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Target Specificity of the CRISPR-Cas9 System in Arabidopsis thaliana, Oryza sativa, and Glycine max Genomes.
Clustered regularly interspaced short palindromic repeats (CRISPR), a class of immune-associated sequences in bacteria, have been developed as a powerful tool for editing eukaryotic genomes in diverse cells and organisms in recent years. The CRISPR-Cas9 system can recognize upstream 20 nucleotides (guide sequence) adjacent to the protospacer-adjacent motif site and trigger double-stranded DNA cleavage as well as DNA repair mechanisms, which eventually result in knockout, knockin, or site-specific mutagenesis. However, off-target effect caused by guide sequence misrecognition is the major drawback and restricts its widespread application. In this study, global analysis of specificities of all guide sequences in Arabidopsis thaliana, Oryza sativa (rice), and Glycine max (soybean) were performed. As a result, a simple pipeline and three genome-wide databases were established and shared for the scientific society. For each target site of CRISPR-Cas9, specificity score and off-target number were calculated and evaluated. The mean values of off-target numbers for A. thaliana, rice, and soybean were determined as 27.5, 57.3, and 174.7, respectively. Comparative analysis among these plants suggested that the frequency of off-target effects was correlated to genome size, chromosomal locus, gene density, and guanine-cytosine (GC) content. Our results contributed to the better understanding of CRISPR-Cas9 system in plants and would help to minimize the off-target effect during its applications in the future.
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Computational Tools and Resources for CRISPR/Cas Genome Editing.
The past decade has witnessed a rapid evolution in identifying more versatile clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) nucleases and their functional variants, as well as in developing precise CRISPR/Cas-derived genome editors. The programmable and robust features of the genome editors provide an effective RNA-guided platform for fundamental life science research and subsequent applications in diverse scenarios, including biomedical innovation and targeted crop improvement. One of the most essential principles is to guide alterations in genomic sequences or genes in the intended manner without undesired off-target impacts, which strongly depends on the efficiency and specificity of single guide RNA (sgRNA)-directed recognition of targeted DNA sequences. Recent advances in empirical scoring algorithms and machine learning models have facilitated sgRNA design and off-target prediction. In this review, we first briefly introduce the different features of CRISPR/Cas tools that should be taken into consideration to achieve specific purposes. Secondly, we focus on the computer-assisted tools and resources that are widely used in designing sgRNAs and analyzing CRISPR/Cas-induced on- and off-target mutations. Thirdly, we provide insights into the limitations of available computational tools that would help researchers of this field for further optimization. Lastly, we suggest a simple but effective workflow for choosing and applying web-based resources and tools for CRISPR/Cas genome editing.
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Protocol for De Novo Gene Targeting Via In Utero Electroporation.
Developments in genome-editing technology, especially CRISPR-Cas9, have revolutionized the way in which genetically engineered animals are generated. However, the process of generation includes microinjection to the one-cell stage embryo and the transfer of the microinjected embryo to the surrogate animals, which requires trained personnel. We recently reported the method includes introduction of CRISPR-Cas9 systems to the developing cerebral cortex via in utero electroporation thus generating gene-targeted neural stem cells in vivo. This technique is widely applicable for gene knockout, monitoring gene expression, and lineage analysis in developmental biology. In this chapter, the detailed protocol of EGFP (enhanced green fluorescent protein) knock-in method via in utero electroporation is described.
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Human pluripotent stem cell engineering with CRISPR-Cas9 for Parkinson's disease.
Parkinson's disease (PD) entails loss of substantia nigra dopamine (DA) neurons and α-synuclein pathology. Currently, no effective disease-modifying therapies have been developed. Human pluripotent stem cells (hPS cells) can generate DA neurons on scale, enabling human genetic PD modeling of mitochondrial, lysosomal and synaptic connection failure that leads to DA neuron degeneration. Clustered regularly interspaced short palindromic repeats (CRISPR) extends this human model by providing causal, isogenic interrogation and transcriptional regulation of PD genes and reporter knock-ins that support purification and high-content screening. hPS cell-based DA cell grafts can restore motor function yet face >90% acute cell death and product heterogeneity in vivo post implantation. CRISPR enabled not only an in vivo cell survival screen to identify the cell death regulators but also a reporter-guided enrichment of DA neurons and chemogenetic control of grafted DA cell function in vivo. Here we summarize this progress and outline a practical road map to accelerate the development of precise human models and advanced hPS cell-based cell therapies for PD.
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The most common disease-causing mutation of factor XIII deficiency is corrected by CRISPR/CAS9 gene editing system.
Factor XIII (FXIII) deficiency is one of the most severe congenital bleeding disorders, with an estimated incidence of one person per one million. Patients with severe FXIII deficiency present a wide range of clinical manifestations, including umbilical cord bleeding, intracranial haemorrhage and recurrent miscarriages. Due to the high rate of life-threatening bleeding, primary prophylaxis is mandatory from the time of diagnosis. Although replacement therapy is the most common therapeutic choice, gene therapy remains the only curative option. In the present study, we assessed the efficacy of the clustered regularly interspaced short palindromic repeats - CRISPR-associated protein 9 (CRISPR/Cas9) system in the correction of the most common FXIII disease-causing mutation (c.562 T > C). A dermal fibroblast was harvested from the human skin biopsy of a young patient with FXIII deficiency. Sanger sequencing was used to confirm the presence of c.562 T>C mutation in the patient and in the harvested fibroblasts. PX459 vector was digested with BbsI restriction enzyme, and after annealing and ligation of two 20-bp guide-RNAs (g-RNAs) close to the PAM (NGG) sequence, the constructed vectors were amplified in Escherichia coli Top 10. Transfection was performed by a nucleofector device, and DNA extraction was performed after puromycin selection and serial dilution from potentially transfected colonies. A 50-bp template oligonucleotide was used to aid homologous repair for correction of the underlying mutation and synonymous mutation as an internal control. The synonymous mutation (AAT to ACT) near the mutation site was used as internal control. Sanger sequencing was done in order to check the gene correction. The c.562 T > C mutation was detected in homozygote state in the primary fibroblasts of the patient and wild-type alleles were confirmed in the normal individual. Colony PCR and sequencing revealed successful cloning of the designed gRNAs. The detected mutation was corrected from a homozygote mutant state (c.562 T > C) to a homozygote wild type in transfected dermal fibroblasts of the patient. The control mutation, as an internal control, was also corrected in the same fibroblasts in the heterozygote manner. The result of the study shows that the CRISPR/CAS9 gene editing system is an effective tool for correction of point mutations in transfected fibroblasts of patients with congenital FXIII deficiency and represents a new, potentially curative, option.
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Repurposing the Endogenous Type I-E CRISPR/Cas System for Gene Repression in Gluconobacter oxydans WSH-003.
Gluconobacter oxydans is well-known for its incomplete oxidizing capacity and has been widely applied in industrial production. However, genetic tools in G. oxydans are still scarce compared with model microorganisms, limiting its metabolic engineering. This study aimed to develop a clustered regularly interspaced short palindromic repeats interference (CRISPRi) system based on the typical type I-E endogenous CRISPR/CRISPR-associated proteins (Cas) system in G. oxydans WSH-003. The nuclease Cas3 in this system was inactivated naturally and hence did not need to be knocked out. Subsequently, the CRISPRi effect was verified by repressing the expression of fluorescent proteins, revealing effective multiplex gene repression. Finally, the endogenous CRISPRi system was used to study the role of the central carbon metabolism pathway, including the pentose phosphate pathway (PPP) and Entner-Doudoroff pathway (EDP), in G. oxydans WSH-003. This was done to demonstrate a metabolic engineering application. The PPP was found to be important for cell growth and the substrate conversion rate. The development of the CRISPRi system enriched the gene regulation tools in G. oxydans and promoted the metabolic engineering modification of G. oxydans to improve its performance. In addition, it might have implications for metabolic engineering modification of other genetically recalcitrant strains.
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Potential of Gene and Cell Therapy for Inner Ear Hair Cells.
Sensorineural hearing loss is caused by the loss of sensory hair cells (HCs) or a damaged afferent nerve pathway to the auditory cortex. The most common option for the treatment of sensorineural hearing loss is hearing rehabilitation using hearing devices. Various kinds of hearing devices are available but, despite recent advancements, their perceived sound quality does not mimic that of the "naïve" cochlea. Damage to crucial cochlear structures is mostly irreversible and results in permanent hearing loss. Cochlear HC regeneration has long been an important goal in the field of hearing research. However, it remains challenging because, thus far, no medical treatment has successfully regenerated cochlear HCs. Recent advances in genetic modulation and developmental techniques have led to novel approaches to generating HCs or protecting against HC loss, to preserve hearing. In this review, we present and review the current status of two different approaches to restoring or protecting hearing, gene therapy, including the newly introduced CRISPR/Cas9 genome editing, and stem cell therapy, and suggest the future direction.
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Extracellular Vesicle-Liposome Hybrid Nanoparticles Delivery of CRISPR/Cas9 Induces a Unique DNA Repair Pattern in the HGF Gene of Stem Cells from Apical Papilla.
Extracellular vesicles (EVs) have been investigated due to their natural biocompatibility and targeting capabilities. The specific approach of combining EVs with liposomes to create hybrid nanoparticles (ELNPs) for the delivery of the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas9) system for deletion of the HGF gene in stem cells, but their effectiveness in encapsulating large nucleic acids is limited due to their small size. This study aimed to knock out the HGF gene by the CRISPR/Cas9 system by ELNPs, and it was expected that the efficiency of the CRISPR/Cas9 system transfer would increase compared to the usual methods of using lipofectamine in stem cells from apical papilla (SCAPs). In this study, gRNA suitable for the HGF gene is designed first, and after insertion into the CRISPR/Cas9 vector, it enters Lipofectamine 2000. In the next step, ELNPs are prepared after collecting EVs and hybridizing them with liposomes containing CRISPR/Cas9 vector. Then, these integrated nanoparticles were presented to SCAPs, and the removal of HGF gene expression was evaluated at the level of RNA and protein. This study showed that the CRISPR/Cas9 system can be efficiently transferred to SCAP cells using ELNPs. Genomic DNA sequencing analyses of SCAP cells showed a unique pattern of mutation, highly likely mediated through EVs. Quantitative PCR and protein staining further showed a decrease in HGF gene expression in the knockout cells. Moreover, cell proliferation analysis showed a decrease in cell proliferation in KO-HGF adipose cells compared to the nonedited counterpart. In summary, this study highlights the supportive role of EVs in facilitating cell transfection and promoting a dominant DNA repair pattern, likely through an RNA-mediated mechanism, rather than the random insertions and deletions typically induced during CRISPR editing of the HGF gene in SCAPs.
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Active genetics comes alive: Exploring the broad applications of CRISPR-based selfish genetic elements (or gene-drives): Exploring the broad applications of CRISPR-based selfish genetic elements (or gene-drives).
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based "active genetic" elements developed in 2015 bypassed the fundamental rules of traditional genetics. Inherited in a super-Mendelian fashion, such selfish genetic entities offered a variety of potential applications including: gene-drives to disseminate gene cassettes carrying desired traits throughout insect populations to control disease vectors or pest species, allelic drives biasing inheritance of preferred allelic variants, neutralizing genetic elements to delete and replace or to halt the spread of gene-drives, split-drives with the core constituent Cas9 endonuclease and guide RNA (gRNA) components inserted at separate genomic locations to accelerate assembly of complex arrays of genetic traits or to gain genetic entry into novel organisms (vertebrates, plants, bacteria), and interhomolog based copying systems in somatic cells to develop tools for treating inherited or infectious diseases. Here, we summarize the substantial advances that have been made on all of these fronts and look forward to the next phase of this rapidly expanding and impactful field.
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NATF (Native and Tissue-Specific Fluorescence): A Strategy for Bright, Tissue-Specific GFP Labeling of Native Proteins in Caenorhabditis elegans.
GFP labeling by genome editing can reveal the authentic location of a native protein, but is frequently hampered by weak GFP signals and broad expression across a range of tissues that may obscure cell-specific localization. To overcome these problems, we engineered a Native And Tissue-specific Fluorescence (NATF) strategy that combines genome editing and split-GFP to yield bright, cell-specific protein labeling. We use clustered regularly interspaced short palindromic repeats CRISPR/Cas9 to insert a tandem array of seven copies of the GFP11 β-strand (gfp11x7 ) at the genomic locus of each target protein. The resultant gfp11x7 knock-in strain is then crossed with separate reporter lines that express the complementing split-GFP fragment (gfp1-10) in specific cell types, thus affording tissue-specific labeling of the target protein at its native level. We show that NATF reveals the otherwise undetectable intracellular location of the immunoglobulin protein OIG-1 and demarcates the receptor auxiliary protein LEV-10 at cell-specific synaptic domains in the Caenorhabditis elegans nervous system.
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