Clustered Regularly Interspaced Palindromic Repeats Research Articles

From baker's yeast to genetically modified budding yeasts: the scientific evolution of bioethanol industry from sugarcane.

The peculiarities of Brazilian fuel ethanol fermentation allow the entry of native yeasts that may dominate over the starter strains of Saccharomyces cerevisiae and persist throughout the sugarcane harvest. The switch from the use of baker's yeast as starter to selected budding yeasts obtained by a selective pressure strategy was followed by a wealth of genomic information that enabled the understanding of the superiority of selected yeast strains. This review describes how the process of yeast selection evolved in the sugarcane-based bioethanol industry, the selection criteria and recent advances in genomics that could advance the fermentation process. The prospective use of genetically modified yeast strains, specially designed for increased robustness and product yield, with special emphasis on those obtained by the CRISPR (clustered regularly interspaced palindromic repeats)-Cas9 (CRISPR-associated protein 9) genome-editing approach, is discussed as a possible solution to confer higher performance and stability to the fermentation process for fuel ethanol production.

Genome-Wide CRISPR Off-Target DNA Break Detection by the BLISS Method.

Clustered regularly interspaced palindromic repeat (CRISPR) systems are revolutionizing many areas of biology and medicine, where they are increasingly utilized as therapeutic tools for correcting disease-causing mutations. From a clinical perspective, unintended off-target (OT) DNA double-strand break (DSB) induction by CRISPR nucleases represents a major concern. Therefore, in recent years considerable effort has been dedicated to developing methods for assessing the OT activity of CRISPR nucleases, which in turn can be used to guide engineering of nucleases with minimal OT activity. Here we describe a detailed protocol for quantifying OT DSBs genome-wide in cultured cells transfected with CRISPR enzymes, based on the breaks labeling in situ and sequencing (BLISS) method that we have previously developed. CRISPR-BLISS is versatile and scalable, and allows assessment of multiple guide RNAs in different cell types and time points following cell transfection or transduction.

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

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.

Modeling pediatric AML FLT3 mutations using CRISPR/Cas12a- mediated gene editing

Clustered regularly interspaced palindromic repeats (CRISPR) with the associated (Cas) nuclease complexes have democratized genetic engineering through their precision and ease-of-use. We have applied a variation of this technology, known as CRISPR-directed mutagenesis (CDM), to reconstruct genetic profiles within the FLT3 gene of AML patients. We took advantage of the versatility of CDM and built expression vectors that, in combination with a specifically designed donor DNA fragment, recapitulate simple and complex mutations within the FLT3 gene. We generate insertions and point mutations including combinations of these mutations originating from individual patient samples. We then analyze how these complex genetic profiles modulate transformation of Ba/F3 cells. Our results show that FLT3 expression plasmids bearing patient-specific single or multiple mutations recapitulate cellular transformation properties induced by FLT3 ITDs and modify their sensitivity or resistance in response to established AML drugs as a function of these complex mutations.

Synthetic Virus-Derived Nanosystems (SVNs) for Delivery and Precision Docking of Large Multifunctional DNA Circuitry in Mammalian Cells.

DNA delivery is at the forefront of current research efforts in gene therapy and synthetic biology. Viral vectors have traditionally dominated the field; however, nonviral delivery systems are increasingly gaining traction. Baculoviruses are arthropod-specific viruses that can be easily engineered and repurposed to accommodate and deliver large sequences of exogenous DNA into mammalian cells, tissues, or ultimately organisms. These synthetic virus-derived nanosystems (SVNs) are safe, readily customized, and can be manufactured at scale. By implementing clustered regularly interspaced palindromic repeats (CRISPR) associated protein (CRISPR/Cas) modalities into this system, we developed SVNs capable of inserting complex DNAs into genomes, at base pair precision. We anticipate a major role for SVNs as an attractive alternative to viral vectors in accelerating genome engineering and gene therapy applications in the future.

Generating and characterizing single- and multigene mutants of the Rubisco small subunit family in Arabidopsis.

The primary CO2-fixing enzyme Rubisco limits the productivity of plants. The small subunit of Rubisco (SSU) can influence overall Rubisco levels and catalytic efficiency, and is now receiving increasing attention as a potential engineering target to improve the performance of Rubisco. However, SSUs are encoded by a family of nuclear rbcS genes in plants, which makes them challenging to engineer and study. Here we have used CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9] and T-DNA insertion lines to generate a suite of single and multiple gene knockout mutants for the four members of the rbcS family in Arabidopsis, including two novel mutants 2b3b and 1a2b3b. 1a2b3b contained very low levels of Rubisco (~3% relative to the wild-type) and is the first example of a mutant with a homogenous Rubisco pool consisting of a single SSU isoform (1B). Growth under near-outdoor levels of light demonstrated Rubisco-limited growth phenotypes for several SSU mutants and the importance of the 1A and 3B isoforms. We also identified 1a1b as a likely lethal mutation, suggesting a key contributory role for the least expressed 1B isoform during early development. The successful use of CRISPR/Cas here suggests that this is a viable approach for exploring the functional roles of SSU isoforms in plants.

Mageb4 , a Testis-Specific Gene, is Dispensable for Mouse Spermatogenesis

Objective: It has recently been shown that the melanoma antigen gene (MAGE) family is expressed in various tumor cell lines but silent in normal tissues, except germ cell lines. Mageb4, a member of the MAGE family, is highly expressed in the testis and homologous in humans and mice. Whole-exome sequencing studies have identified Mageb4 as a possible X-linked cause of inherited male infertility. However, the function of Mageb4 protein remains largely unknown. Methods: Using clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) 9 technology, we generated a Mageb4 knockout mouse model (Mageb4−/Y) to explore the role of this gene in spermatogenesis. Results: First, immunostaining of testicular cells showed that Mageb4 is localized in the cytoplasm of spermatogonia. Second, Mageb4−/Y male mice displayed significant increases in apoptosis. However, Mageb4−/Y male mice showed normal fertility, including normal sperm concentration, sperm motility, and testicular and epididymal histology. Conclusions: These findings suggest that, despite testis-exclusive expression, Mageb4 is dispensable for mouse spermatogenesis. Future research should focus on the role of this gene in apoptosis, aiming to provide clinical guidance regarding male infertility.

ABA-responsive transcription factor bZIP1 is involved in modulating biosynthesis of phenolic acids and tanshinones in Salvia miltiorrhiza.

Phenolic acids and tanshinones are major bioactive ingredients in Salvia miltiorrhiza, which possess pharmacological activities with great market demand. However, transcriptional regulation of phenolic acid and tanshinone biosynthesis remains poorly understood. Here, a basic leucine zipper transcription factor (TF) named SmbZIP1 was screened from the abscisic acid (ABA)-induced transcriptome library. Overexpression of SmbZIP1 positively promoted phenolic acid biosynthesis by enhancing expression of biosynthetic genes such as cinnamate-4-hydroxylase (C4H1). Furthermore, biochemical experiments revealed that SmbZIP1 bound the G-Box-like1 element in the promoter of the C4H1 gene. Meanwhile, SmbZIP1 inhibited accumulation of tanshinones mainly by suppressing the expression of biosynthetic genes including geranylgeranyl diphosphate synthase (GGPPS) which was confirmed as a target gene by in vitro and in vivo experiments. In contrast, the phenolic acid content was reduced and tanshinone was enhanced in CRISPR/Cas9 [clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9]-mediated knockout lines. In addition, the previously reported positive regulator of tanshinone biosynthesis, SmERF1L1, was found to be inhibited in SmbZIP1 overexpression lines indicated by RNA sequencing, and was proven to be the target of SmbZIP1. In summary, this work uncovers a novel regulator and deepens our understanding of the transcriptional and regulatory mechanisms of phenolic acid and tanshinone biosynthesis, and also sheds new light on metabolic engineering in S. miltiorrhiza.

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

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

Construction and immunogenicity of a gE/gI/TK-deleted PRV based on porcine pseudorabies virus variant

Pseudorabies (PR) caused by re-emerging pseudorabies virus (PRV) variant has outbroken among PRV vaccine-immunized swine herds on many Chinese pig farms, with severe socioeconomic consequences since late 2011. Here, a gE/gI/TK-deleted recombinant virus (rPRV NY-gE−/gI−/TK−) was constructed based on PRV NY strain from 2012 through homologous DNA recombination and gene-editing technology termed clustered regularly interspaced palindromic repeats (CRISPR)/associated (Cas9) system. The rPRV NY-gE−/gI−/TK− strain showed similar growth kinetics to the parental PRV NY strain in vitro, and was safe for mice. Sixty mice were injected subcutaneously (s.c.) twice with 106.0 TCID50 of rPRV NY-gE−/gI−/TK− and DMEM, respectively, with two-week interval. The levels of PRV gB antibodies and neutralizing antibodies against PRV NY in mice immunized with rPRV NY-gE−/gI−/TK− were higher than those in the DMEM control group. The number of T lymphocyte subclasses CD3+, CD4+ and CD8+ in rPRV NY-gE−/gI−/TK−-immunized mice was higher than that in DMEM-injected mice. After challenge with 106.0 TCID50 PRV NY at 42 dpi, all rPRV NY-gE−/gI−/TK−-immunized mice survived without exhibiting any pathological lesions in different tissues and intranuclear eosinophilic inclusions of the brain, and the viral genomic copy numbers in various organs of mice were obviously lower than DMEM group. These results showed the rPRV NY-gE−/gI−/TK− could be a promising next-generation vaccine to control now epidemic PR in China.

Genome Editing Fidelity in the Context of DNA Sequence and Chromatin Structure.

Genome editing by Clustered Regularly Inter Spaced Palindromic Repeat (CRISPR) associated (Cas) systems has revolutionized medical research and holds enormous promise for correcting genetic diseases. Understanding how these Cas nucleases work and induce mutations, as well as identifying factors that affect their efficiency and fidelity is key to developing this technology for therapeutic uses. Here, we discuss recent studies that reveal how DNA sequence and chromatin structure influences the different steps of genome editing. These studies also demonstrate that a deep understanding of the balance between error prone and error free DNA repair pathways is crucial for making genome editing a safe clinical tool, which does not induce further mutations to the genome.

A CRISPR-Cas9-mediated domain-specific base-editing screen enables functional assessment of ACCase variants in rice.

A CRISPR-Cas9-mediated domain-specific base-editing screen enables functional assessment of ACCase variants in rice.

CRISPR/Cas9-Based Transcriptional Activation of Endogenous IL-10 Gene in the Donor Lung for Transplantation

Gene therapy of donor lungs has shown promise in lung transplantation (LTx). The discovery of the clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has advanced gene therapy by enabling precise and highly flexible modulation of endogenous genes. CRISPR-based gene modulation of a whole organ, which remains challenging, will allow us to optimize donor lungs by modulating any gene. To establish CRISPR-based epigenome editing for LTx, we started with activating the endogenous IL-10 gene, an anti-inflammatory gene, in rat lungs. We developed a specific vector targeting the IL-10 gene and tested in rat model. Adenoviral vectors expressing a Cas9 activator (Cas9-VPR) with or without a guide RNA (gRNA) designed to target the promoter region of the rat IL-10 gene were administered to Lewis rats via the airway at a dose of 5 × 108 PFU/rat. Rats were divided into three groups (n=6 each): 1) No gRNA - received adenovirus expressing Cas9-VPR alone; 2) gRNA - received adenovirus expressing Cas9-VPR and: 3) diluent - received buffer without any viruses. The lung was assessed for IL-10 gene expression and inflammation after 72 hours. The Cas9-VPR was detected in lung tissue of the gRNA group and the No gRNA group using immunohistochemistry (Fig A). The gRNA group showed increased IL-10 (5.85±2.05 vs 2.85±1.89 pg/mg protein, p=0.046) compared to the No gRNA group (Fig B) whereas TNFa and IFNg showed comparable level in both groups. Furthermore, the gRNA group showed improved oxygenation (587±41 vs 435±55 mmHg, p<0.0001, Fig C) and Wet/Dry ratio (5.04±0.14 vs 5.63±0.33, p=0.001, Fig D), demonstrating an anti-inflammatory effect of IL-10 gene activation on vector-induced inflammation in the donor lung. Using CRISPR/Cas9-based transcriptional modulation we have achieved targeted activation of the endogenous IL-10 gene in rat donor lungs. This proof of concept study opens the door to targeted gene modification of donor organs to improve outcomes after LTx.

Efficient genome editing in the olive fruit fly, Bactrocera oleae.

The olive fruit fly, Bactrocera oleae, causes great damage to the quality and quantity of olive production worldwide. Pest management approaches have proved difficult for a variety of reasons, a fact that has brought about a need for alternative tools and approaches. Here we report for the first time in B. oleae the development of the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) gene editing tool, using the well-known eye colour marker gene scarlet. Two synthetic guide RNAs targeting the coding region of the scarlet gene were synthesized and shown to work efficiently in vitro. These reagents were then microinjected along with purified Cas9 protein into early-stage embryos. Successful CRISPR-induced mutations of both copies of the scarlet gene showed a striking yellow eye phenotype, indicative of gene disruption. Multiple successful CRISPR events were confirmed by PCR and sequencing. The establishment of an efficient CRISPR-based gene editing tool in B. oleae will enable the study of critical molecular mechanisms in olive fruit fly biology and physiology, including the analysis of insecticide resistance mechanisms and the discovery of novel insecticide targets, as well as facilitate the development of novel biotechnology-based pest control strategies.

The CRISP(Y) Future of Pediatric Soft Tissue Sarcomas.

The RNA-guided clustered regularly interspaced palindromic repeats (CRISPR)/associated nuclease 9 (Cas9)-based genome editing technology has increasingly become a recognized method for translational research. In oncology, the ease and versatility of CRISPR/Cas9 has made it possible to obtain many results in the identification of new target genes and in unravel mechanisms of resistance to therapy. The majority of the studies have been made on adult tumors so far. In this mini review we present an overview on the major aspects of CRISPR/Cas9 technology with a focus on a group of rare pediatric malignancies, soft tissue sarcomas, on which this approach is having promising results.

CRISPR/Cas9-mediated mutation of asparagine-linked glycosylation 13 transcript variant 1 causes epilepsy in mice

Abstract Objective: The asparagine-linked glycosylation 13 homolog (Alg13) has been identified as causative for congenital disorders of glycosylation type I with epilepsy. The aim of this study was to determine whether mice carrying a mutated version of Alg13 could be used as a model for epileptic encephalopathies or congenital disorders of glycosylation type I. Methods: A model of epileptic encephalopathy was established in C57BL/6 mice by introducing mutations in Alg13 via the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) system. All surgical procedures were approved by the Institutional Animal Care and Use Committee of Shanghai Jiao Tong University (A2016084) on October 8, 2016. Results: Mice with 3 different mutations, Alg13 –54nt , Alg13 –5nt and Alg13 –4nt , all of which are located in Alg13 transcript variant 1, were created. The Alg13 –5nt mice exhibited spontaneous seizures similar to patients with Alg13 mutations, suggesting that they could be used as a model for epilepsy. Western blot analysis demonstrated that Alg13 –5nt mice had lower levels of Alg13 expression than wild-type mice. Video observations showed that two of the 17 Alg13 –5nt mice had stage 5 seizures involving jumping and falling, while 12 had stage 3 seizures with head nodding. Conclusion: The Alg13 mouse model provides an outstanding tool for studying epileptic encephalopathies and investigating different aspects of defects in glycosylation or other post-translational modification that cannot be assessed in patients or cell culture systems.

Construction of a CRISPR‐based paired‐sgRNA library for chromosomal deletion of long non‐coding RNAs

BackgroundDerived from an adaptive bacterial immune system, the clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR‐associated 9 (Cas9) system has shown great potential in high‐throughput functional genomic screening, especially for protein‐coding genes. However, it is still challenging to apply the similar strategy to study non‐coding genomic elements such as long non‐coding RNAs (lncRNAs) or clusters of microRNAs, because short insertions or deletions may not be sufficient to generate loss‐of‐function phenotypes.MethodsHere, we presented a systematic strategy for designing a CRISPR‐based paired‐sgRNA library for high‐throughput screening in non‐coding regions. Due to the abundance of lncRNAs and their diverse regulatory roles in vivo, we repurposed microarray datasets to select 600 highly expressed lncRNAs in non‐small‐cell lung cancer and designed two schemes for lncRNA deletion with ~20 paired‐sgRNAs for each lncRNA. Through Golden‐Gate assembly, we generated a pooled CRISPR‐based library with a total of 12,878 sgRNA pairs.ResultsOver 80% of paired‐sgRNAs were recovered from final pooled library with a relatively even distribution. Cleavage efficiency of sgRNA pairs was validated through experiments of transient transfection and viral infection. Moreover, randomly selected paired‐sgRNAs showed that efficient deletion of genomic DNA could be achieved with a deletion size within the range of 500 to 3000 bp.ConclusionsIn summary, we have demonstrated a strategy to design and construct a pooled paired‐sgRNA library to generate genomic deletion in the lncRNA regions, validated their deletion efficiency and explored the relationship of deletion efficiency with respect to deletion size. This method would be also suitable for investigation of other uncharacterized non‐coding genomic regions in mammalian cells in an efficient and cost‐effective manner.

Improving Cancer Immunotherapy with CRISPR-Based Technology.

The rapidly evolving field of immunotherapy has attracted great attention in the field of cancer research and already revolutionized the clinical practice standard for treating cancer. Genetically engineered T cells expressing either T cell receptors or chimeric antigen receptors represent novel treatment modalities and are considered powerful weapons to fight cancer. The immune checkpoint blockade, which harnesses the negative control signaling behind the anti-tumor immune response with therapeutic antibodies by blocking cytotoxic T lymphocyte-associated protein 4 or the programmed cell death 1 pathways are another mainstream direction for cancer immunotherapy. In addition to cytotoxic T cells, other immune cell types such as nature killer cells and macrophages also possess the ability to eradicate cancer cells, which may serve as the basis to develop novel cancer immunotherapies. The advent of cutting-edge genome editing technology, especially clustered regularly interspaced palindromic repeats (CRISPR)-based tools, has greatly expedited many biomedical research areas, including cancer immunology and immunotherapy. In this review, the contribution of current CRISPR techniques to basic and translational cancer immunology research is discussed, and the future for cancer immunotherapy in the age of CRISPR is predicted.

EMT signaling: potential contribution of CRISPR/Cas gene editing.

Epithelial to mesenchymal transition (EMT) is a complex plastic and reversible cellular process that has critical roles in diverse physiological and pathological phenomena. EMT is involved in embryonic development, organogenesis and tissue repair, as well as in fibrosis, cancer metastasis and drug resistance. In recent years, the ability to edit the genome using the clustered regularly interspaced palindromic repeats (CRISPR) and associated protein (Cas) system has greatly contributed to identify or validate critical genes in pathway signaling. This review delineates the complex EMT networks and discusses recent studies that have used CRISPR/Cas technology to further advance our understanding of the EMT process.

Novel Approaches of CRISPR-Cas Technology in Airway Diseases.

Clustered regularly interspaced palindromic repeats (CRISPR) technique plays a vital role in preclinical modelling of many respiratory diseases. Diseases such as chronic obstructive pulmonary disease (COPD), asthma, acute tracheal bronchitis, pneumonia, tuberculosis, lung cancer, and influenza infection continue to significantly impact human health. CRISPR associated (Cas) proteins, isolated from the immune system of prokaryotes, are one component of a very useful technique to manipulate gene sequences or editing and gene expression with significant implications for respiratory research in the field of molecular biology. CRISPR technology is a promising tool that is easily adaptable for specific editing of DNA sequences of interest with a goal towards modifying or eliminating gene function. Among its many potential applications, CRISPR can be applied to correcting genetic defects as well as for therapeutic approaches for treatment. This review elucidates recent advances in CRISPR-Cas technology in airway diseases.