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CRISPR/Cas9 mediated targeted mutagenesis of the fast growing cyanobacterium Synechococcus elongatus UTEX 2973.
BACKGROUND: As autotrophic prokaryotes, cyanobacteria are ideal chassis organisms for sustainable production of various useful compounds. The newly characterized cyanobacterium Synechococcus elongatus UTEX 2973 is a promising candidate for serving as a microbial cell factory because of its unusually rapid growth rate. Here, we seek to develop a genetic toolkit that enables extensive genomic engineering of Synechococcus 2973 by implementing a CRISPR/Cas9 editing system. We targeted the nblA gene because of its important role in biological response to nitrogen deprivation conditions. RESULTS: First, we determined that the Streptococcus pyogenes Cas9 enzyme is toxic in cyanobacteria, and conjugational transfer of stable, replicating constructs containing the cas9 gene resulted in lethality. However, after switching to a vector that permitted transient expression of the cas9 gene, we achieved markerless editing in 100 % of cyanobacterial exconjugants after the first patch. Moreover, we could readily cure the organisms of antibiotic resistance, resulting in a markerless deletion strain. CONCLUSIONS: High expression levels of the Cas9 protein in Synechococcus 2973 appear to be toxic and result in cell death. However, introduction of a CRISPR/Cas9 genome editing system on a plasmid backbone that leads to transient cas9 expression allowed for efficient markerless genome editing in a wild type genetic background.
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DNA Repair Pathway Choices in CRISPR-Cas9-Mediated Genome Editing.
Many clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-based genome editing technologies take advantage of Cas nucleases to induce DNA double-strand breaks (DSBs) at desired locations within a genome. Further processing of the DSBs by the cellular DSB repair machinery is then necessary to introduce desired mutations, sequence insertions, or gene deletions. Thus, the accuracy and efficiency of genome editing are influenced by the cellular DSB repair pathways. DSBs are themselves highly genotoxic lesions and as such cells have evolved multiple mechanisms for their repair. These repair pathways include homologous recombination (HR), classical nonhomologous end joining (cNHEJ), microhomology-mediated end joining (MMEJ) and single-strand annealing (SSA). In this review, we briefly highlight CRISPR-Cas9 and then describe the mechanisms of DSB repair. Finally, we summarize recent findings of factors that can influence the choice of DNA repair pathway in response to Cas9-induced DSBs.
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CRISPR/Cas9-Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells.
Human induced pluripotent stem cells (hiPSCs) can be used to mass produce surrogates of human tissues, enabling new advances in drug screening, disease modeling, and cell therapy. Recent developments in clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome editing technology use homology-directed repair (HDR) to efficiently generate custom hiPSC lines harboring a variety of genomic insertions and deletions. Thus, hiPSCs that encode an endogenous protein fused to a fluorescent reporter protein can be rapidly created by employing CRISPR/Cas9 genome editing, enhancing HDR efficiency and optimizing homology arm length. These fluorescently tagged hiPSCs can be used to visualize protein function and dynamics in real time as cells proliferate and differentiate. Given that nearly any intracellular protein can be fluorescently tagged, this system serves as a powerful tool to facilitate new discoveries across many biological disciplines. In this unit, we present protocols for the design, generation, and monoclonal expansion of genetically customized hiPSCs encoding fluorescently tagged endogenous proteins. © 2018 by John Wiley & Sons, Inc.
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Hidden prevalence of deletion-inversion bi-alleles in CRISPR-mediated deletions of tandemly arrayed genes in plants.
Tandemly arrayed genes (TAGs) with functional redundancy and chromosomal linkage constitute 14 ~ 35% in sequenced plant genomes. The multiplex CRISPR system is the tool of choice for creating targeted TAG deletions. Here, we show that up to ~80% of CRISPR-mediated TAG knockout alleles in Arabidopsis and rice are deletion-inversion (delinver) bi-alleles, which are easily misidentified as homozygous deletion alleles by routine PCR-based genotyping. This can lead to misinterpretation of experimental data and production of progenies with genetic heterogeneity in an unnoticed manner. In ~2,650 transgenic events, delinver mutation frequencies are predominantly correlated with deletion frequencies but unrelated to chromosomal locations or deletion sizes. Delinver mutations also occur frequently at genomic non-TAG loci during multiplexed CRISPR editing. Our work raises the alarm about delinver mutations as common unwanted products of targeted TAG deletions in plants and helps prevent false interpretation of plant TAG functions due to this hidden genotype issue.
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Rapid and efficient analysis of gene function using CRISPR-Cas9 in Xenopus tropicalis founders.
Recent advances in genome editing using programmable nucleases, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system, have facilitated reverse genetics in Xenopus tropicalis. To establish a practical workflow for analyzing genes of interest using CRISPR-Cas9, we examined various experimental procedures and conditions. We first compared the efficiency of gene disruption between Cas9 protein and mRNA injection by analyzing genotype and phenotype frequency, and toxicity. Injection of X. tropicalis embryos with Cas9 mRNA resulted in high gene-disrupting efficiency comparable with that produced by Cas9 protein injection. To exactly evaluate the somatic mutation rates of on-target sites, amplicon sequencing and restriction fragment length polymorphism analysis using a restriction enzyme or recombinant Cas9 were performed. Mutation rates of two target genes (slc45a2 and ltk) required for pigmentation were estimated to be over 90% by both methods in animals exhibiting severe phenotypes, suggesting that targeted somatic mutations were biallelically introduced in almost all somatic cells of founder animals. Using a heteroduplex mobility assay, we also showed that off-target mutations were induced at a low rate. Based on our results, we propose a CRISPR-Cas9-mediated gene disruption workflow for a rapid and efficient analysis of gene function using X. tropicalis founders.
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CRISPR-Cas9, A Promising Therapeutic Tool for Cancer Therapy: A Review.
Cancer is one of the most leading causes of mortality all over the world and remains a foremost social and economic burden. Mutations in the genome of individuals are taking place more frequently due to the excessive progress of xenobiotics and industrialization in the present world. With the progress in the field of molecular biology, it is possible to alter the genome and to observe the functional changes derived from genetic modulation using gene-editing technologies. Several therapies have been applied for the treatment of malignancy which affect the normal body cells; however, more effort is required to develop vsome latest therapeutic approaches for cancer biology and oncology exploiting these molecular biology advances. Recently, the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated protein 9 (Cas9) system has emerged as a powerful technology for cancer therapy because of its great accuracy and efficiency. Genome editing technologies have demonstrated a plethora of benefits to the biological sciences. CRISPR- Cas9, a versatile gene editing tool, has become a robust strategy for making alterations to the genome of organisms and a potent weapon in the arsenal of tumor treatment. It has revealed an excellent clinical potential for cancer therapy by discovering novel targets and has provided the researchers with the perception about how tumors respond to drug therapy. Stern efforts are in progress to enhance its efficiency of sequence specific targeting and consequently repressing offtarget effects. CRISPR-Cas9 uses specific proteins to convalesce mutations at genetic level. In CRISPR-Cas9 system, RNA-guided Cas9 endonuclease harnesses gene mutation, DNA deletion or insertion, transcriptional activation or repression, multiplex targeting only by manipulating 20-nucleotide components of RNA. Originally, CRISPR-Cas9 system was used by bacteria for their defense against different bacteriophages, and recently this system is receiving noteworthy appreciation due to its emerging role in the treatment of genetic disorders and carcinogenesis. CRISPR-Cas9 can be employed to promptly engineer oncolytic viruses and immune cells for cancer therapeutic applications. More notably, it has the ability to precisely edit genes not only in model organisms but also in human being that permits its use in therapeutic analysis. It also plays a significant role in the development of complete genomic libraries for cancer patients. In this review, we have highlighted the involvement of CRISPR-Cas9 system in cancer therapy accompanied by its prospective applications in various types of malignancy and cancer biology. In addition, some other conspicuous functions of this unique system have also been discussed beyond genome editing.
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Perspectives on new opportunities for nano-enabled strategies for gene delivery to plants using nanoporous materials.
Engineered nanocarriers have great potential to deliver different genetic cargos to plant cells and increase the efficiency of plant genetic engineering. Genetic engineering has improved the quality and quantity of crops by introducing desired DNA sequences into the plant genome. Traditional transformation strategies face constraints such as low transformation efficiency, damage to plant tissues, and genotype dependency. Smart nanovehicle-based delivery is a newly emerged method for direct DNA delivery to plant genomes. The basis of this new approach of plant genetic transformation, nanomaterial-mediated gene delivery, is the appropriate protection of transferred DNA from the nucleases present in the cell cytoplasm through the nanocarriers. The conjugation of desired nucleic acids with engineered nanocarriers can solve the problem of genetic manipulation in some valuable recalcitrant plant genotypes. Combining nano-enabled genetic transformation with the new and powerful technique of targeted genome editing, CRISPR (clustered regularly interspaced short palindromic repeats), can create new protocols for efficient improvement of desired plants. Silica-based nanoporous materials, especially mesoporous silica nanoparticles (MSNs), are currently regarded as exciting nanoscale platforms for genetic engineering as they possess several useful properties including ordered and porous structure, biocompatibility, biodegradability, and surface chemistry. These specific features have made MSNs promising candidates for the design of smart, controlled, and targeted delivery systems in agricultural sciences. In the present review, we discuss the usability, challenges, and opportunities for possible application of nano-enabled biomolecule transformation as part of innovative approaches for target delivery of genes of interest into plants.
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Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells.
The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide RNA (sgRNA) that confers specificity, the Cas9 protein cleaves both DNA strands at the targeted locus. The DNA break can trigger either non-homologous end joining (NHEJ) or homology directed repair (HDR). NHEJ can introduce small deletions or insertions which lead to frame-shift mutations, while HDR allows for larger and more precise perturbations. Here, we present protocols for generating knockout cell lines by coupling established CRISPR/Cas9 methods with two options for downstream selection/screening. The NHEJ approach uses a single sgRNA cut site and selection-independent screening, where protein production is assessed by dot immunoblot in a high-throughput manner. The HDR approach uses two sgRNA cut sites that span the gene of interest. Together with a provided HDR template, this method can achieve deletion of tens of kb, aided by the inserted selectable resistance marker. The appropriate applications and advantages of each method are discussed.
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Periodontal Disease Pathogens, Pathogenesis, and Therapeutics: The CRISPR-Cas Effect.
Periodontal disease (PD) is an immune-inflammatory disease affecting the supporting structures of the teeth, which results in progressive destruction of the hard and soft tissues surrounding teeth, ultimately resulting in tooth loss. The primary etiological factor for this disease is the presence of pathogenic microorganisms. Pathogenic bacteria face antagonistic conditions and foreign DNA components during the infection stage and depend on defense mechanisms such as clustered regularly interspaced short palindromic repeats (CRISPR)-Cas to counter them. Virulence genes regulated by the CRISPR-Cas system are often expressed by bacteria as part of the stress response to the presence of stress conditions and foreign elements. There is ever-growing evidence regarding the role of CRISPR-Cas in virulence of periodontal pathogens. The same CRISPR-Cas system may also be targeted to reduce bacterial virulence and it may also be utilized to develop diagnostic and therapeutic strategies for prevention and control of PD progression. This review article describes the CRISPR-Cas systems in the periodontal dysbiotic microbial communities, their role in the virulence of periodontal pathogens, and their potential role in understanding the pathogenesis of periodontitis and treatment of PD.
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Applications of CRISPR/Cas13-Based RNA Editing in Plants.
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is widely used as a genome-editing tool in various organisms, including plants, to elucidate the fundamental understanding of gene function, disease diagnostics, and crop improvement. Among the CRISPR/Cas systems, Cas9 is one of the widely used nucleases for DNA modifications, but manipulation of RNA at the post-transcriptional level is limited. The recently identified type VI CRISPR/Cas systems provide a platform for precise RNA manipulation without permanent changes to the genome. Several studies reported efficient application of Cas13 in RNA studies, such as viral interference, RNA knockdown, and RNA detection in various organisms. Cas13 was also used to produce virus resistance in plants, as most plant viruses are RNA viruses. However, the application of CRISPR/Cas13 to studies of plant RNA biology is still in its infancy. This review discusses the current and prospective applications of CRISPR/Cas13-based RNA editing technologies in plants.
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