovascular Diseases.

Angewandte Chemie (International ed. in English), 58(36):12404-12408.

A high level of low-density lipoprotein cholesterol (LDL-C) in the blood is a major risk factor for coronary heart disease. Herein, we present a triple-targeting strategy to generate a loss-of-function mutation in Pcsk9, which regulates plasma cholesterol levels, using a nanocarrier-delivered CRISPR/Cas9 system. Nuclear localization signal (NLS)-tagged Cas9 and Pcsk9-targeted single guide RNA (sgPcsk9) were complexed with gold nanoclusters (GNCs) modified with cationic HIV-1-transactivating transcriptor (TAT) peptide and further encapsulated in a galactose-modified lipid layer to target the nanoclusters to the liver. The resulting nanoclusters had an in vitro Pcsk9-editing efficiency of about 60 % and resulting in a decrease in plasma LDL-C in mice of approximately 30%. No off-target mutagenesis was detected in 10 sites with high similarity. This approach may have therapeutic potential for the prevention and treatment of cardiovascular disease without side effects.

RevDate: 2020-09-23
CmpDate: 2020-09-23

Ju A, Lee SW, Lee YE, et al (2019)

A carrier-free multiplexed gene editing system applicable for suspension cells.

Biomaterials, 217:119298.

Genetically engineered cells via CRISPR/Cas9 system can serve as powerful sources for cancer immunotherapeutic applications. Furthermore, multiple genetic alterations are necessary to overcome tumor-induced immune-suppressive mechanisms. However, one of the major obstacles is the technical difficulty with efficient multiple gene manipulation of suspension cells due to the low transfection efficacy. Herein, we established a carrier-free multiplexed gene editing platform in a simplified method, which can enhance the function of cytotoxic CD8+ T cells by modulating suspension cancer cells. Our multiple Cas9 ribonucleoproteins (RNPs) enable simultaneous disruption of two programmed cell death 1 (PD-1) ligands, functioning as negative regulators in the immune system, by accessing engineered Cas9 proteins with abilities of complexation and cellular penetration. In addition, combination with electroporation enhanced multiple gene editing efficacy, compared with that by treatment of multiple Cas9 RNPs alone. This procedure resulted in high gene editing at multiple loci of suspension cells. The treatment of multiple Cas9 RNPs targeting both ligands strongly improved Th1-type cytokine production of cytotoxic CD8+ T cells, resulting in synergistic cytotoxic effects against cancer. Simultaneous suppression of PD-L1 and PD-L2 on cancer cells via our developed editing system allows effective anti-tumor immunity. Furthermore, the treatment of multiple Cas9 RNPs targeting PD-L1, PD-L2, and TIM-3 had approximately 70-90% deletion efficacy. Thus, our multiplexed gene editing strategy endows potential clinical utilities in cancer immunotherapy.

RevDate: 2020-09-23
CmpDate: 2020-09-23

Zhang Y, Shen S, Zhao G, et al (2019)

In situ repurposing of dendritic cells with CRISPR/Cas9-based nanomedicine to induce transplant tolerance.

Biomaterials, 217:119302.

Organ transplantation is the only effective method to treat end-stage organ failure. However, it is continuously plagued by immune rejection, which is mostly caused by T cell-mediated reactions. Dendritic cells (DCs) are professional antigen-presenting cells, and blocking the costimulatory signaling molecule CD40 in DCs inhibits T cell activation and induces transplant tolerance. In this study, to relieve graft rejection, Cas9 mRNA (mCas9) and a guide RNA targeting the costimulatory molecule CD40 (gCD40) were prepared and encapsulated into poly(ethylene glycol)-block-poly(lactide-co-glycolide) (PEG-b-PLGA)-based cationic lipid-assisted nanoparticles (CLAN), denoted CLANmCas9/gCD40. CLAN effectively delivered mCas9/gCD40 into DCs and disrupted CD40 in DCs at the genomic level both in vitro and in vivo. After intravenous injection into an acute mouse skin transplant model, CLANmCas9/gCD40-mediated CD40 disruption significantly inhibited T cell activation, which reduced graft damage and prolonged graft survival. This work provides a promising strategy for reprogramming DCs with nanoparticles carrying the CRISPR/Cas9 system to abate transplant rejection.

RevDate: 2020-09-23
CmpDate: 2020-09-23

Connelly JP, SM Pruett-Miller (2019)

CRIS.py: A Versatile and High-throughput Analysis Program for CRISPR-based Genome Editing.

Scientific reports, 9(1):4194 pii:10.1038/s41598-019-40896-w.

CRISPR-Cas9 technology allows the creation of user-defined genomic modifications in cells and whole organisms. However, quantifying editing rates in pools of cells or identifying correctly edited clones is tedious. Targeted next-generation sequencing provides a high-throughput platform for optimizing editing reagents and identifying correctly modified clones, but the large amount of data produced can be difficult to analyze. Here, we present CRIS.py, a simple and highly versatile python-based program which concurrently analyzes next-generation sequencing data for both knock-out and multiple user-specified knock-in modifications from one or many edited samples. Compared to available NGS analysis programs for CRISPR based-editing, CRIS.py has many advantages: (1) the ability to analyze from one to thousands of samples at once, (2) the capacity to check each sample for multiple sequence modifications, including those induced by base-editors, (3) an output in an easily searchable file format enabling users to quickly sort through and identify correctly targeted clones.

RevDate: 2020-09-23
CmpDate: 2020-09-23

Slesarev A, Viswanathan L, Tang Y, et al (2019)

CRISPR/CAS9 targeted CAPTURE of mammalian genomic regions for characterization by NGS.

Scientific reports, 9(1):3587.

The robust detection of structural variants in mammalian genomes remains a challenge. It is particularly difficult in the case of genetically unstable Chinese hamster ovary (CHO) cell lines with only draft genome assemblies available. We explore the potential of the CRISPR/Cas9 system for the targeted capture of genomic loci containing integrated vectors in CHO-K1-based cell lines followed by next generation sequencing (NGS), and compare it to popular target-enrichment sequencing methods and to whole genome sequencing (WGS). Three different CRISPR/Cas9-based techniques were evaluated; all of them allow for amplification-free enrichment of target genomic regions in the range from 5 to 60 fold, and for recovery of ~15 kb-long sequences with no sequencing artifacts introduced. The utility of these protocols has been proven by the identification of transgene integration sites and flanking sequences in three CHO cell lines. The long enriched fragments helped to identify Escherichia coli genome sequences co-integrated with vectors, and were further characterized by Whole Genome Sequencing (WGS). Other advantages of CRISPR/Cas9-based methods are the ease of bioinformatics analysis, potential for multiplexing, and the production of long target templates for real-time sequencing.

RevDate: 2020-09-23
CmpDate: 2020-09-23

Kostyushev D, Kostyusheva A, Brezgin S, et al (2019)

Suppressing the NHEJ pathway by DNA-PKcs inhibitor NU7026 prevents degradation of HBV cccDNA cleaved by CRISPR/Cas9.

Scientific reports, 9(1):1847.

Chronic hepatitis B is a severe liver disease caused by hepatitis B virus (HBV) infection. Covalently closed circular DNA (cccDNA), a super-spiralized, double-stranded form of the HBV genome, is the major determinant of viral persistence. CRISPR/Cas9 nucleases have been recently shown to introduce double-stranded DNA breaks into HBV cccDNA. The inflicted damage results predominantly in erroneous repair of cccDNA by non-homologous end-joining (NHEJ). NHEJ has been suggested to enhance anti-HBV activity of CRISPR/Cas9 and increase cccDNA mutation. In this study, we assessed anti-HBV activity of CRISPR/Cas9 and cccDNA repair outcomes in an altered NHEJ/HR environment. NU7026, a strong inhibitor of NHEJ, prevented CRISPR/Cas9-mediated degradation of cccDNA and resulted in frequent on-target deletions. We conclude that CRISPR/Cas9 is a highly effective tool to degrade cccDNA and first demonstrate that inhibiting NHEJ impairs cccDNA degradation.

RevDate: 2020-09-22

Mitsui R, Yamada R, Matsumoto T, et al (2020)

Construction of lactic acid-tolerant Saccharomyces cerevisiae by using CRISPR-Cas-mediated genome evolution for efficient D-lactic acid production.

Applied microbiology and biotechnology pii:10.1007/s00253-020-10906-3 [Epub ahead of print].

Lactic acid (LA) is chemically synthesized or fermentatively produced using glucose as substrate, mainly using lactic acid bacteria. Polylactic acid is used as a biodegradable bioplastic for packaging materials, medical materials, and filaments for 3D printers. In this study, we aimed to construct a LA-tolerant yeast to reduce the neutralization cost in LA production. The pHLA2-51 strain was obtained through a previously developed genome evolution strategy, and transcriptome analysis revealed the gene expression profile of the mutant yeast. Furthermore, the expression of the genes associated with glycolysis and the LA synthesis pathway in the LA-tolerant yeast was comprehensively and randomly modified to construct a D-LA-producing, LA-tolerant yeast. In detail, DNA fragments expressing thirteen genes, HXT7, HXK2, PGI1, PFK1, PFK2, FBA1, TPI1, TDH3, PGK1, GPM1, ENO2, and PYK2, and D-lactate dehydrogenase (D-LDH) from Leuconostoc mesenteroides were randomly integrated into the genomic DNA in the LA-tolerant yeast. The resultant engineered yeast produced about 33.9 g/L of D-LA from 100 g/L glucose without neutralizing agents in a non-neutralized condition and 52.2 g/L of D-LA from 100 g/L glucose with 20 g/L CaCO3 in a semi-neutralized condition. Our research provides valuable insights into non-neutralized fermentative production of LA. KEY POINTS: • Lactic acid (LA) tolerance of yeast was improved by genome evolution. • The transcription levels of 751 genes were changed under LA stress. • Rapid LA production with semi-neutralization was achieved by modifying glycolysis. • A versatile yeast strain construction method based on the CRISPR system was proposed.

RevDate: 2020-09-22

Lin J, Fuglsang A, Kjeldsen AL, et al (2020)

DNA targeting by subtype I-D CRISPR-Cas shows type I and type III features.

Nucleic acids research pii:5909925 [Epub ahead of print].

Prokaryotic CRISPR-Cas immune systems are classified into six types based on their effector complexes which cleave dsDNA specifically (types I, II and V), ssRNA exclusively (type VI) or both ssRNA via a ruler mechanism and ssDNA unspecifically (type III). To date, no specific cleavage of ssDNA target has been reported for CRISPR-Cas. Here, we demonstrate dual dsDNA and ssDNA cleavage activities of a subtype I-D system which carries a type III Cas10-like large subunit, Cas10d. In addition to a specific dsDNA cleavage activity dependent on the HD domain of Cas10d, the helicase Cas3' and a compatible protospacer adjacent motif (PAM), the subtype I-D effector complex can cleave ssDNA that is complementary in sequence to the crRNA. Significantly, the ssDNA cleavage sites occur at 6-nt intervals and the cleavage is catalysed by the backbone subunit Csc2 (Cas7), similar to the periodic cleavage of ssRNA by the backbone subunit of type III effectors. The typical type I cleavage of dsDNA combined with the exceptional 6-nt spaced cleavage of ssDNA and the presence of a type III like large subunit provide strong evidence for the subtype I-D system being an evolutionary intermediate between type I and type III CRISPR-Cas systems.

RevDate: 2020-09-22

Zhang B (2020)

CRISPR/Cas gene therapy.

Journal of cellular physiology [Epub ahead of print].

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated enzyme (Cas) is a naturally occurring genome editing tool adopted from the prokaryotic adaptive immune defense system. Currently, CRISPR/Cas9-based genome editing has been becoming one of the most promising tools for treating human genetic diseases, including cardiovascular diseases, neuro-disorders, and cancers. As the quick modification of the CRISPR/Cas9 system, including delivery system, CRISPR/Cas9-based gene therapy has been extensively studied in preclinic and clinic treatments. CRISPR/Cas genome editing is also a robust tool to create animal genetic models for studying and treating human genetic disorders, particularly diseases associated with point mutations. However, significant challenges also remain before CRISPR/Cas technology can be routinely employed in the clinic for treating different genetic diseases, which include toxicity and immune response of treated cells to CRISPR/Cas component, highly throughput delivery method, and potential off-target impact. The off-target effect is one of the major concerns for CRISPR/Cas9 gene therapy, more research should be focused on limiting this impact by designing high specific gRNAs and using high specificity of Cas enzymes. Modifying the CRISPR/Cas9 delivery method not only targets a specific tissue/cell but also potentially limits the off-target impact.

RevDate: 2020-09-22

Yu J, Tu L, Subburaj S, et al (2020)

Simultaneous targeting of duplicated genes in Petunia protoplasts for flower color modification via CRISPR-Cas9 ribonucleoproteins.

Plant cell reports pii:10.1007/s00299-020-02593-1 [Epub ahead of print].

KEY MESSAGE: We obtained a complete mutant line of Petunia having mutations in both F3H genes via Cas9-ribonucleoproteins delivery, which exhibited a pale purplish pink flower color. The CRISPR-Cas system is now revolutionizing agriculture by allowing researchers to generate various desired mutations in plants at will. In particular, DNA-free genome editing via Cas9-ribonucleoproteins (RNPs) delivery has many advantages in plants; it does not require codon optimization or specific promoters for expression in plant cells; furthermore, it can bypass GMO regulations in some countries. Here, we have performed site-specific mutagenesis in Petunia to engineer flower color modifications. We determined that the commercial Petunia cultivar 'Madness Midnight' has two F3H coding genes and designed one guide RNA that targets both F3H genes at once. Among 67 T0 plants regenerated from Cas9-RNP transfected protoplasts, we obtained seven mutant lines that contain mutations in either F3HA or F3HB gene and one complete mutant line having mutations in both F3H genes without any selectable markers. It is noteworthy that only the f3ha f3hb exhibited a clearly modified, pale purplish pink flower color (RHS 69D), whereas the others, including the single copy gene knock-out plants, displayed purple violet (RHS 93A) flowers similar to the wild-type Petunia. To the best of our knowledge, we demonstrated a precedent of ornamental crop engineering by DNA-free CRISPR method for the first time, which will greatly accelerate a transition from a laboratory to a farmer's field.

RevDate: 2020-09-22
CmpDate: 2020-09-22

Wang J, Zhang X, Cheng L, et al (2020)

An overview and metanalysis of machine and deep learning-based CRISPR gRNA design tools.

RNA biology, 17(1):13-22.

The CRISPR-Cas9 system has become the most promising and versatile tool for genetic manipulation applications. Albeit the technology has been broadly adopted by both academic and pharmaceutic societies, the activity (on-target) and specificity (off-target) of CRISPR-Cas9 are decisive factors for any application of the technology. Several in silico gRNA activity and specificity predicting models and web tools have been developed, making it much more convenient and precise for conducting CRISPR gene editing studies. In this review, we present an overview and comparative analysis of machine and deep learning (MDL)-based algorithms, which are believed to be the most effective and reliable methods for the prediction of CRISPR gRNA on- and off-target activities. As an increasing number of sequence features and characteristics are discovered and are incorporated into the MDL models, the prediction outcome is getting closer to experimental observations. We also introduced the basic principle of CRISPR activity and specificity and summarized the challenges they faced, aiming to facilitate the CRISPR communities to develop more accurate models for applying.

RevDate: 2020-09-22
CmpDate: 2020-09-22

Wang S, Min Z, Ji Q, et al (2020)

Rescue of premature aging defects in Cockayne syndrome stem cells by CRISPR/Cas9-mediated gene correction.

Protein & cell, 11(1):1-22.

Cockayne syndrome (CS) is a rare autosomal recessive inherited disorder characterized by a variety of clinical features, including increased sensitivity to sunlight, progressive neurological abnormalities, and the appearance of premature aging. However, the pathogenesis of CS remains unclear due to the limitations of current disease models. Here, we generate integration-free induced pluripotent stem cells (iPSCs) from fibroblasts from a CS patient bearing mutations in CSB/ERCC6 gene and further derive isogenic gene-corrected CS-iPSCs (GC-iPSCs) using the CRISPR/Cas9 system. CS-associated phenotypic defects are recapitulated in CS-iPSC-derived mesenchymal stem cells (MSCs) and neural stem cells (NSCs), both of which display increased susceptibility to DNA damage stress. Premature aging defects in CS-MSCs are rescued by the targeted correction of mutant ERCC6. We next map the transcriptomic landscapes in CS-iPSCs and GC-iPSCs and their somatic stem cell derivatives (MSCs and NSCs) in the absence or presence of ultraviolet (UV) and replicative stresses, revealing that defects in DNA repair account for CS pathologies. Moreover, we generate autologous GC-MSCs free of pathogenic mutation under a cGMP (Current Good Manufacturing Practice)-compliant condition, which hold potential for use as improved biomaterials for future stem cell replacement therapy for CS. Collectively, our models demonstrate novel disease features and molecular mechanisms and lay a foundation for the development of novel therapeutic strategies to treat CS.

RevDate: 2020-09-22
CmpDate: 2020-09-22

Rezza A, Jacquet C, Le Pillouer A, et al (2019)

Unexpected genomic rearrangements at targeted loci associated with CRISPR/Cas9-mediated knock-in.

Scientific reports, 9(1):3486.

The CRISPR/Cas9 gene editing tool enables accessible and efficient modifications which (re)ignited molecular research in certain species. However, targeted integration of large DNA fragments using CRISPR/Cas9 can still be challenging in numerous models. To systematically compare CRISPR/Cas9's efficiency to classical homologous recombination (cHR) for insertion of large DNA fragments, we thoroughly performed and analyzed 221 experiments targeting 128 loci in mouse ES cells. Although both technologies proved efficient, CRISPR/Cas9 yielded significantly more positive clones as detected by overlapping PCRs. It also induced unexpected rearrangements around the targeted site, ultimately rendering CRISPR/Cas9 less efficient than cHR for the production of fully validated clones. These data show that CRISPR/Cas9-mediated recombination can induce complex long-range modifications at targeted loci, thus emphasizing the need for thorough characterization of any genetically modified material obtained through CRISPR-mediated gene editing before further functional studies or therapeutic use.

RevDate: 2020-09-18
CmpDate: 2020-09-18

Yildiz O, Downes GB, CG Sagerström (2019)

Zebrafish prdm12b acts independently of nkx6.1 repression to promote eng1b expression in the neural tube p1 domain.

Neural development, 14(1):5.

BACKGROUND: Functioning of the adult nervous system depends on the establishment of neural circuits during embryogenesis. In vertebrates, neurons that make up motor circuits form in distinct domains along the dorsoventral axis of the neural tube. Each domain is characterized by a unique combination of transcription factors (TFs) that promote a specific fate, while repressing fates of adjacent domains. The prdm12 TF is required for the expression of eng1b and the generation of V1 interneurons in the p1 domain, but the details of its function remain unclear.

METHODS: We used CRISPR/Cas9 to generate the first germline mutants for prdm12 and employed this resource, together with classical luciferase reporter assays and co-immunoprecipitation experiments, to study prdm12b function in zebrafish. We also generated germline mutants for bhlhe22 and nkx6.1 to examine how these TFs act with prdm12b to control p1 formation.

RESULTS: We find that prdm12b mutants lack eng1b expression in the p1 domain and also possess an abnormal touch-evoked escape response. Using luciferase reporter assays, we demonstrate that Prdm12b acts as a transcriptional repressor. We also show that the Bhlhe22 TF binds via the Prdm12b zinc finger domain to form a complex. However, bhlhe22 mutants display normal eng1b expression in the p1 domain. While prdm12 has been proposed to promote p1 fates by repressing expression of the nkx6.1 TF, we do not observe an expansion of the nkx6.1 domain upon loss of prdm12b function, nor is eng1b expression restored upon simultaneous loss of prdm12b and nkx6.1.

CONCLUSIONS: We conclude that prdm12b germline mutations produce a phenotype that is indistinguishable from that of morpholino-mediated loss of prdm12 function. In terms of prdm12b function, our results indicate that Prdm12b acts as transcriptional repressor and interacts with both EHMT2/G9a and Bhlhe22. However, bhlhe22 function is not required for eng1b expression in vivo, perhaps indicating that other bhlh genes can compensate during embryogenesis. Lastly, we do not find evidence for nkx6.1 and prdm12b acting as a repressive pair in formation of the p1 domain - suggesting that prdm12b is not solely required to repress non-p1 fates, but is specifically needed to promote p1 fates.

RevDate: 2020-09-21

Abdullah , Jiang Z, Hong X, et al (2020)

CRISPR base editing and prime editing: DSB and template-free editing systems for bacteria and plants.

Synthetic and systems biotechnology, 5(4):277-292 pii:S2405-805X(20)30062-4.

CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated) has been extensively exploited as a genetic tool for genome editing. The RNA guided Cas nucleases generate DNA double-strand break (DSB), triggering cellular repair systems mainly Non-homologous end-joining (NHEJ, imprecise repair) or Homology-directed repair (HDR, precise repair). However, DSB typically leads to unexpected DNA changes and lethality in some organisms. The establishment of bacteria and plants into major bio-production platforms require efficient and precise editing tools. Hence, in this review, we focus on the non-DSB and template-free genome editing, i.e., base editing (BE) and prime editing (PE) in bacteria and plants. We first highlight the development of base and prime editors and summarize their studies in bacteria and plants. We then discuss current and future applications of BE/PE in synthetic biology, crop improvement, evolutionary engineering, and metabolic engineering. Lastly, we critically consider the challenges and prospects of BE/PE in PAM specificity, editing efficiency, off-targeting, sequence specification, and editing window.

RevDate: 2020-09-21

Molla KA, Qi Y, Karmakar S, et al (2020)

Base Editing Landscape Extends to Perform Transversion Mutation.

Trends in genetics : TIG pii:S0168-9525(20)30237-7 [Epub ahead of print].

Base editors have drawn considerable academic and industrial attention in recent years because of their ability to alter single DNA bases with precision. However, the existing cytosine and adenine base editors can only install transition mutations. Three recent studies (Kurt et al.,Zhao et al., and Chen et al.) expand the base editing toolbox by developing cytosine transversion base editors.

RevDate: 2020-09-21
CmpDate: 2020-09-21

Sato M (2020)

[Manipulating Living Systems by Light].

Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan, 140(8):993-1000.

The human genome consists of more than 20000 genes and is essential for all biological phenomena. To understand these biological phenomena, including diseases, and to be able to modify them, approaches that enable optical control of the genome may be useful. Recently, we developed an optogenetic tool, named photoactivatable Cas9 (PA-Cas9). We divided Cas9 nuclease from the CRISPR-Cas9 system into two fragments and connected photo-inducible dimerization proteins, named Magnet system, to the fragments, leading to the development of PA-Cas9 of which nuclease activity is switchable with light. PA-Cas9 allows direct editing of DNA sequences by light stimulation. Additionally, we developed a light-inducible, RNA-guided programmable system for endogenous gene activation based on the CRISPR-Cas9 system. We demonstrated that this optogenetic tool allows rapid and reversible targeted gene activation by light. Using this tool, we exemplified optical control of neuronal differentiation of human induced pluripotent stem cells (iPSCs). The CRISPR-Cas9-based, photoactivatable transcription system offers a simple and versatile approach to precise gene activation. In addition to the CRISPR-Cas9-based optogenetic tools, we developed a photoactivatable Cre-loxP system. This tool allows optical control of DNA recombination reaction in an internal organ even by external, noninvasive illumination using LED light source. To date, genome engineering technology and optogenetics technology have emerged separately as different applications. Our studies described above merge these emerging research fields together.

RevDate: 2020-09-21
CmpDate: 2020-09-21

Pavani G, Laurent M, Fabiano A, et al (2020)

Ex vivo editing of human hematopoietic stem cells for erythroid expression of therapeutic proteins.

Nature communications, 11(1):3778.

Targeted genome editing has a great therapeutic potential to treat disorders that require protein replacement therapy. To develop a platform independent of specific patient mutations, therapeutic transgenes can be inserted in a safe and highly transcribed locus to maximize protein expression. Here, we describe an ex vivo editing approach to achieve efficient gene targeting in human hematopoietic stem/progenitor cells (HSPCs) and robust expression of clinically relevant proteins by the erythroid lineage. Using CRISPR-Cas9, we integrate different transgenes under the transcriptional control of the endogenous α-globin promoter, recapitulating its high and erythroid-specific expression. Erythroblasts derived from targeted HSPCs secrete different therapeutic proteins, which retain enzymatic activity and cross-correct patients' cells. Moreover, modified HSPCs maintain long-term repopulation and multilineage differentiation potential in transplanted mice. Overall, we establish a safe and versatile CRISPR-Cas9-based HSPC platform for different therapeutic applications, including hemophilia and inherited metabolic disorders.

RevDate: 2020-09-21
CmpDate: 2020-09-21

Cortez JT, Montauti E, Shifrut E, et al (2020)

CRISPR screen in regulatory T cells reveals modulators of Foxp3.

Nature, 582(7812):416-420.

Regulatory T (Treg) cells are required to control immune responses and maintain homeostasis, but are a significant barrier to antitumour immunity1. Conversely, Treg instability, characterized by loss of the master transcription factor Foxp3 and acquisition of proinflammatory properties2, can promote autoimmunity and/or facilitate more effective tumour immunity3,4. A comprehensive understanding of the pathways that regulate Foxp3 could lead to more effective Treg therapies for autoimmune disease and cancer. The availability of new functional genetic tools has enabled the possibility of systematic dissection of the gene regulatory programs that modulate Foxp3 expression. Here we developed a CRISPR-based pooled screening platform for phenotypes in primary mouse Treg cells and applied this technology to perform a targeted loss-of-function screen of around 500 nuclear factors to identify gene regulatory programs that promote or disrupt Foxp3 expression. We identified several modulators of Foxp3 expression, including ubiquitin-specific peptidase 22 (Usp22) and ring finger protein 20 (Rnf20). Usp22, a member of the deubiquitination module of the SAGA chromatin-modifying complex, was revealed to be a positive regulator that stabilized Foxp3 expression; whereas the screen suggested that Rnf20, an E3 ubiquitin ligase, can serve as a negative regulator of Foxp3. Treg-specific ablation of Usp22 in mice reduced Foxp3 protein levels and caused defects in their suppressive function that led to spontaneous autoimmunity but protected against tumour growth in multiple cancer models. Foxp3 destabilization in Usp22-deficient Treg cells could be rescued by ablation of Rnf20, revealing a reciprocal ubiquitin switch in Treg cells. These results reveal previously unknown modulators of Foxp3 and demonstrate a screening method that can be broadly applied to discover new targets for Treg immunotherapies for cancer and autoimmune disease.

RevDate: 2020-09-21
CmpDate: 2020-09-21

Leist SR, AS Cockrell (2020)

Genetically Engineering a Susceptible Mouse Model for MERS-CoV-Induced Acute Respiratory Distress Syndrome.

Methods in molecular biology (Clifton, N.J.), 2099:137-159.

Since 2012, monthly cases of Middle East respiratory syndrome coronavirus (MERS-CoV) continue to cause severe respiratory disease that is fatal in ~35% of diagnosed individuals. The ongoing threat to global public health and the need for novel therapeutic countermeasures have driven the development of animal models that can reproducibly replicate the pathology associated with MERS-CoV in human infections. The inability of MERS-CoV to replicate in the respiratory tracts of mice, hamsters, and ferrets stymied initial attempts to generate small animal models. Identification of human dipeptidyl peptidase IV (hDPP4) as the receptor for MERS-CoV infection opened the door for genetic engineering of mice. Precise molecular engineering of mouse DPP4 (mDPP4) with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology maintained inherent expression profiles, and limited MERS-CoV susceptibility to tissues that naturally express mDPP4, notably the lower respiratory tract wherein MERS-CoV elicits severe pulmonary pathology. Here, we describe the generation of the 288-330+/+ MERS-CoV mouse model in which mice were made susceptible to MERS-CoV by modifying two amino acids on mDPP4 (A288 and T330), and the use of adaptive evolution to generate novel MERS-CoV isolates that cause fatal respiratory disease. The 288-330+/+ mice are currently being used to evaluate novel drug, antibody, and vaccine therapeutic countermeasures for MERS-CoV. The chapter starts with a historical perspective on the emergence of MERS-CoV and animal models evaluated for MERS-CoV pathogenesis, and then outlines the development of the 288-330+/+ mouse model, assays for assessing a MERS-CoV pulmonary infection in a mouse model, and describes some of the challenges associated with using genetically engineered mice.

RevDate: 2020-09-21
CmpDate: 2020-09-21

Liu W, Rudis MR, Cheplick MH, et al (2020)

Lipofection-mediated genome editing using DNA-free delivery of the Cas9/gRNA ribonucleoprotein into plant cells.

Plant cell reports, 39(2):245-257.

KEY MESSAGE: A novel and robust lipofection-mediated transfection approach for the use of DNA-free Cas9/gRNA RNP for gene editing has demonstrated efficacy in plant cells. Precise genome editing has been revolutionized by CRISPR/Cas9 systems. DNA-based delivery of CRISPR/Cas9 is widely used in various plant species. However, protein-based delivery of the in vitro translated Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) complex into plant cells is still in its infancy even though protein delivery has several advantages. These advantages include DNA-free delivery, gene-edited host plants that are not transgenic, ease of use, low cost, relative ease to be adapted to high-throughput systems, and low off-target cleavage rates. Here, we show a novel lipofection-mediated transfection approach for protein delivery of the preassembled Cas9/gRNA RNP into plant cells for genome editing. Two lipofection reagents, Lipofectamine 3000 and RNAiMAX, were adapted for successful delivery into plant cells of Cas9/gRNA RNP. A green fluorescent protein (GFP) reporter was fused in-frame with the C-terminus of the Cas9 protein and the fusion protein was successfully delivered into non-transgenic tobacco cv. 'Bright Yellow-2' (BY2) protoplasts. The optimal efficiencies for Lipofectamine 3000- and RNAiMAX-mediated protein delivery were 66% and 48%, respectively. Furthermore, we developed a biolistic method for protein delivery based on the known proteolistics technique. A transgenic tobacco BY2 line expressing an orange fluorescence protein reporter pporRFP was targeted for knockout. We found that the targeted mutagenesis frequency for our Lipofectamine 3000-mediated protein delivery was 6%. Our results showed that the newly developed lipofection-mediated transfection approach is robust for the use of the DNA-free Cas9/gRNA technology for genome editing in plant cells.

RevDate: 2020-09-21
CmpDate: 2020-09-21

Lee J, Le QV, Yang G, et al (2019)

Cas9-edited immune checkpoint blockade PD-1 DNA polyaptamer hydrogel for cancer immunotherapy.

Biomaterials, 218:119359.

Immune checkpoint inhibitors have been widely studied in immunotherapy. Although antibodies have been more widely used to block immune checkpoints, DNA aptamers have unique advantages for this purpose. Here, we designed a DNA polyaptamer hydrogel that can be precisely cut by Cas9/sgRNA for programmed release of an immune checkpoint-blocking DNA aptamer. As a representative immune checkpoint inhibitor, we used a PD-1 DNA aptamer. Rolling-circle amplification was used to generate a hydrogel comprising DNA with PD-1 aptamer and an sgRNA-targeting sequence. When mixed with Cas9/sgRNA, the PD-1 DNA aptamer hydrogel (PAH) lost its gel property and liberated the PD-1 aptamer sequence. The precise Cas9/sgRNA-mediated release of the PD-1 DNA aptamer, which was confirmed by gel electrophoresis, was found to effectively activate the cytokine-secretion function of splenocytes. In vivo, molecular imaging revealed that PD-1 DNA polyaptamer hydrogel co-injected with Cas9/sgRNA (Cas9/PAH) remained at the injection site longer than free aptamer and yielded significantly higher antitumor effects and survival than hydrogel or free aptamer. Moreover, increased immune cell filtration was observed at tumor tissues treated with Cas9/PAH. These results suggest that our Cas9/sgRNA-edited immune checkpoint-blocking aptamer hydrogel has strong potential for anticancer immunotherapy.

RevDate: 2020-09-21
CmpDate: 2020-09-21

Hu J, Jiang M, Liu R, et al (2019)

Label-Free CRISPR/Cas9 Assay for Site-Specific Nucleic Acid Detection.

Analytical chemistry, 91(16):10870-10878.

The development of the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has become a revolutionary step for genome engineering because it enables modification of target genomes. However, many biological applications with the CRISPR/Cas9 system are impeded by off-target effects and loci-dependent nuclease activity with various sgRNAs. Commonly used label-strategy-based CRISPR/Cas9 assays often suffer from possible disturbances to Cas9 activity and a time-consuming labeling procedure. Herein, we for the first time propose a DNA-templated CuNPs-based label-free CRISPR/Cas9 assay, with a low LOD of 0.13 nM and rapid detection in 35 min after CRISPR/Cas9 cleavage. Additionally, the site specificity of the DNA substrate was demonstrated. Through the proposed label-free strategy, a single-base change at a specific loci could lead to a significant reduction of the Cas9 cleavage effect, while the other common genetic modifications might be accepted by the CRIPR/Cas9 system. Therefore, the proposed label-free Cas9 assay may provide a new paradigm for the a priori in vitro CRISPR/Cas9 assay and exploration for in vivo biological applications.

RevDate: 2020-09-21
CmpDate: 2020-09-21

De Caneva A, Porro F, Bortolussi G, et al (2019)

Coupling AAV-mediated promoterless gene targeting to SaCas9 nuclease to efficiently correct liver metabolic diseases.

JCI insight, 5:.

Non-integrative AAV-mediated gene therapy in the liver is effective in adult patients, but faces limitations in pediatric settings due to episomal DNA loss during hepatocyte proliferation. Gene targeting is a promising approach by permanently modifying the genome. We previously rescued neonatal lethality in Crigler-Najjar mice by inserting a promoterless human uridine glucuronosyl transferase A1 (UGT1A1) cDNA in exon 14 of the albumin gene, without the use of nucleases. To increase recombination rate and therapeutic efficacy, here we used CRISPR/SaCas9. Neonatal mice were transduced with two AAVs: one expressing the SaCas9 and sgRNA, and one containing a promoterless cDNA flanked by albumin homology regions. Targeting efficiency increased ~26-fold with an eGFP reporter cDNA, reaching up to 24% of eGFP-positive hepatocytes. Next, we fully corrected the diseased phenotype of Crigler-Najjar mice by targeting the hUGT1A1 cDNA. Treated mice had normal plasma bilirubin up to 10 months after administration, hUGT1A1 protein levels were ~6-fold higher than in WT liver, with a 90-fold increase in recombination rate. Liver histology, inflammatory markers, and plasma albumin were normal in treated mice, with no off-targets in predicted sites. Thus, the improved efficacy and reassuring safety profile support the potential application of the proposed approach to other liver diseases.

RevDate: 2020-09-21
CmpDate: 2020-09-21

Cal L, Suarez-Bregua P, Comesaña P, et al (2019)

Countershading in zebrafish results from an Asip1 controlled dorsoventral gradient of pigment cell differentiation.

Scientific reports, 9(1):3449.

Dorso-ventral (DV) countershading is a highly-conserved pigmentary adaptation in vertebrates. In mammals, spatially regulated expression of agouti-signaling protein (ASIP) generates the difference in shading by driving a switch between the production of chemically-distinct melanins in melanocytes in dorsal and ventral regions. In contrast, fish countershading seemed to result from a patterned DV distribution of differently-coloured cell-types (chromatophores). Despite the cellular differences in the basis for counter-shading, previous observations suggested that Agouti signaling likely played a role in this patterning process in fish. To test the hypotheses that Agouti regulated counter-shading in fish, and that this depended upon spatial regulation of the numbers of each chromatophore type, we engineered asip1 homozygous knockout mutant zebrafish. We show that loss-of-function asip1 mutants lose DV countershading, and that this results from changed numbers of multiple pigment cell-types in the skin and on scales. Our findings identify asip1 as key in the establishment of DV countershading in fish, but show that the cellular mechanism for translating a conserved signaling gradient into a conserved pigmentary phenotype has been radically altered in the course of evolution.

RevDate: 2020-09-15
CmpDate: 2020-09-15

Roghanian M, Semsey S, Løbner-Olesen A, et al (2019)

(p)ppGpp-mediated stress response induced by defects in outer membrane biogenesis and ATP production promotes survival in Escherichia coli.

Scientific reports, 9(1):2934.

Cellular growth requires a high level of coordination to ensure that all processes run in concert. The role of the nucleotide alarmone (p)ppGpp has been extensively studied in response to external stresses, such as amino acid starvation, in Escherichia coli, but much less is known about the involvement of (p)ppGpp in response to perturbations in intracellular processes. We therefore employed CRISPRi to transcriptionally repress essential genes involved in 14 vital processes and investigated whether a (p)ppGpp-mediated response would be induced. We show that (p)ppGpp is produced and required for a pertinent stress response during interference with outer membrane biogenesis and ADP synthesis specifically. When these processes were perturbed via the transcriptional repression of essential genes, wild type E. coli MG1655 ceased growing and entered a semi-dormant state, whereas isogenic (p)ppGpp0 cells continued to grow uncontrollably to the point of lysis. Furthermore, in vivo measurements revealed that the ATP levels were intrinsically offset in (p)ppGpp0 cells, further indicating a role for the alarmone in cellular energy homeostasis. In summary, our investigation suggests that (p)ppGpp acts as a coordinator of cell growth in response to imbalances in outer membrane biogenesis and adenosine ribonucleotide synthesis, elucidating novel roles for (p)ppGpp in bacterial physiology.

RevDate: 2020-09-18

Teixeira LPR, Lopes FEM, Antunes ASLM, et al (2020)

Application of a cost-effective DNA extraction protocol for screening transgenic and CRISPR-edited primary goat cells.

PloS one, 15(9):e0239435 pii:PONE-D-20-17199.

The genotyping of genetically-modified cells is a crucial step in studies of transgenics and genomic editing with systems such as CRISPR/Cas. The detection of genome editing events can be directly related to the genotyping methodology used, which is influenced by its costs, since many experiments require the analysis of a large number of samples. The aim of this study was to compare the performance of direct lysis methods of genomic DNA (gDNA) extraction for the detection of knockins and knockouts in primary goat cells. Initially, three gDNA extraction protocols (protocol A, heat denaturation/freeze-thaw in water; protocol B, heat denaturation/proteinase K; and protocol C, CellsDirect Kit) were tested using different quantities (1,000, 5,000 and 10,000 cells) and types of goat primary cells (fibroblasts and goat mammary epithelial cells-GMECs) for subsequent validation by PCR amplification of small (GAPDH) and large amplicons (hLF transgene). All protocols were successful in the detection of the small amplicon; however, in GMECs, only protocol B resulted efficient amplification (protocol A-0%, protocol B-93%, protocol C-13.33%, P <0.05). In a proof-of-principle experiment, the TP53 gene was knocked out in GMECs by CRISPR/Cas9-mediated deletion while constructs containing the anti-VEGF monoclonal antibody (pBC-anti-VEGF) and bacterial L-Asparaginase (pBC-ASNase) transgenes were knocked-in separately in fibroblasts. Detection of successful editing was performed using protocol B and PCR. The integration rates of the pBC-ASNase and pBC-anti-VEGF transgenes were 93.6% and 72%, respectively, as per PCR. The efficiency of biallelic editing in GMECs using CRISPR/Cas9 for the TP53 deletion was 5.4%. Our results suggest that protocol B (heat denaturation/proteinase K) can be used as an inexpensive and quick methodology for detecting genetic modifications in different types of primary goat cells, with efficiency rates consistent with values previously described in the literature when using extraction kits or more complex proteinase K formulations.

RevDate: 2020-09-18

Gratacap RL, Jin YH, Mantsopoulou M, et al (2020)

Efficient Genome Editing in Multiple Salmonid Cell Lines Using Ribonucleoprotein Complexes.

Marine biotechnology (New York, N.Y.) pii:10.1007/s10126-020-09995-y [Epub ahead of print].

Infectious and parasitic diseases have major negative economic and animal welfare impacts on aquaculture of salmonid species. Improved knowledge of the functional basis of host response and genetic resistance to these diseases is key to developing preventative and treatment options. Cell lines provide valuable models to study infectious diseases in salmonids, and genome editing using CRISPR/Cas systems provides an exciting avenue to evaluate the function of specific genes in those systems. While CRISPR/Cas editing has been successfully performed in a Chinook salmon cell line (CHSE-214), there are no reports to date of editing of cell lines derived from the most commercially relevant salmonid species Atlantic salmon and rainbow trout, which are difficult to transduce and therefore edit using lentivirus-mediated methods. In the current study, a method of genome editing of salmonid cell lines using ribonucleoprotein (RNP) complexes was optimised and tested in the most commonly used salmonid fish cell lines: Atlantic salmon (SHK-1 and ASK cell lines), rainbow trout (RTG-2) and Chinook salmon (CHSE-214). Electroporation of RNP based on either Cas9 or Cas12a was efficient at targeted editing of all the tested lines (typically > 90% cells edited), and the choice of enzyme expands the number of potential target sites for editing within the genomes of these species. These optimised protocols will facilitate functional genetic studies in salmonid cell lines, which are widely used as model systems for infectious diseases in aquaculture.

RevDate: 2020-09-18

Vu A, PB McCray (, Jr) (2020)

New Directions in Pulmonary Gene Therapy.

Human gene therapy, 31(17-18):921-939.

The lung has long been a target for gene therapy, yet efficient delivery and phenotypic disease correction has remained challenging. Although there have been significant advancements in gene therapies of other organs, including the development of several ex vivo therapies, in vivo therapeutics of the lung have been slower to transition to the clinic. Within the past few years, the field has witnessed an explosion in the development of new gene addition and gene editing strategies for the treatment of monogenic disorders. In this review, we will summarize current developments in gene therapy for cystic fibrosis, alpha-1 antitrypsin deficiency, and surfactant protein deficiencies. We will explore the different gene addition and gene editing strategies under investigation and review the challenges of delivery to the lung.

RevDate: 2020-09-18
CmpDate: 2020-09-18

Liu MS, Gong S, Yu HH, et al (2020)

Engineered CRISPR/Cas9 enzymes improve discrimination by slowing DNA cleavage to allow release of off-target DNA.

Nature communications, 11(1):3576.

CRISPR/Cas9 is a programmable genome editing tool widely used for biological applications and engineered Cas9s have increased discrimination against off-target cleavage compared with wild-type Streptococcus pyogenes (SpCas9) in vivo. To understand the basis for improved discrimination against off-target DNA containing important mismatches at the distal end of the guide RNA, we performed kinetic analyses on the high-fidelity (Cas9-HF1) and hyper-accurate (HypaCas9) engineered Cas9 variants. We show that DNA cleavage is impaired by more than 100- fold for the high-fidelity variants. The high-fidelity variants improve discrimination by slowing the observed rate of cleavage without increasing the rate of DNA rewinding and release. The kinetic partitioning favors release rather than cleavage of a bound off-target substrate only because the cleavage rate is so low. Further improvement in discrimination may require engineering increased rates of dissociation of off-target DNA.

RevDate: 2020-09-18
CmpDate: 2020-09-18

Lin P, Qin S, Pu Q, et al (2020)

CRISPR-Cas13 Inhibitors Block RNA Editing in Bacteria and Mammalian Cells.

Molecular cell, 78(5):850-861.e5.

Cas13 has demonstrated unique and broad utility in RNA editing, nucleic acid detection, and disease diagnosis; however, a constantly active Cas enzyme may induce unwanted effects. Bacteriophage- or prophage-region-encoded anti-CRISPR (acr) gene molecules provide the potential to control targeting specificity and potency to allow for optimal RNA editing and nucleic acid detection by spatiotemporally modulating endonuclease activities. Using integrated approaches to screen acrVI candidates and evaluate their effects on Cas13 function, we discovered a series of acrVIA1-7 genes that block the activities of Cas13a. These VI-A CRISPR inhibitors substantially attenuate RNA targeting and editing by Cas13a in human cells. Strikingly, type VI-A anti-CRISPRs (AcrVIAs) also significantly muffle the single-nucleic-acid editing ability of the dCas13a RNA-editing system. Mechanistically, AcrVIA1, -4, -5, and -6 bind LwaCas13a, while AcrVIA2 and -3 can only bind the LwaCas13-crRNA (CRISPR RNA) complex. These identified acr molecules may enable precise RNA editing in Cas13-based application and study of phage-bacterium interaction.

RevDate: 2020-09-18
CmpDate: 2020-09-18

Li X, Liu Q, Sun W, et al (2020)

Improving cellulases production by Myceliophthora thermophila through disruption of protease genes.

Biotechnology letters, 42(2):219-229.

OBJECTIVE: To identify main protease genes for the proteolytic degradation of cellulases in M. thermophila and generate a lower-proteases fungal host that can be used for further metabolic engineering to increase cellulase production and heterologous protein expression.

RESULTS: Systematic transcriptomic analysis were conducted on the expression of proteases genes in M. thermophila genome and five highly expressed genes encoding extracellular proteases were selected for mutation analyses. A series of single- and multi-gene mutants of these five selected genes was constructed using the CRISPR-Cas9 technique. Compared with WT, the ΔMtalp1 and the quintuple mutant showed significantly lower protease activity (decreased 52.7% and 58.4%, respectively) and at least double enhanced cellulase production.

CONCLUSIONS: The results indicated that Mtalp1 is a critical protease gene in cellulase degradation in M. thermophila and disruption of protease genes showed significantly decreased protease activity and obviously enhanced cellulase production in the fermentation broth of ΔMtalp1 and the quintuple mutant.

RevDate: 2020-09-18
CmpDate: 2020-09-18

She J, Wu Y, Lou B, et al (2019)

Genetic compensation by epob in pronephros development in epoa mutant zebrafish.

Cell cycle (Georgetown, Tex.), 18(20):2683-2696.

Zebrafish erythropoietin a (epoa) is a well characterized regulator of red blood cell formation. Recent morpholino mediated knockdown data have also identified epoa being essential for physiological pronephros development in zebrafish, which is driven by blocking apoptosis in developing kidneys. Yet, zebrafish mutants for epoa have not been described so far. In order to compare a transient knockdown vs. permanent knockout for epoa in zebrafish on pronephros development, we used CRISPR/Cas9 technology to generate epoa knockout zebrafish mutants and we performed structural and functional studies on pronephros development. In contrast to epoa morphants, epoa-/- zebrafish mutants showed normal pronephros structure; however, a previously uncharacterized gene in zebrafish, named epob, was identified and upregulated in epoa-/- mutants. epob knockdown altered pronephros development, which was further aggravated in epoa-/- mutants. Likewise, epoa and epob morphants regulated similar and differential gene signatures related to kidney development in zebrafish. In conclusion, stable loss of epoa during embryonic development can be compensated by epob leading to phenotypical discrepancies in epoa knockdown and knockout zebrafish embryos.

RevDate: 2020-09-17
CmpDate: 2020-09-17

Daniel-Moreno A, Lamsfus-Calle A, Raju J, et al (2019)

CRISPR/Cas9-modified hematopoietic stem cells-present and future perspectives for stem cell transplantation.

Bone marrow transplantation, 54(12):1940-1950.

Allogeneic hematopoietic stem cell transplantation (HSCT) is a standard therapeutic intervention for hematological malignancies and several monogenic diseases. However, this approach has limitations related to lack of a suitable donor, graft-versus-host disease and infectious complications due to immune suppression. On the contrary, autologous HSCT diminishes the negative effects of allogeneic HSCT. Despite the good efficacy, earlier gene therapy trials with autologous HSCs and viral vectors have raised serious safety concerns. However, the CRISPR/Cas9-edited autologous HSCs have been proposed to be an alternative option with a high safety profile. In this review, we summarized the possibility of CRISPR/Cas9-mediated autologous HSCT as a potential treatment option for various diseases supported by preclinical gene-editing studies. Furthermore, we discussed future clinical perspectives and possible clinical grade improvements of CRISPR/cas9-mediated autologous HSCT.

RevDate: 2020-09-17

Zhou-Li Y, Boisnard S, Enache-Angoulvant A, et al (2020)

Genome editing in the yeast Nakaseomyces delphensis and description of its complete sexual cycle.

Yeast (Chichester, England) [Epub ahead of print].

The environmental yeast Nakaseomyces delphensis is, phylogenetically, the closest known species to Candida glabrata, a major fungal pathogen of humans. C. glabrata is haploid and described as asexual, while N. delphensis is also haploid, but has been described as competent for mating and meiosis. Both genomes contain homologs of all the genes necessary for sexual reproduction, and also the genes for Ho-dependent mating-type switching, like Saccharomyces cerevisiae. We first report the construction of genetically-engineered strains of N. delphensis, including by CRISPR-Cas 9 gene-editing. We also report the description of the sexual cycle of N. delphensis. We show that it undergoes Ho-dependent mating-type switching in culture, and that deletion of the HO gene prevents such switching and allows maintenance of stable, separate, MATa and MATalpha haploid strains. Rare, genetically selected diploids can be obtained through mating of haploid strains, mutated or not for the HO gene. In contrast to HO/HO diploids, which behave as expected, Δho/Δho diploids exhibit unusual profiles in flow-cytometry. Both types of diploids can produce recombined haploid cells, which grow like the original haploid type strain. Our experiments thus allow the genetic manipulation of N. delphensis and the reconstruction, in the laboratory, of its entire life-cycle.

RevDate: 2020-09-17

Kot W, Olsen NS, Nielsen TK, et al (2020)

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

Nucleic acids research pii:5907968 [Epub ahead of print].

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

RevDate: 2020-09-17

Jolany Vangah S, Katalani C, Booneh HA, et al (2020)

CRISPR-Based Diagnosis of Infectious and Noninfectious Diseases.

Biological procedures online, 22:22 pii:135.

Interest in CRISPR technology, an instrumental component of prokaryotic adaptive immunity which enables prokaryotes to detect any foreign DNA and then destroy it, has gained popularity among members of the scientific community. This is due to CRISPR's remarkable gene editing and cleaving abilities. While the application of CRISPR in human genome editing and diagnosis needs to be researched more fully, and any potential side effects or ambiguities resolved, CRISPR has already shown its capacity in an astonishing variety of applications related to genome editing and genetic engineering. One of its most currently relevant applications is in diagnosis of infectious and non-infectious diseases. Since its initial discovery, 6 types and 22 subtypes of CRISPR systems have been discovered and explored. Diagnostic CRISPR systems are most often derived from types II, V, and VI. Different types of CRISPR-Cas systems which have been identified in different microorganisms can target DNA (e.g. Cas9 and Cas12 enzymes) or RNA (e.g. Cas13 enzyme). Viral, bacterial, and non-infectious diseases such as cancer can all be diagnosed using the cleavage activity of CRISPR enzymes from the aforementioned types. Diagnostic tests using Cas12 and Cas13 enzymes have already been developed for detection of the emerging SARS-CoV-2 virus. Additionally, CRISPR diagnostic tests can be performed using simple reagents and paper-based lateral flow assays, which can potentially reduce laboratory and patient costs significantly. In this review, the classification of CRISPR-Cas systems as well as the basis of the CRISPR/Cas mechanisms of action will be presented. The application of these systems in medical diagnostics with emphasis on the diagnosis of COVID-19 will be discussed.

RevDate: 2020-09-17

Liu Y, Dai L, Dong J, et al (2020)

Covalent modifications of bacteriophage genome confer a degree of resistance to bacterial CRISPR systems.

Journal of virology pii:JVI.01630-20 [Epub ahead of print].

The interplay between defense and counter-defense systems of bacteria and bacteriophages has been driving the evolution of both the organisms leading to their great genetic diversity on the planet. Restriction-modification systems are well-studied defense mechanisms of bacteria, where phages have evolved covalent modifications as a counter-defense mechanism to protect their genomes against restriction. Here, we present evidence that these genome modifications might also have been selected to counter, broadly, the CRISPR-Cas systems, an adaptive bacterial defense mechanism. We found that the phage T4 genome modified by cytosine hydroxymethylation and glucosylation (ghmC) exhibits various degrees of resistance to the type V CRISPR-Cas12a system, producing orders of magnitude more progeny than the T4(C) mutant that contains unmodified cytosines. Furthermore, the progeny accumulated CRISPR-escape mutations allowing rapid evolution of mutant phages under CRISPR pressure. A synergistic effect on phage restriction was observed when two CRISPR-Cas12a complexes were targeted to independent sites on the phage genome, another potential counter mechanism by bacteria to more effectively defend themselves against modified phages. These studies suggest that the defense-counter defense mechanisms by bacteria and phages while affording protection against one another also provide evolutionary benefits for both.IMPORTANCE Restriction-modification (R-M) and CRISPR-Cas systems are two well-known defense mechanisms of bacteria. Both recognize and cleave phage DNA at specific sites while protecting their own genomes. It is well-accepted that T4 and other phages have evolved counter-defense mechanisms to protect their genomes from R-M cleavage by covalent modifications such as the hydroxymethylation and glucosylation of cytosine. However, it is unclear whether such genome modifications also provide broad protection against the CRIPSR-Cas systems. Our results suggest that genome modifications indeed afford resistance against CRISPR systems. However, the resistance is not complete, and also variable, allowing rapid evolution of mutant phages that escape CRISPR pressure. Bacteria in turn could target more than one site on the phage genome to more effectively restrict the infection of ghmC-modified phage. Such defense-counter defense strategies seem to confer survival advantages to both the organisms, one of the possible reasons for their great diversity on the planet.

RevDate: 2020-09-17

Szulc B, Sosicka P, Maszczak-Seneczko D, et al (2020)

Biosynthesis of GlcNAc-rich N- and O-glycans in the Golgi apparatus does not require the nucleotide sugar transporter SLC35A3.

The Journal of biological chemistry pii:RA119.012362 [Epub ahead of print].

Nucleotide sugar transporters, encoded by the SLC35 gene family, deliver nucleotide sugars throughout the cell for various glycosyltransferase-catalyzed glycosylation reactions. N-acetylglucosamine, in the form of UDP-GlcNAc, and galactose, as UDP-Gal, are delivered into the Golgi apparatus by SLC35A3 and SLC35A2 transporters, respectively. However, although the UDP-Gal transporting activity of SLC35A2 has been clearly demonstrated, UDP-GlcNAc delivery by SLC35A3 is not fully understood. Therefore, we analyzed a panel of CHO, HEK293T and HepG2 cell lines including wild type cells, SLC35A2 knockouts, SLC35A3 knockouts, and double knock-out cells. Cells lacking SLC35A2 displayed significant changes in N- and O-glycan synthesis. However, in SLC35A3-knock-out CHO cells, only limited changes were observed - GlcNAc was still incorporated into N-glycans but complex type N-glycan branching was impaired, although UDP-GlcNAc transport into Golgi vesicles was not decreased. In SLC35A3-knock-out HEK293T cells, UDP-GlcNAc transport was significantly decreased, but not completely abolished. However, N-glycan branching was not impaired in these cells. In CHO and HEK293T cells the effect of SLC35A3 deficiency on N-glycan branching was potentiated in the absence of SLC35A2. Moreover, in SLC35A3-knock-out HEK293T and HepG2 cells GlcNAc was still incorporated into O-glycans. However, in the case of HepG2 cells, no qualitative changes in N-glycans between wild type and SLC35A3 knock-out cells, as well as between SLC35A2 knock-out and double knock-out cells were observed. These findings suggest that SLC35A3 may not be the primary UDP-GlcNAc transporter and/or different mechanisms of UDP-GlcNAc transport into the Golgi apparatus may exist.

RevDate: 2020-09-17
CmpDate: 2020-09-17

Baker M (2020)

When antibodies mislead: the quest for validation.

Nature, 585(7824):313-314.

RevDate: 2020-09-17
CmpDate: 2020-09-17

Bennett NK, Nguyen MK, Darch MA, et al (2020)

Defining the ATPome reveals cross-optimization of metabolic pathways.

Nature communications, 11(1):4319.

Disrupted energy metabolism drives cell dysfunction and disease, but approaches to increase or preserve ATP are lacking. To generate a comprehensive metabolic map of genes and pathways that regulate cellular ATP-the ATPome-we conducted a genome-wide CRISPR interference/activation screen integrated with an ATP biosensor. We show that ATP level is modulated by distinct mechanisms that promote energy production or inhibit consumption. In our system HK2 is the greatest ATP consumer, indicating energy failure may not be a general deficiency in producing ATP, but rather failure to recoup the ATP cost of glycolysis and diversion of glucose metabolites to the pentose phosphate pathway. We identify systems-level reciprocal inhibition between the HIF1 pathway and mitochondria; glycolysis-promoting enzymes inhibit respiration even when there is no glycolytic ATP production, and vice versa. Consequently, suppressing alternative metabolism modes paradoxically increases energy levels under substrate restriction. This work reveals mechanisms of metabolic control, and identifies therapeutic targets to correct energy failure.

RevDate: 2020-09-17
CmpDate: 2020-09-17

Xu H, Han M, Zhou S, et al (2020)

Chromosome drives via CRISPR-Cas9 in yeast.

Nature communications, 11(1):4344.

Self-propagating drive systems are capable of causing non-Mendelian inheritance. Here, we report a drive system in yeast referred to as a chromosome drive that eliminates the target chromosome via CRISPR-Cas9, enabling the transmission of the desired chromosome. Our results show that the entire Saccharomyces cerevisiae chromosome can be eliminated efficiently through only one double-strand break around the centromere via CRISPR-Cas9. As a proof-of-concept experiment of this CRISPR-Cas9 chromosome drive system, the synthetic yeast chromosome X is completely eliminated, and the counterpart wild-type chromosome X harboring a green fluorescent protein gene or the components of a synthetic violacein pathway are duplicated by sexual reproduction. We also demonstrate the use of chromosome drive to preferentially transmit complex genetic traits in yeast. Chromosome drive enables entire chromosome elimination and biased inheritance on a chromosomal scale, facilitating genomic engineering and chromosome-scale genetic mapping, and extending applications of self-propagating drives.

RevDate: 2020-09-17
CmpDate: 2020-09-17

Singh SP, Thomason PA, Lilla S, et al (2020)

Cell-substrate adhesion drives Scar/WAVE activation and phosphorylation by a Ste20-family kinase, which controls pseudopod lifetime.

PLoS biology, 18(8):e3000774.

The Scar/WAVE complex is the principal catalyst of pseudopod and lamellipod formation. Here we show that Scar/WAVE's proline-rich domain is polyphosphorylated after the complex is activated. Blocking Scar/WAVE activation stops phosphorylation in both Dictyostelium and mammalian cells, implying that phosphorylation modulates pseudopods after they have been formed, rather than controlling whether they are initiated. Unexpectedly, phosphorylation is not promoted by chemotactic signaling but is greatly stimulated by cell:substrate adhesion and diminished when cells deadhere. Phosphorylation-deficient or phosphomimetic Scar/WAVE mutants are both normally functional and rescue the phenotype of knockout cells, demonstrating that phosphorylation is dispensable for activation and actin regulation. However, pseudopods and patches of phosphorylation-deficient Scar/WAVE last substantially longer in mutants, altering the dynamics and size of pseudopods and lamellipods and thus changing migration speed. Scar/WAVE phosphorylation does not require ERK2 in Dictyostelium or mammalian cells. However, the MAPKKK homologue SepA contributes substantially-sepA mutants have less steady-state phosphorylation, which does not increase in response to adhesion. The mutants also behave similarly to cells expressing phosphorylation-deficient Scar, with longer-lived pseudopods and patches of Scar recruitment. We conclude that pseudopod engagement with substratum is more important than extracellular signals at regulating Scar/WAVE's activity and that phosphorylation acts as a pseudopod timer by promoting Scar/WAVE turnover.

RevDate: 2020-09-17
CmpDate: 2020-09-17

Aryan A, Anderson MAE, Biedler JK, et al (2020)

Nix alone is sufficient to convert female Aedes aegypti into fertile males and myo-sex is needed for male flight.

Proceedings of the National Academy of Sciences of the United States of America, 117(30):17702-17709.

A dominant male-determining locus (M-locus) establishes the male sex (M/m) in the yellow fever mosquito, Aedes aegyptiNix, a gene in the M-locus, was shown to be a male-determining factor (M factor) as somatic knockout of Nix led to feminized males (M/m) while transient expression of Nix resulted in partially masculinized females (m/m), with male reproductive organs but retained female antennae. It was not clear whether any of the other 29 genes in the 1.3-Mb M-locus are also needed for complete sex-conversion. Here, we report the generation of multiple transgenic lines that express Nix under the control of its own promoter. Genetic and molecular analyses of these lines provided insights unattainable from previous transient experiments. We show that the Nix transgene alone, in the absence of the M-locus, was sufficient to convert females into males with all male-specific sexually dimorphic features and male-like gene expression. The converted m/m males are flightless, unable to perform the nuptial flight required for mating. However, they were able to father sex-converted progeny when presented with cold-anesthetized wild-type females. We show that myo-sex, a myosin heavy-chain gene also in the M-locus, was required for male flight as knockout of myo-sex rendered wild-type males flightless. We also show that Nix-mediated female-to-male conversion was 100% penetrant and stable over many generations. Therefore, Nix has great potential for developing mosquito control strategies to reduce vector populations by female-to-male sex conversion, or to aid in a sterile insect technique that requires releasing only non-biting males.

RevDate: 2020-09-17
CmpDate: 2020-09-17

McCarthy MW (2020)

Harnessing the potential of CRISPR-based platforms to advance the field of hospital medicine.

Expert review of anti-infective therapy, 18(8):799-805.

INTRODUCTION: Clustered regularly interspaced short palindromic repeats (CRISPR) are segments of nucleic acid that play a role in prokaryotic defense and form the basis of a genome editing technology that allows permanent alteration of genetic material. This methodology, known as CRISPR-Cas9, is poised to revolutionize molecular biology, but no literature yet exists on how these advances will affect hospitalists.

AREAS COVERED: These specialists in inpatient medicine care for a wide variety of hospitalized patients, including those with infectious disease, cancer, cardiovascular disease, autoimmune disease, hematologic disease, and a variety of other conditions that may soon be impacted by advances in gene-modifying technology provided by CRISPR-Cas9. A Literature search was performed using PubMed [1 December 2019-17 April 2020].

EXPERT OPINION: This paper reviews the remarkable diagnostic and therapeutic potential of the CRISPR-Cas9 platform and concludes with a look at ethical issues and technical hurdles pertaining to the implementation of permanent gene modification in the practice of Hospital Medicine.

RevDate: 2020-09-17
CmpDate: 2020-09-17

Guzzo M, Castro LK, Reisch CR, et al (2020)

A CRISPR Interference System for Efficient and Rapid Gene Knockdown in Caulobacter crescentus.

mBio, 11(1):.

CRISPR interference (CRISPRi) is a powerful new tool used in different organisms that provides a fast, specific, and reliable way to knock down gene expression. Caulobacter crescentus is a well-studied model bacterium, and although a variety of genetic tools have been developed, it currently takes several weeks to delete or deplete individual genes, which significantly limits genetic studies. Here, we optimized a CRISPRi approach to specifically downregulate the expression of genes in C. crescentus Although the Streptococcus pyogenes CRISPRi system commonly used in other organisms does not work efficiently in Caulobacter, we demonstrate that a catalytically dead version of Cas9 (dCas9) derived from the type II CRISPR3 module of Streptococcus thermophilus or from Streptococcus pasteurianus can each be effectively used in Caulobacter We show that these CRISPRi systems can be used to rapidly and inducibly deplete ctrA or gcrA, two essential well-studied genes in Caulobacter, in either asynchronous or synchronized populations of cells. Additionally, we demonstrate the ability to multiplex CRISPRi-based gene knockdowns, opening new possibilities for systematic genetic interaction studies in CaulobacterIMPORTANCECaulobacter crescentus is a major model organism for understanding cell cycle regulation and cellular asymmetry. The current genetic tools for deleting or silencing the expression of individual genes, particularly those essential for viability, are time-consuming and labor-intensive, which limits global genetic studies. Here, we optimized CRISPR interference (CRISPRi) for use in Caulobacter Using Streptococcus thermophilus CRISPR3 or Streptococcus pasteurianus CRISPR systems, we show that the coexpression of a catalytically dead form of Cas9 (dCas9) with a single guide RNA (sgRNA) containing a seed region that targets the promoter region of a gene of interest efficiently downregulates the expression of the targeted gene. We also demonstrate that multiple sgRNAs can be produced in parallel to enable the facile silencing of multiple genes, opening the door to systematic genetic interaction studies. In sum, our work now provides a rapid, specific, and powerful new tool for silencing gene expression in C. crescentus and possibly other alphaproteobacteria.

RevDate: 2020-09-17
CmpDate: 2020-09-17

Li T, Wang S, Luo F, et al (2020)

MultiGuideScan: a multi-processing tool for designing CRISPR guide RNA libraries.

Bioinformatics (Oxford, England), 36(3):920-921.

SUMMARY: The recent advance in genome engineering technologies based on CRISPR/Cas9 system is enabling people to systematically understand genomic functions. A short RNA string (the CRISPR guide RNA) can guide the Cas9 endonuclease to specific locations in complex genomes to cut DNA double-strands. The CRISPR guide RNA is essential for gene editing systems. Recently, the GuideScan software is developed to design CRISPR guide RNA libraries, which can be used for genome editing of coding and non-coding genomic regions effectively. However, GuideScan is a serial program and computationally expensive for designing CRISPR guide RNA libraries from large genomes. Here, we present an efficient guide RNA library designing tool (MultiGuideScan) by implementing multiple processes of GuideScan. MultiGuideScan speeds up the guide RNA library designing about 9-12 times on a 32-process mode comparing to GuideScan. MultiGuideScan makes it possible to design guide RNA libraries from large genomes.


SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

RevDate: 2020-09-17
CmpDate: 2020-09-17

Lorenzo D, Esquerda M, Grupo Interdisciplinar en Bioética (2019)

Map of ethical conflicts of the CRISPR-Cas9 gene edition technique.

Medicina clinica, 153(9):357-359.

RevDate: 2020-09-16

Zhao R, Yang Y, Zheng F, et al (2020)

A Membrane-Associated DHH-DHHA1 Nuclease Degrades Type III CRISPR Second Messenger.

Cell reports, 32(11):108133.

Type III CRISPR-Cas systems initiate an intracellular signaling pathway to confer immunity. The signaling pathway includes synthesis of cyclic oligo-adenylate (cOA) and activation of the RNase activity of type III accessory ribonuclease Csm6/Csx1 by cOA. After the immune response, cOA should be cleared on time to avoid constant cellular RNA degradation. In this study, we find a metal-dependent cOA degradation activity in Sulfolobus islandicus. The activity is associated with the cell membrane and able to accelerate cOA clearance at a high cOA level. Further, we show that a metal-dependent and membrane-associated DHH-DHHA1 family nuclease (MAD) rapidly cleaves cOA and deactivates Csx1 ribonuclease. The cOA degradation efficiency of MAD is much higher than the cellular ring nuclease. However, the subcellular organization may prevent it from degrading nascent cOA. Together, the data suggest that MAD acts as the second cOA degrader after the ring nuclease to remove diffused redundant cOA.

RevDate: 2020-09-16

Gholizadeh P, Aghazadeh M, Ghotaslou R, et al (2020)

CRISPR-cas system in the acquisition of virulence genes in dental-root canal and hospital-acquired isolates of Enterococcus faecalis.

Virulence, 11(1):1257-1267.

Enterococcus faecalis is one of the important causative agents of nosocomial and life-threatening infections in human. Several studies have demonstrated that the presence of CRISPR-cas is associated with antibiotic susceptibility and lack of virulence traits. In this study, we aimed to assess the phenotypic and genotypic virulence determinants in relation to CRISPR elements from the dental-root canals and hospital-acquired isolates of E. faecalis. Eighty-eight hospital-acquired and 73 dental-root canal isolates of E. faecalis were assessed in this study. Phenotypic screening of the isolates included biofilm formation, and gelatinase and hemolysis activities. Genotypical screening using PCR was further used to evaluate the presence of CRISPR elements and different virulence-associated genes such as efaA, esp, cylA, hyl, gelE, ace, ebpR, and asa1. Biofilm formation, gelatinase, and hemolysis activities were detected in 93.8%, 29.2%, and 19.2% of the isolates, respectively. The most prevalent virulence-associated gene was ace, which was followed by efaA, whereas cylA was the least identified. The presence of CRISPR1-cas, orphan CRISPR2, and CRISPR3-cas was determined in 13%, 55.3%, and 17.4% of the isolates, respectively. CRISPR elements were significantly more prevalent in the dental-root canal isolates. An inverse significant correlation was found between CRISPR-cas loci, esp, and gelE, while direct correlations were observed in the case of cylA, hyl, gelE (among CRISPR-loci 1 and 3), asa1, ace, biofilm formation, and hemolysis activity. Findings, therefore, indicate that CRISPR-cas might prevent the acquisition of some respective pathogenicity factors in some isolates, though not all; so selective forces could not influence pathogenic traits. Abbreviations: BHI: brain-heart infusion agar; CRISPRs: Clustered regularly interspaced short palindromic repeats; Esp: Cell wall-associated protein; ENT: ear-nose-throat; ICU: intensive care units; OD: optical densities; PCR: polymerase chain reaction; SDS: sodium dodecyl sulfate; UTI: urinary tract infection.

RevDate: 2020-09-15
CmpDate: 2020-09-15

Aubert M, Strongin DE, Roychoudhury P, et al (2020)

Gene editing and elimination of latent herpes simplex virus in vivo.

Nature communications, 11(1):4148.

We evaluate gene editing of HSV in a well-established mouse model, using adeno-associated virus (AAV)-delivered meganucleases, as a potentially curative approach to treat latent HSV infection. Here we show that AAV-delivered meganucleases, but not CRISPR/Cas9, mediate highly efficient gene editing of HSV, eliminating over 90% of latent virus from superior cervical ganglia. Single-cell RNA sequencing demonstrates that both HSV and individual AAV serotypes are non-randomly distributed among neuronal subsets in ganglia, implying that improved delivery to all neuronal subsets may lead to even more complete elimination of HSV. As predicted, delivery of meganucleases using a triple AAV serotype combination results in the greatest decrease in ganglionic HSV loads. The levels of HSV elimination observed in these studies, if translated to humans, would likely significantly reduce HSV reactivation, shedding, and lesions. Further optimization of meganuclease delivery and activity is likely possible, and may offer a pathway to a cure for HSV infection.

RevDate: 2020-09-16
CmpDate: 2020-09-16

Dhoonmoon A, Schleicher EM, Clements KE, et al (2020)

Genome-wide CRISPR synthetic lethality screen identifies a role for the ADP-ribosyltransferase PARP14 in DNA replication dynamics controlled by ATR.

Nucleic acids research, 48(13):7252-7264.

The DNA damage response is essential to maintain genomic stability, suppress replication stress, and protect against carcinogenesis. The ATR-CHK1 pathway is an essential component of this response, which regulates cell cycle progression in the face of replication stress. PARP14 is an ADP-ribosyltransferase with multiple roles in transcription, signaling, and DNA repair. To understand the biological functions of PARP14, we catalogued the genetic components that impact cellular viability upon loss of PARP14 by performing an unbiased, comprehensive, genome-wide CRISPR knockout genetic screen in PARP14-deficient cells. We uncovered the ATR-CHK1 pathway as essential for viability of PARP14-deficient cells, and identified regulation of DNA replication dynamics as an important mechanistic contributor to the synthetic lethality observed. Our work shows that PARP14 is an important modulator of the response to ATR-CHK1 pathway inhibitors.

RevDate: 2020-09-15
CmpDate: 2020-09-15

Zhang X, Chen L, Zhu B, et al (2020)

Increasing the efficiency and targeting range of cytidine base editors through fusion of a single-stranded DNA-binding protein domain.

Nature cell biology, 22(6):740-750.

Cytidine base editors are powerful genetic tools that catalyse cytidine to thymidine conversion at specific genomic loci, and further improvement of the editing range and efficiency is critical for their broader applications. Through insertion of a non-sequence-specific single-stranded DNA-binding domain from Rad51 protein between Cas9 nickase and the deaminases, serial hyper cytidine base editors were generated with substantially increased activity and an expanded editing window towards the protospacer adjacent motif in both cell lines and mouse embryos. Additionally, hyeA3A-BE4max selectively catalysed cytidine conversion in TC motifs with a broader editing range and much higher activity (up to 257-fold) compared with eA3A-BE4max. Moreover, hyeA3A-BE4max specifically generated a C-to-T conversion without inducing bystander mutations in the haemoglobin gamma gene promoter to mimic a naturally occurring genetic variant for amelioration of β-haemoglobinopathy, suggesting the therapeutic potential of the improved base editors.

RevDate: 2020-09-16
CmpDate: 2020-09-16

Chang YJ, Bae J, Zhao Y, et al (2020)

In vivo multiplex gene targeting with Streptococcus pyogens and Campylobacter jejuni Cas9 for pancreatic cancer modeling in wild-type animal.

Journal of veterinary science, 21(2):e26.

Pancreatic ductal adenocarcinoma is a lethal cancer type that is associated with multiple gene mutations in somatic cells. Genetically engineered mouse is hardly applicable for developing a pancreatic cancer model, and the xenograft model poses a limitation in the reflection of early stage pancreatic cancer. Thus, in vivo somatic cell gene engineering with clustered regularly interspaced short palindromic repeats is drawing increasing attention for generating an animal model of pancreatic cancer. In this study, we selected Kras, Trp53, Ink4a, Smad4, and Brca2 as target genes, and applied Campylobacter jejuni Cas9 (CjCas9) and Streptococcus pyogens Cas9 (SpCas9) for developing pancreatic cancer using adeno associated virus (AAV) transduction. After confirming multifocal and diffuse transduction of AAV2, we generated SpCas9 overexpression mice, which exhibited high double-strand DNA breakage (DSB) in target genes and pancreatic intraepithelial neoplasia (PanIN) lesions with two AAV transductions; however, wild-type (WT) mice with three AAV transductions did not develop PanIN. Furthermore, small-sized Cjcas9 was applied to WT mice with two AAV system, which, in addition, developed high extensive DSB and PanIN lesions. Histological changes and expression of cancer markers such as Ki67, cytokeratin, Mucin5a, alpha smooth muscle actin in duct and islet cells were observed. In addition, the study revealed several findings such as 1) multiple DSB potential of AAV-CjCas9, 2) peri-ductal lymphocyte infiltration, 3) multi-focal cancer marker expression, and 4) requirement of > 12 months for initiation of PanIN in AAV mediated targeting. In this study, we present a useful tool for in vivo cancer modeling that would be applicable for other disease models as well.

RevDate: 2020-09-16
CmpDate: 2020-09-16

Yin H, Li Z, Zhang J, et al (2020)

Construction of a US7/US8/UL23/US3-deleted recombinant pseudorabies virus and evaluation of its pathogenicity in dogs.

Veterinary microbiology, 240:108543.

Since 2011, to control the spread of pseudorabies (PR), US7/US8/UL23-deleted recombinant PRV (rPRV) vaccines based on current variants have been developed. The vaccines can provide effective immune protection to pigs, but fur-bearing animals, such as dogs, foxes, and minks, are increasingly infected by PRV due to consuming contaminated raw meat or offal from immunized pigs. It is suspected that the attenuated PRV vaccine strain is not safe for these fur-bearing animals. To confirm this, we construct a US7/US8/UL23-deleted and a US7/US8/UL23/US3-deleted rPRV based on PRV GL isolated from fox using the CRISPR/Cas9 method. Growth kinetics in vitro and pathogenicity in dogs were compared between the wild type and both rPRVs. The results showed that the growth kinetics of wild-type PRV and US7/US8/UL23-deleted rPRV were faster than those of US7/US8/UL23/US3-deleted recombinant PRV from 24 h to 48 h post infection. Moreover, PRV GL- and rPRVdelUS7/US8/UL23-infected cells formed cell-cell fusion, but the rPRVdelUS7/US8/UL23/US3-infected cells did not. Dogs challenged with wild-type PRV or US7/US8/UL23-deleted rPRV showed obvious nervous symptoms, and all the dogs died, but the group challenged with the US7/US8/UL23/US3-deleted rPRV did not show any nervous symptoms, and all the dogs survived for the duration of the experiment. Tissue viral load analyses also showed that the virulence of the US7/US8/UL23/US3-deleted rPRV was significantly reduced in dogs. This study provides evidence that the US7/US8/UL23-deleted rPRV variant still exhibits high virulence for dogs and also highlights the role of the US3 gene in the pathogenicity of PRV in dogs and provides a strategy for developing a safer vaccine.

RevDate: 2020-09-16
CmpDate: 2020-09-16

Cancellieri S, Canver MC, Bombieri N, et al (2020)

CRISPRitz: rapid, high-throughput and variant-aware in silico off-target site identification for CRISPR genome editing.

Bioinformatics (Oxford, England), 36(7):2001-2008.

MOTIVATION: Clustered regularly interspaced short palindromic repeats (CRISPR) technologies allow for facile genomic modification in a site-specific manner. A key step in this process is the in silico design of single guide RNAs to efficiently and specifically target a site of interest. To this end, it is necessary to enumerate all potential off-target sites within a given genome that could be inadvertently altered by nuclease-mediated cleavage. Currently available software for this task is limited by computational efficiency, variant support or annotation, and assessment of the functional impact of potential off-target effects.

RESULTS: To overcome these limitations, we have developed CRISPRitz, a suite of software tools to support the design and analysis of CRISPR/CRISPR-associated (Cas) experiments. Using efficient data structures combined with parallel computation, we offer a rapid, reliable, and exhaustive search mechanism to enumerate a comprehensive list of putative off-target sites. As proof-of-principle, we performed a head-to-head comparison with other available tools on several datasets. This analysis highlighted the unique features and superior computational performance of CRISPRitz including support for genomic searching with DNA/RNA bulges and mismatches of arbitrary size as specified by the user as well as consideration of genetic variants (variant-aware). In addition, graphical reports are offered for coding and non-coding regions that annotate the potential impact of putative off-target sites that lie within regions of functional genomic annotation (e.g. insulator and chromatin accessible sites from the ENCyclopedia Of DNA Elements [ENCODE] project).

The software is freely available at: https://github.com/pinellolab/CRISPRitzhttps://github.com/InfOmics/CRISPRitz.

SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

RevDate: 2020-09-15
CmpDate: 2020-09-15

Towers CG, A Thorburn (2019)

Circumventing autophagy inhibition.

Cell cycle (Georgetown, Tex.), 18(24):3421-3431.

Autophagy is cellular recycling process that plays a complex role in cancer. Pre-clinical studies indicating a pro-tumorigenic role of autophagy have led to the launch of dozens of clinical trials combining autophagy inhibition with other standard of care therapies in different tumor types. A recent publication utilized a novel, acute, CRISPR/Cas9 assay to identify cancer cell lines that are exquisitely sensitive to loss of core autophagy genes within the first 7 days. However, weeks later, rare populations of originally autophagy dependent cells were found that could circumvent autophagy inhibition. Analysis of these rare clones revealed that in the process of circumventing loss of autophagy, the cells upregulated NRF2 signaling to maintain protein homeostasis and consequently become more sensitive to proteasome inhibition as well as knock down of NRF2. This review highlights recent publications regarding the role of autophagy in cancer and potential mechanisms cancer cells may be able to commandeer to circumvent autophagy inhibition. We hope to make significant clinical advances by understanding if and when cancer cells will become resistant to autophagy inhibition, and pre-clinical studies may be able to provide insight into the best combinatorial therapies to prevent tumor relapse while on autophagy inhibitors.

RevDate: 2020-09-16
CmpDate: 2020-09-16

Bergadà-Pijuan J, Pulido-Quetglas C, Vancura A, et al (2020)

CASPR, an analysis pipeline for single and paired guide RNA CRISPR screens, reveals optimal target selection for long non-coding RNAs.

Bioinformatics (Oxford, England), 36(6):1673-1680.

MOTIVATION: CRISPR-Cas9 loss-of-function (LOF) pooled screening promises to identify which long non-coding RNAs (lncRNAs), amongst the many thousands to have been annotated so far, are capable of mediating cellular functions. The two principal LOF perturbations, CRISPR-inhibition and CRISPR-deletion, employ one and two guide RNAs, respectively. However, no software solution has the versatility to identify hits across both modalities, and the optimal design parameters for such screens remain poorly understood.

RESULTS: Here, we present CRISPR Analysis for Single and Paired RNA-guides (CASPR), a user-friendly, end-to-end screen analysis tool. CASPR is compatible with both CRISPRi and CRISPR-del screens, and balances sensitivity and specificity by generating consensus predictions from multiple algorithms. Benchmarking on ground-truth sets of cancer-associated lncRNAs demonstrates CASPR's improved sensitivity with respect to existing methods. Applying CASPR to published screens, we identify two parameters that predict lncRNA hits: expression and annotation quality of the transcription start site. Thus, CASPR is a versatile and complete solution for lncRNA CRISPR screen analysis, and reveals principles for including lncRNAs in screening libraries.


SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

RevDate: 2020-09-16
CmpDate: 2020-09-16

Freeman AJ, Vervoort SJ, Ramsbottom KM, et al (2019)

Natural Killer Cells Suppress T Cell-Associated Tumor Immune Evasion.

Cell reports, 28(11):2784-2794.e5.

Despite the clinical success of cancer immunotherapies, the majority of patients fail to respond or develop resistance through disruption of pathways that promote neo-antigen presentation on MHC I molecules. Here, we conducted a series of unbiased, genome-wide CRISPR/Cas9 screens to identify genes that limit natural killer (NK) cell anti-tumor activity. We identified that genes associated with antigen presentation and/or interferon-γ (IFN-γ) signaling protect tumor cells from NK cell killing. Indeed, Jak1-deficient melanoma cells were sensitized to NK cell killing through attenuated NK cell-derived IFN-γ-driven transcriptional events that regulate MHC I expression. Importantly, tumor cells that became resistant to T cell killing through enrichment of MHC I-deficient clones were highly sensitive to NK cell killing. Taken together, we reveal the genes targeted by tumor cells to drive checkpoint blockade resistance but simultaneously increase their vulnerability to NK cells, unveiling NK cell-based immunotherapies as a strategy to antagonize tumor immune escape.

RevDate: 2020-09-16
CmpDate: 2020-09-16

Ou XY, Wu XL, Peng F, et al (2019)

Metabolic engineering of a robust Escherichia coli strain with a dual protection system.

Biotechnology and bioengineering, 116(12):3333-3348.

Considerable attention has been given to the development of robust fermentation processes, but microbial contamination and phage infection remain deadly threats that need to be addressed. In this study, a robust Escherichia coli BL21(DE3) strain was successfully constructed by simultaneously introducing a nitrogen and phosphorus (N&P) system in combination with a CRISPR/Cas9 system. The N&P metabolic pathways were able to express formamidase and phosphite dehydrogenase in the host cell, thus enabled cell growth in auxotrophic 3-(N-morpholino)propanesulfonic acid medium with formamide and phosphite as nitrogen and phosphorus sources, respectively. N&P metabolic pathways also allowed efficient expression of heterologous proteins, such as green fluorescent protein (GFP) and chitinase, while contaminating bacteria or yeast species could hardly survive in this medium. The host strain was further engineered by exploiting the CRISPR/Cas9 system to enhance the resistance against phage attack. The resultant strain was able to grow in the presence of T7 phage at a concentration of up to 2 × 107 plaque-forming units/ml and produce GFP with a yield of up to 30 μg/109 colony-forming units, exhibiting significant advantages over conventional engineered E. coli. This newly engineered, robust E. coli BL21(DE3) strain therefore shows great potential for future applications in industrial fermentation.

RevDate: 2020-09-16
CmpDate: 2020-09-16

Wu X, Zha J, Koffas MAG, et al (2019)

Reducing Staphylococcus aureus resistance to lysostaphin using CRISPR-dCas9.

Biotechnology and bioengineering, 116(12):3149-3159.

Bacteriolytic enzymes (cell lytic enzymes) are promising alternatives to antibiotics especially in killing drug-resistant bacteria. However, some bacteria slowly become resistant to various classes of peptidoglycan hydrolases, for reasons not well studied, in the presence of growth-supporting nutrients, which are prevalent at sites of infection. Here, we show that Staphylococcus aureus, a human and animal pathogen, while susceptible to the potent staphylolytic enzyme lysostaphin (Lst) in buffered saline, is highly resistant in the rich medium tryptic soy broth (TSB). Through a series of biochemical analysis, we identified that the resistance was due to prevention of Lst-cell binding mediated by the wall teichoic acids (WTAs) present on the cell surface. Inhibition or deletion of the gene tarO responsible for the first step of WTA biosynthesis greatly reduced S. aureus resistance to Lst in TSB. To overcome the resistance, we took advantage of the gene regulation potential of CRISPR-dCas9 and demonstrated that downregulation of tarO, tarH, and/or tarG gene expression, the latter two encoding enzymes that anchor WTAs in the outer layer of cell wall peptidoglycan, sensitized S. aureus to Lst and enabled eradication of the bacterium in TSB in 24 hr. As a result, we elucidate a key mechanism of Lst resistance in metabolically active S. aureus and provide a potential approach for treating life-threatening or hard-to-treat infections caused by Gram-positive pathogens.

RevDate: 2020-09-16
CmpDate: 2020-09-16

Pazmiño-Ibarra V, Mengual-Martí A, Targovnik AM, et al (2019)

Improvement of baculovirus as protein expression vector and as biopesticide by CRISPR/Cas9 editing.

Biotechnology and bioengineering, 116(11):2823-2833.

The clustered regularly interspaced short palindromic repeats (CRISPR) system-associated Cas9 endonuclease is a molecular tool that enables specific sequence editing with high efficiency. In this study, we have explored the use of CRISPR/Cas9 system for the engineering of baculovirus. We have shown that the delivering of Cas9-single guide RNA ribonucleoprotein (RNP) complex with or without DNA repair template into Sf21 insect cells through lipofection might be efficient to produce knockouts as well as knock-ins into the baculovirus. To evaluate potential application of our CRISPR/Cas9 method to improve baculovirus as protein expression vector and as biopesticide, we attempted to knockout several genes from a recombinant AcMNPV form used in the baculovirus expression system as well as in a natural occurring viral isolate from the same virus. We have additionally confirmed the adaptation of this methodology for the generation of viral knock-ins in specific regions of the viral genome. Analysis of the generated mutants revealed that the editing efficiency and the type of changes was variable but relatively high. Depending on the targeted gene, the editing rate ranged from 10% to 40%. This study established the first report revealing the potential of CRISPR/Cas9 for genome editing in baculovirus, contributing to the engineering of baculovirus as a protein expression vector as well as a biological control agent.

RevDate: 2020-09-15
CmpDate: 2020-09-15

Vergnes B, Gazanion E, Mariac C, et al (2019)

A single amino acid substitution (H451Y) in Leishmania calcium-dependent kinase SCAMK confers high tolerance and resistance to antimony.

The Journal of antimicrobial chemotherapy, 74(11):3231-3239.

BACKGROUND: For almost a century, antimonials have remained the first-line drugs for the treatment of leishmaniasis. However, little is known about their mode of action and clinical resistance mechanisms.

OBJECTIVES: We have previously shown that Leishmania nicotinamidase (PNC1) is an essential enzyme for parasite NAD+ homeostasis and virulence in vivo. Here, we found that parasites lacking the pnc1 gene (Δpnc1) are hypersusceptible to the active form of antimony (SbIII) and used these mutant parasites to better understand antimony's mode of action and the mechanisms leading to resistance.

METHODS: SbIII-resistant WT and Δpnc1 parasites were selected in vitro by a stepwise selection method. NAD(H)/NADP(H) dosages and quantitative RT-PCR experiments were performed to explain the susceptibility differences observed between strains. WGS and a marker-free CRISPR/Cas9 base-editing approach were used to identify and validate the role of a new resistance mutation.

RESULTS: NAD+-depleted Δpnc1 parasites were highly susceptible to SbIII and this phenotype could be rescued by NAD+ precursor or trypanothione precursor supplementation. Δpnc1 parasites could become resistant to SbIII by an unknown mechanism. WGS revealed a unique amino acid substitution (H451Y) in an EF-hand domain of an orphan calcium-dependent kinase, recently named SCAMK. When introduced into a WT reference strain by base editing, the H451Y mutation allowed Leishmania parasites to survive at extreme concentrations of SbIII, potentiating the rapid emergence of resistant parasites.

CONCLUSIONS: These results establish that Leishmania SCAMK is a new central hub of antimony's mode of action and resistance development, and uncover the importance of drug tolerance mutations in the evolution of parasite drug resistance.

RevDate: 2020-09-16
CmpDate: 2020-09-16

Yu M, Nguyen ND, Huang Y, et al (2019)

Mitochondrial fusion exploits a therapeutic vulnerability of pancreatic cancer.

JCI insight, 5:.

Pancreatic ductal adenocarcinoma (PDAC) requires mitochondrial oxidative phosphorylation (OXPHOS) to fuel its growth, however, broadly inhibiting this pathway might also disrupt essential mitochondrial functions in normal tissues. PDAC cells exhibit abnormally fragmented mitochondria that are essential to its oncogenicity, but it was unclear if this mitochondrial feature was a valid therapeutic target. Here, we present evidence that normalizing the fragmented mitochondria of pancreatic cancer via the process of mitochondrial fusion reduces OXPHOS, which correlates with suppressed tumor growth and improved survival in preclinical models. Mitochondrial fusion was achieved by genetic or pharmacologic inhibition of dynamin related protein-1 (Drp1) or through overexpression of mitofusin-2 (Mfn2). Notably, we found that oral leflunomide, an FDA-approved arthritis drug, promoted a two-fold increase in Mfn2 expression in tumors and was repurposed as a chemotherapeutic agent, improving the median survival of mice with spontaneous tumors by 50% compared to vehicle. We found that the chief tumor suppressive mechanism of mitochondrial fusion was enhanced mitophagy, which proportionally reduced mitochondrial mass and ATP production. These data suggest that mitochondrial fusion is a specific and druggable regulator of pancreatic cancer growth that could be rapidly translated to the clinic.

RevDate: 2020-09-16
CmpDate: 2020-09-16

Dowrey T, Schwager EE, Duong J, et al (2019)

Satb2 regulates proliferation and nuclear integrity of pre-osteoblasts.

Bone, 127:488-498.

Special AT-rich sequence binding protein 2 (Satb2) is a matrix attachment region (MAR) binding protein. Satb2 impacts skeletal development by regulating gene transcription required for osteogenic differentiation. Although its role as a high-order transcription factor is well supported, other roles for Satb2 in skeletal development remain unclear. In particular, the impact of dosage sensitivity (heterozygous mutations) and variance on phenotypic severity is still not well understood. To further investigate molecular and cellular mechanisms of Satb2-mediated skeletal defects, we used the CRISPR/Cas9 system to generate Satb2 mutations in MC3T3-E1 cells. Our data suggest that, in addition to its role in differentiation, Satb2 regulates progenitor proliferation. We also find that mutations in Satb2 cause chromatin defects including nuclear blebbing and donut-shaped nuclei. These defects may contribute to a slight increase in apoptosis in mutant cells, but apoptosis is insufficient to explain the proliferation defects. Satb2 expression exhibits population-level variation and is most highly expressed from late G1 to late G2. Based on these data, we hypothesize that Satb2 may regulate proliferation through two separate mechanisms. First, Satb2 may regulate the expression of genes necessary for cell cycle progression in pre-osteoblasts. Second, similar to other MAR-binding proteins, Satb2 may participate in DNA replication. We also hypothesize that variation in the severity or penetrance of Satb2-mediated proliferation defects is due to stochastic variation in Satb2 binding to DNA, which may be buffered in some genetic backgrounds. Further elucidation of the role of Satb2 in proliferation has potential impacts on our understanding of both skeletal defects and cancer.

RevDate: 2020-09-16
CmpDate: 2020-09-16

Wang Y, Liu Y, Li J, et al (2019)

Expanding targeting scope, editing window, and base transition capability of base editing in Corynebacterium glutamicum.

Biotechnology and bioengineering, 116(11):3016-3029.

CRISPR/Cas9-guided cytidine deaminase enables C:G to T:A base editing in bacterial genome without introduction of lethal double-stranded DNA break, supplement of foreign DNA template, or dependence on inefficient homologous recombination. However, limited by genome-targeting scope, editing window, and base transition capability, the application of base editing in metabolic engineering has not been explored. Herein, four Cas9 variants accepting different protospacer adjacent motif (PAM) sequences were used to increase the genome-targeting scope of bacterial base editing. After a comprehensive evaluation, we demonstrated that PAM requirement of bacterial base editing can be relaxed from NGG to NG using the Cas9 variants, providing 3.9-fold more target loci for gene inactivation in Corynebacterium glutamicum. Truncated or extended guide RNAs were employed to expand the canonical 5-bp editing window to 7-bp. Bacterial adenine base editing was also achieved with Cas9 fused to adenosine deaminase. With these updates, base editing can serve as an enabling tool for fast metabolic engineering. To demonstrate its potential, base editing was used to deregulate feedback inhibition of aspartokinase via amino acid substitution for lysine overproduction. Finally, a user-friendly online tool named gBIG was provided for designing guide RNAs for base editing-mediated inactivation of given genes in any given sequenced genome (www.ibiodesign.net/gBIG).

RevDate: 2020-09-16
CmpDate: 2020-09-16

Lynch MR, Tran MT, Ralto KM, et al (2019)

TFEB-driven lysosomal biogenesis is pivotal for PGC1α-dependent renal stress resistance.

JCI insight, 5:.

Because injured mitochondria can accelerate cell death through the elaboration of oxidative free radicals and other mediators, it is striking that proliferator gamma coactivator 1-alpha (PGC1α), a stimulator of increased mitochondrial abundance, protects stressed renal cells instead of potentiating injury. Here we report that PGC1α's induction of lysosomes via transcription factor EB (TFEB) may be pivotal for kidney protection. CRISPR and stable gene transfer showed that PGC1α knockout tubular cells were sensitized to the genotoxic stressor cisplatin whereas transgenic cells were protected. The biosensor mtKeima unexpectedly revealed that cisplatin blunts mitophagy both in cells and mice. PGC1α not only counteracted this effect but also raised basal mitophagy, as did the downstream mediator nicotinamide adenine dinucleotide (NAD+). PGC1α did not consistently affect known autophagy pathways modulated by cisplatin. Instead RNA sequencing identified coordinated regulation of lysosomal biogenesis via TFEB. This effector pathway was sufficiently important that inhibition of TFEB or lysosomes unveiled a striking harmful effect of excess PGC1α in cells and conditional mice. These results uncover an unexpected effect of cisplatin on mitophagy and PGC1α's exquisite reliance on lysosomes for kidney protection. Finally, the data illuminate TFEB as a novel target for renal tubular stress resistance.

RevDate: 2020-09-16
CmpDate: 2020-09-16

Saito T, Wada I, N Inoue (2019)

Alternative splicing of the Izumo1 gene ensures triggering gamete fusion in mice.

Scientific reports, 9(1):3151.

IZUMO1 is a sperm acrosomal membrane protein that is essential for mammalian fertilization through recognition of JUNO on the oocyte surface and accompanying IZUMO1-JUNO complex formation. Here, we report a new Izumo1 gene splicing variant (IZUMO1_v2) with a unique 52-amino-acid-long signal sequence transcribed from Exon 1b. Although the mRNA amount of Izumo1_v2 is 76 times lower than that of the original Izumo1 (IZUMO1_v1) in the testis, the cell-oocyte assay indicates that IZUMO1_v2-expressing COS-7 cells have the ability to attach to the oocyte equivalent of IZUMO1_v1. To clarify the physiological function of IZUMO1_v2, we produced an IZUMO1_v1-specific knockout mouse line with a nine-base deletion adjacent to the initial methionine codon of IZUMO1_v1 by the CRISPR/Cas9 system. The IZUMO1_v1 knockout male mice carry 0.19-fold lower level of IZUMO1 protein in the spermatozoon; however, reduction in fertility was only minimally affected compared to the wild-type mice, suggesting that only a small fraction of IZUMO1 is sufficient for triggering sperm-egg fusion. We propose that the alternative splicing generating IZUMO1_v2 might function as a fail-safe in mouse for when splicing is disturbed.

RevDate: 2020-09-14

Shin HR, Kweon J, Y Kim (2021)

Gene Manipulation Using Fusion Guide RNAs for Cas9 and Cas12a.

Methods in molecular biology (Clifton, N.J.), 2162:185-193.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) protein has emerged as a genome engineering tool for various organisms. Known as the CRISPR-Cas system, Cas endonucleases such as Cas9 and Cas12a (also known as Cpf1) and guide RNA (gRNA) complexes recognize and cleave the target DNA, allowing for targeted gene manipulation. Along with the Cas protein engineering, gRNA engineering has broadened the applications of the CRISPR-Cas system. Recently, we have developed fusion guide RNAs (fgRNAs) for orthogonal gene manipulation using Cas9 and Cas12a. Here, we describe the methods for designing and generating fgRNAs-expression constructs to achieve multiplex genome editing and gene manipulation in human cells.

RevDate: 2020-09-14

Hwang GH, Kim JS, S Bae (2021)

Web-Based CRISPR Toolkits: Cas-OFFinder, Cas-Designer, and Cas-Analyzer.

Methods in molecular biology (Clifton, N.J.), 2162:23-33.

The CRISPR-Cas system facilitates highly efficient genome editing; thus, it has been applied in many research fields such as biological science, medicine, and gene therapy. However, CRISPR nucleases can cleave off-target sites as well as on-target sites, causing unwanted mutations. Furthermore, after CRISPR treatments are delivered into cells or organisms, it is important to estimate the resulting mutation rates and to determine the patterns of mutations, but these tasks can be difficult. To address these issues, we have developed a tool for identifying potential off-target sites (Cas-OFFinder), a tool for designing CRISPR targets (Cas-Designer), and an assessment tool (Cas-Analyzer). These programs are all implemented on our website so that researchers can easily design CRISPR guide RNAs and assess the resulting mutations by simply clicking on the appropriate buttons; no login process is required.

RevDate: 2020-09-14

Heigwer F, M Boutros (2021)

Cloud-Based Design of Short Guide RNA (sgRNA) Libraries for CRISPR Experiments.

Methods in molecular biology (Clifton, N.J.), 2162:3-22.

CRISPR/Cas-based genome editing in any biological application requires the evaluation of suitable genomic target sites to design efficient reagents. Considerations for the design of short guide (sg) RNAs include the assessment of possible off-target activities, the prediction of on-target efficacies and mutational outcome. Manual design of sgRNAs taking into account these parameters, however, remains a difficult task. Thus, computational tools to design sgRNA reagents from small scale to genome-wide libraries have been developed that assist during all steps of the design process. Here, we will describe practical guidance for the sgRNA design process using the web-based tool E-CRISP used in the design of individual sgRNAs. E-CRISP (www.e-crisp.org) has been the first web-based sgRNA design tool and uniquely features simple, yet efficient, scoring schemes in combination with fast evaluation and simple usage. We will also discuss the installation of a dockerized version of CRISPR Library Designer (CLD) that can be deployed locally or in the cloud to support the end-to-end design of sgRNA libraries for more than 50 different organisms. CLD was built upon E-CRISP to further increase the scope of sgRNA design to more experimental modalities (CRISPRa/i, Cas12a, all possible protospacer adjacency motifs) offering the same flexibility as E-CRISP, plus the scalability through local and cloud installation. Together, these tools facilities the design of small and large-scale CRISPR/Cas experiments.

RevDate: 2020-09-14

Song X, Zhang XY, Xiong ZQ, et al (2020)

CRISPR-Cas-mediated gene editing in lactic acid bacteria.

Molecular biology reports pii:10.1007/s11033-020-05820-w [Epub ahead of print].

The high efficiency, convenience and diversity of clustered regular interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems are driving a technological revolution in the gene editing of lactic acid bacteria (LAB). Cas-RNA cassettes have been adopted as tools to perform gene deletion, insertion and point mutation in several species of LAB. In this article, we describe the basic mechanisms of the CRISPR-Cas system, and the current gene editing methods available, focusing on the CRISPR-Cas models developed for LAB. We also compare the different types of CRISPR-Cas-based genomic manipulations classified according to the different Cas proteins and the type of recombineering, and discuss the rapidly evolving landscape of CRISPR-Cas application in LAB.

RevDate: 2020-09-13

Riedl A, Gruber S, Z Ruzsics (2020)

Novel conditional plasmids regulated by chemical switches provide versatile tools for genetic engineering in Escherichia coli.

Plasmid pii:S0147-619X(20)30043-3 [Epub ahead of print].

Engineering bacterial genomes or foreign DNA cloned as bacterial artificial chromosomes (BACs) relies on usage of helper plasmids, which deliver the desired tools transiently into the bacteria to be modified. After the anticipated action is completed the helper plasmids need to be cured. To make this efficient, plasmids are used that are maintained by conditional amplicons or carry a counter-selection marker. Here, we describe new conditional plasmids that can be maintained or cured by using chemical induction or repression. Our method is based on the dependency of plasmids carrying ori6Kγ origin of replication on the presence of protein Π. Ori6Kγ based plasmids are tightly regulated conditional constructs, but they require usually special E. coli strains to operate. To avoid this, we placed the Π protein expression under the control of a co-expressed conditional repressor. Regulating the maintenance of plasmids with administration or removal of chemicals is fully compatible with any other conditional amplicons applied to date. Here, we describe methods for inducing sites specific recombination of BACs as an example. However, the same strategy might be used to construct appropriate helper plasmids for any other transient components of genome editing methodologies such as λred recombinases or CRISPR/Cas components.

RevDate: 2020-09-14
CmpDate: 2020-09-14

Mazzara PG, Muggeo S, Luoni M, et al (2020)

Frataxin gene editing rescues Friedreich's ataxia pathology in dorsal root ganglia organoid-derived sensory neurons.

Nature communications, 11(1):4178.

Friedreich's ataxia (FRDA) is an autosomal-recessive neurodegenerative and cardiac disorder which occurs when transcription of the FXN gene is silenced due to an excessive expansion of GAA repeats into its first intron. Herein, we generate dorsal root ganglia organoids (DRG organoids) by in vitro differentiation of human iPSCs. Bulk and single-cell RNA sequencing show that DRG organoids present a transcriptional signature similar to native DRGs and display the main peripheral sensory neuronal and glial cell subtypes. Furthermore, when co-cultured with human intrafusal muscle fibers, DRG organoid sensory neurons contact their peripheral targets and reconstitute the muscle spindle proprioceptive receptors. FRDA DRG organoids model some molecular and cellular deficits of the disease that are rescued when the entire FXN intron 1 is removed, and not with the excision of the expanded GAA tract. These results strongly suggest that removal of the repressed chromatin flanking the GAA tract might contribute to rescue FXN total expression and fully revert the pathological hallmarks of FRDA DRG neurons.

RevDate: 2020-09-14
CmpDate: 2020-09-14

Banik SM, Pedram K, Wisnovsky S, et al (2020)

Lysosome-targeting chimaeras for degradation of extracellular proteins.

Nature, 584(7820):291-297.

The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein-for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)1,2 and others (for example, dTAGs3, Trim-Away4, chaperone-mediated autophagy targeting5 and SNIPERs6) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins-the products of 40% of all protein-encoding genes7-are key agents in cancer, ageing-related diseases and autoimmune disorders8, and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified-but essential-component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics.

RevDate: 2020-09-14
CmpDate: 2020-09-14

Tong Y, Whitford CM, Blin K, et al (2020)

CRISPR-Cas9, CRISPRi and CRISPR-BEST-mediated genetic manipulation in streptomycetes.

Nature protocols, 15(8):2470-2502.

Streptomycetes are prominent sources of bioactive natural products, but metabolic engineering of the natural products of these organisms is greatly hindered by relatively inefficient genetic manipulation approaches. New advances in genome editing techniques, particularly CRISPR-based tools, have revolutionized genetic manipulation of many organisms, including actinomycetes. We have developed a comprehensive CRISPR toolkit that includes several variations of 'classic' CRISPR-Cas9 systems, along with CRISPRi and CRISPR-base editing systems (CRISPR-BEST) for streptomycetes. Here, we provide step-by-step protocols for designing and constructing the CRISPR plasmids, transferring these plasmids to the target streptomycetes, and identifying correctly edited clones. Our CRISPR toolkit can be used to generate random-sized deletion libraries, introduce small indels, generate in-frame deletions of specific target genes, reversibly suppress gene transcription, and substitute single base pairs in streptomycete genomes. Furthermore, the toolkit includes a Csy4-based multiplexing option to introduce multiple edits in a single experiment. The toolkit can be easily extended to other actinomycetes. With our protocol, it takes <10 d to inactivate a target gene, which is much faster than alternative protocols.

RevDate: 2020-09-14
CmpDate: 2020-09-14

Labeau A, Simon-Loriere E, Hafirassou ML, et al (2020)

A Genome-Wide CRISPR-Cas9 Screen Identifies the Dolichol-Phosphate Mannose Synthase Complex as a Host Dependency Factor for Dengue Virus Infection.

Journal of virology, 94(7):.

Dengue virus (DENV) is a mosquito-borne flavivirus responsible for dengue disease, a major human health concern for which no specific therapies are available. Like other viruses, DENV relies heavily on the host cellular machinery for productive infection. In this study, we performed a genome-wide CRISPR-Cas9 screen using haploid HAP1 cells to identify host genes important for DENV infection. We identified DPM1 and -3, two subunits of the endoplasmic reticulum (ER) resident dolichol-phosphate mannose synthase (DPMS) complex, as host dependency factors for DENV and other related flaviviruses, such as Zika virus (ZIKV). The DPMS complex catalyzes the synthesis of dolichol-phosphate mannose (DPM), which serves as mannosyl donor in pathways leading to N-glycosylation, glycosylphosphatidylinositol (GPI) anchor biosynthesis, and C- or O-mannosylation of proteins in the ER lumen. Mutation in the DXD motif of DPM1, which is essential for its catalytic activity, abolished DPMS-mediated DENV infection. Similarly, genetic ablation of ALG3, a mannosyltransferase that transfers mannose to lipid-linked oligosaccharide (LLO), rendered cells poorly susceptible to DENV. We also established that in cells deficient for DPMS activity, viral RNA amplification is hampered and truncated oligosaccharides are transferred to the viral prM and E glycoproteins, affecting their proper folding. Overall, our study provides new insights into the host-dependent mechanisms of DENV infection and supports current therapeutic approaches using glycosylation inhibitors to treat DENV infection.IMPORTANCE Dengue disease, which is caused by dengue virus (DENV), has emerged as the most important mosquito-borne viral disease in humans and is a major global health concern. DENV encodes only few proteins and relies on the host cell machinery to accomplish its life cycle. The identification of the host factors important for DENV infection is needed to propose new targets for antiviral intervention. Using a genome-wide CRISPR-Cas9 screen, we identified DPM1 and -3, two subunits of the DPMS complex, as important host factors for the replication of DENV as well as other related viruses such as Zika virus. We established that DPMS complex plays dual roles during viral infection, both regulating viral RNA replication and promoting viral structural glycoprotein folding/stability. These results provide insights into the host molecules exploited by DENV and other flaviviruses to facilitate their life cycle.

RevDate: 2020-09-14
CmpDate: 2020-09-14

Khanzadi MN, AA Khan (2020)

CRISPR/Cas9: Nature's gift to prokaryotes and an auspicious tool in genome editing.

Journal of basic microbiology, 60(2):91-102.

Clustered regularly interspaced short palindromic repeats (CRISPR) is a family of DNA direct repeats found in many prokaryotic genomes. It was discovered in bacteria as their (adaptive) immune system against invading viruses. Cas9 is an endonuclease enzyme linked with the CRISPR system in bacteria. Bacteria use the Cas9 enzyme to chop viral DNA sequences by unwinding it and then finding the complementary base pairs to the guide RNA. CRISPR/Cas9 is a modern and powerful molecular biology approach that is widely used in genome engineering (to activate/repress gene expression). It can be used in vivo to cause targeted genome modifications with better efficiency as compared to meganucleases, zinc-finger nucleases and transcription activator-like effector nucleases. CRISPR/Cas9 is a simple, reliable, and rapid method for causing gene alterations that open new horizons of gene editing in a variety of living organisms, including humans, for the treatment of several diseases. In this short review, we explored the basic mechanisms underlying its working principles along with some of its current applications in a number of diverse fields.

RevDate: 2020-09-14
CmpDate: 2020-09-14

van den Boogaard M, van Weerd JH, Bawazeer AC, et al (2019)

Identification and Characterization of a Transcribed Distal Enhancer Involved in Cardiac Kcnh2 Regulation.

Cell reports, 28(10):2704-2714.e5.

The human ether-a-go-go-related gene KCNH2 encodes the voltage-gated potassium channel underlying IKr, a current critical for the repolarization phase of the cardiac action potential. Mutations in KCNH2 that cause a reduction of the repolarizing current can result in cardiac arrhythmias associated with long-QT syndrome. Here, we investigate the regulation of KCNH2 and identify multiple active enhancers. A transcribed enhancer ∼85 kbp downstream of Kcnh2 physically contacts the promoters of two Kcnh2 isoforms in a cardiac-specific manner in vivo. Knockdown of its ncRNA transcript results in reduced expression of Kcnh2b and two neighboring mRNAs, Nos3 and Abcb8, in vitro. Genomic deletion of the enhancer, including the ncRNA transcription start site, from the mouse genome causes a modest downregulation of both Kcnh2a and Kcnh2b in the ventricles. These findings establish that the regulation of Kcnh2a and Kcnh2b is governed by a complex regulatory landscape that involves multiple partially redundantly acting enhancers.

RevDate: 2020-09-14
CmpDate: 2020-09-14

Zhang R, Earnest JT, Kim AS, et al (2019)

Expression of the Mxra8 Receptor Promotes Alphavirus Infection and Pathogenesis in Mice and Drosophila.

Cell reports, 28(10):2647-2658.e5.

Mxra8 is a recently described receptor for multiple alphaviruses, including Chikungunya (CHIKV), Mayaro (MAYV), Ross River (RRV), and O'nyong nyong (ONNV) viruses. To determine its role in pathogenesis, we generated mice with mutant Mxra8 alleles: an 8-nucleotide deletion that produces a truncated, soluble form (Mxra8Δ8/Δ8) and a 97-nucleotide deletion that abolishes Mxra8 expression (Mxra8Δ97/Δ97). Mxra8Δ8/Δ8 and Mxra8Δ97/Δ97 fibroblasts show reduced CHIKV infection in culture, and Mxra8Δ8/Δ8 and Mxra8Δ97/Δ97 mice have decreased infection of musculoskeletal tissues with CHIKV, MAYV, RRV, or ONNV. Less foot swelling is observed in CHIKV-infected Mxra8 mutant mice, which correlated with fewer infiltrating neutrophils and cytokines. A recombinant E2-D71A CHIKV with diminished binding to Mxra8 is attenuated in vivo in wild-type mice. Ectopic Mxra8 expression is sufficient to enhance CHIKV infection and lethality in transgenic flies. These studies establish a role for Mxra8 in the pathogenesis of multiple alphaviruses and suggest that targeting this protein may mitigate disease in humans.

RevDate: 2020-09-08
CmpDate: 2020-09-08

Shao J, Lu J, Zhu W, et al (2019)

Derepression of LOXL4 inhibits liver cancer growth by reactivating compromised p53.

Cell death and differentiation, 26(11):2237-2252.

TP53 is the most frequently mutated gene in human cancer, whereas tumors with wild-type TP53 develop alternative strategies to survive. Identifying new regulators of p53 reactivation would greatly contribute to the development of cancer therapies. After screening the entire genome in liver cancer cells, we identified lysyl oxidase-like 4 (LOXL4) as a novel regulator for p53 activation. We found that 5-azacytidine (5-aza-CR) induces LOXL4 upregulation, with LOXL4 subsequently binding the basic domain of p53 via its low-isoelectric point region. The interaction between LOXL4 and p53 induces the reactivation of compromised p53, resulting in cell death. Furthermore, the nude mouse xenograft model showed that the 5-aza-CR-dependent LOXL4-p53 axis reduces tumor growth. A positive correlation between LOXL4 expression and overall survival in liver cancer patients with wild-type p53 tumors was observed. In conclusion, we found that 5-aza-CR-induced LOXL4 upregulation reactivates wild-type p53 and triggers cell death, which blocks liver cancer development.

RevDate: 2020-09-12

Feng S, Hu L, Zhang Q, et al (2020)

CRISPR/Cas technology promotes the various application of Dunaliella salina system.

Applied microbiology and biotechnology pii:10.1007/s00253-020-10892-6 [Epub ahead of print].

Dunaliella salina (D. salina) has been widely applied in various fields because of its inherent advantages, such as the study of halotolerant mechanism, wastewater treatment, recombinant proteins expression, biofuel production, preparation of natural materials, and others. However, owing to the existence of low yield or in the laboratory exploration stage, D. salina system has been greatly restricted for practical production of various components. In past decade, significant progresses have been achieved for research of D. salina in these fields. Among them, D. salina as a novel expression system demonstrated a bright prospect, especially for large-scale production of foreign proteins, like the vaccines, antibodies, and other therapeutic proteins. Due to the low efficiency, application of traditional regulation tools is also greatly limited for exploration of D. salina system. The emergence of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system offers a precise editing tool to overcome the obstacles of D. salina system. This review not only comprehensively summarizes the recent progresses of D. salina in domain of gene engineering but also gives a deep analysis of problems and deficiencies in different fields of D. salina. Moreover, further prospects of CRISPR/Cas system and its significant challenges have been discussed in various aspects of D. salina. It provides a great referencing value for speeding up the maturity of D. salina system, and also supplies practical guiding significance to expand the new application fields for D. salina. KEY POINTS: • The review provides recent research progresses of various applications of D. salina. • The problems and deficiencies in different fields of D. salina were deeply analyzed. • The further prospects of CRISPR/Cas technology in D. salina system were predicted. • CRISPR/Cas system will promote the new application fields and maturity for D. salina.

RevDate: 2020-09-11

Watry HL, Feliciano CM, Gjoni K, et al (2020)

Rapid, precise quantification of large DNA excisions and inversions by ddPCR.

Scientific reports, 10(1):14896 pii:10.1038/s41598-020-71742-z.

The excision of genomic sequences using paired CRISPR-Cas nucleases is a powerful tool to study gene function, create disease models and holds promise for therapeutic gene editing. However, our understanding of the factors that favor efficient excision is limited by the lack of a rapid, accurate measurement of DNA excision outcomes that is free of amplification bias. Here, we introduce ddXR (droplet digital PCR eXcision Reporter), a method that enables the accurate and sensitive detection of excisions and inversions independent of length. The method can be completed in a few hours without the need for next-generation sequencing. The ddXR method uncovered unexpectedly high rates of large (> 20 kb) excisions and inversions, while also revealing a surprisingly low dependence on linear distance, up to 170 kb. We further modified the method to measure precise repair of excision junctions and allele-specific excision, with important implications for disease modeling and therapeutic gene editing.

RevDate: 2020-09-11

Pattharaprachayakul N, Lee M, Incharoensakdi A, et al (2020)

Current understanding of the cyanobacterial CRISPR-Cas systems and development of the synthetic CRISPR-Cas systems for cyanobacteria.

Enzyme and microbial technology, 140:109619.

Cyanobacteria are photosynthetic microorganisms that are capable of converting CO2 to value-added chemicals. Engineering of cyanobacteria with synthetic biology tools, including the CRISPR-Cas system, has allowed an opportunity for biological CO2 utilization. Here, we described natural CRISPR-Cas systems for understanding cyanobacterial genomics and synthetic CRISPR-Cas systems for metabolic engineering applications. The natural CRISPR-Cas systems in cyanobacteria have been identified as Class 1, with type I and III, and some Class 2, with type V, as an adaptive immune system against viral invasion. As synthetic tools, CRISPR-Cas9 and -Cas12a have been successfully established in cyanobacteria to delete a target gene without a selection marker. Deactivated Cas9 and Cas12a have also been used to repress genes for metabolic engineering. In addition, a perspective on how advanced CRISPR-Cas systems and a pool of the guide RNAs can be advantageous for precise genome engineering and understanding of unknown functions was discussed for advanced engineering of cyanobacteria.

RevDate: 2020-09-11

Gabr H, El Ghamrawy MK, Almaeen AH, et al (2020)

CRISPR-mediated gene modification of hematopoietic stem cells with beta-thalassemia IVS-1-110 mutation.

Stem cell research & therapy, 11(1):390 pii:10.1186/s13287-020-01876-4.

BACKGROUND: β-Thalassemias represent a group of genetic disorders caused by human hemoglobin beta (HBB) gene mutations. The radical curative approach is to correct the mutations causing the disease. CRISPR-CAS9 is a novel gene-editing technology that can be used auspiciously for the treatment of these disorders. The study aimed to investigate the utility of CRISPR-CAS9 for gene modification of hematopoietic stem cells in β-thalassemia with IVS-1-110 mutation.

METHODS AND RESULTS: We successfully isolated CD34+ cells from peripheral blood of β-thalassemia patients with IVS-1-110 mutation. The cells were transfected with Cas9 endonuclease together with guide RNA to create double-strand breaks and knock out the mutation. The mutation-corrected CD34+ cells were subjected to erythroid differentiation by culturing in complete media containing erythropoietin.

CONCLUSION: CRISPR/Cas-9 is an effective tool for gene therapy that will broaden the spectrum of therapy and potentially improve the outcomes of β-thalassemia.


RJR Experience and Expertise


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.


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.


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.


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.


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.


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.


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.


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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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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E-mail: RJR8222@gmail.com

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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