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Advances in genome editing for improved animal breeding: A review.
Since centuries, the traits for production and disease resistance are being targeted while improving the genetic merit of domestic animals, using conventional breeding programs such as inbreeding, outbreeding, or introduction of marker-assisted selection. The arrival of new scientific concepts, such as cloning and genome engineering, has added a new and promising research dimension to the existing animal breeding programs. Development of genome editing technologies such as transcription activator-like effector nuclease, zinc finger nuclease, and clustered regularly interspaced short palindromic repeats systems begun a fresh era of genome editing, through which any change in the genome, including specific DNA sequence or indels, can be made with unprecedented precision and specificity. Furthermore, it offers an opportunity of intensification in the frequency of desirable alleles in an animal population through gene-edited individuals more rapidly than conventional breeding. The specific research is evolving swiftly with a focus on improvement of economically important animal species or their traits all of which form an important subject of this review. It also discusses the hurdles to commercialization of these techniques despite several patent applications owing to the ambiguous legal status of genome-editing methods on account of their disputed classification. Nonetheless, barring ethical concerns gene-editing entailing economically important genes offers a tremendous potential for breeding animals with desirable traits.
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Nanotechnology and CRISPR/Cas-Mediated Gene Therapy Strategies: Potential Role for Treating Genetic Disorders.
Gene therapy has made substantial progress in the treatment of the genetic diseases, focussing on the reduction of characteristics of recessive/dominant disorders, as well as various cancers. Extensive research has been conducted in the past few decades to investigate the application of nanotechnology and CRISPR/Cas technology in gene therapy. Nanotechnology due to attributes such has targeted drug delivery, controlled release, scalability and low toxicity has gained attention of the medical world. CRISPR/Cas9 system is considered as an impactful genome editing tool in the area of next-generation therapeutics and molecular diagnostics. CRISPR technology emphasises on gene editing, gene regulation modulation, and formulation of defined genetic changes. Its applications in treatment of the genetic disorders are extended beyond traditional therapies. These techniques are being explored as treatment of several genetic disorders including Duchenne muscular dystrophy, cystic fibrosis, Alzheimer's disease, Parkinson's disease, and Huntington disease. Despite considerable therapeutic potential of gene therapy, several obstacles must be addressed before it can be widely adopted in clinical practice, particularly in terms of ensuring safety and effectiveness. As research advances in this captivating field, these therapies will become the primary treatments and will have significant beneficial effects on the lives of patients with genetic disorders.
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Compositional Optimization of CRISPR/Cas9 Lipid Nanoparticles for Efficient Knockdown of Target Genes.
Efficient and safe delivery systems remain a major barrier to the clinical translation of CRISPR/Cas9 gene-editing technologies; among these, formulation optimization of lipid nanoparticles (LNPs) is a key approach to improve delivery performance. Here, we constructed an orthogonal formulation library of LNPs using the ionizable lipids 4A2C2C6-A8 and 4A2C2C8-A8 at varied molar ratios and screened for optimal compositions. We identified formulations that combined high editing efficiency with low cytotoxicity; in HeLa-Luc cells, the optimized formulation achieved >80% knockout of the luciferase reporter. Further physicochemical and functional investigations showed that LNPs with relatively high zeta potential, mean diameters near 200 nm, and appropriate internal hydrophobicity-when paired with superior cellular uptake and endosomal escape capabilities-synergistically enhanced delivery efficiency. Using the compositionally optimized LNP to codeliver Cas9 mRNA and an sgRNA targeting HSP47 (whose overexpression has been implicated in fibrosis), we achieved efficient protein-level knockdown of HSP47 in L929 cells. These results provide important guidance for formulation optimization of CRISPR/Cas9 LNPs and support their potential application in antifibrotic therapies.
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Russian 'CRISPR-baby' scientist has started editing genes in human eggs with goal of altering deaf gene.
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Differential expression of genes in pre-blastoderm embryos of oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) microinjected with white locus CRISPR/Cas9 ribo nucleo protein (RNP) complex.
CRISPR/Cas9-based genome editing is currently revolutionizing applications in agriculture, human health, and other domains. Targeted gene editing employing CRISPR/Cas9 is achieved by delivering the various components viz. guide RNA, traCrRNA, and the Cas9 protein in DNA or RNA or RNP format. RNP enables fast, DNA-free editing without genomic integration. While innate immunity to guide RNA is studied in humans and plants, similar research in insects remains unexplored. In the present study, the RNP complex (sgRNA+Cas9) was administered via microinjection of embryos (eggs) in the pre-blastoderm (G0) stage. Transcriptome sequencing was performed using the Illumina Novoseq 6000 platform to understand the prevalence of innate immunity against the delivered guide RNA for the white locus of Bactrocera dorsalis. 33 up- and 67 down-regulated genes were found in the differential gene expression analysis from the RNA-Seq data. The correlation between RT-qPCR and RNA-Seq gene expression levels showed a strong coefficient of determination (R2 = 0.984). The genes related to stress, intron removal, and effector recognition were overexpressed. However, the genes responsible for growth and development were significantly downregulated. Thus, the present study identified the possible reasons that could play a vital role in influencing the editing outcomes employed through the RNP complex.
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Ribonucleoprotein Transfection for CRISPR/Cas9-Mediated Gene Knockout in Primary T Cells.
CRISPR/Cas9 has enabled the rapid and efficient generation of gene knockouts across various cell types of several species. T cells are central players in adaptive immune responses. Gene editing in primary T cells not only represents a valuable research tool, but is also critical for next generation immunotherapies, such as CAR T cells. Broad application of CRIPSR/Cas9 for gene editing in primary T cells has been hampered by limitations in transfection efficiency and the requirement for TCR stimulation. In this article, we provide a detailed protocol for Cas9/gRNA ribonucleoprotein (RNP) transfection of primary mouse and human T cells without the need for TCR stimulation that achieves near complete loss of target gene expression at the population level. This approach enables rapid target discovery and validation in both mouse and human primary T cells. © 2018 by John Wiley & Sons, Inc.
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A non-cationic nucleic acid nanogel for the delivery of the CRISPR/Cas9 gene editing tool.
Herein, we report a non-cationic DNA-crosslinked nanogel for intracellular delivery of a Cas9 and single guide RNA (Cas9/sgRNA) complex. A DNA-grafted polycaprolactone brush (DNA-g-PCL) is first loaded with the Cas9/sgRNA complex and then crosslinked by DNA linkers via nucleic acid hybridization to form a nanosized hydrogel, in which the gene editing tools are embedded and protected inside. With compact architecture, the Cas9/sgRNA complex-containing nanogel exhibited excellent physiological stability against nuclease digestion and enhanced cellular uptake efficiency, making the delivery system a promising tool for target genome editing.
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Multiple site-directed mutagenesis via simple cloning by prolonged overlap extension.
We describe the application of simple cloning by prolonged overlap extension for multiple site-directed mutagenesis in the same plasmid. We show that it is possible to use this technique with very short PCR templates. The technique is ideally suited for the generation of longer donor DNA sequences for CRISPR/Cas9-mediated homologous repair.
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Efficient Genome Editing in Apple Using a CRISPR/Cas9 system.
Genome editing is a powerful technique for genome modification in molecular research and crop breeding, and has the great advantage of imparting novel desired traits to genetic resources. However, the genome editing of fruit tree plantlets remains to be established. In this study, we describe induction of a targeted gene mutation in the endogenous apple phytoene desaturase (PDS) gene using the CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed and stably transformed with Cas9 separately in apple. Clear and partial albino phenotypes were observed in 31.8% of regenerated plantlets for one gRNA, and bi-allelic mutations in apple PDS were confirmed by DNA sequencing. In addition, an 18-bp gRNA also induced a targeted mutation. These CRIPSR/Cas9 induced-mutations in the apple genome suggest activation of the NHEJ pathway, but with some involvement also of the HR pathway. Our results demonstrate that genome editing can be practically applied to modify the apple genome.
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Genome editing for inborn errors of metabolism: advancing towards the clinic.
Inborn errors of metabolism (IEM) include many disorders for which current treatments aim to ameliorate disease manifestations, but are not curative. Advances in the field of genome editing have recently resulted in the in vivo correction of murine models of IEM. Site-specific endonucleases, such as zinc-finger nucleases and the CRISPR/Cas9 system, in combination with delivery vectors engineered to target disease tissue, have enabled correction of mutations in disease models of hemophilia B, hereditary tyrosinemia type I, ornithine transcarbamylase deficiency, and lysosomal storage disorders. These in vivo gene correction studies, as well as an overview of genome editing and future directions for the field, are reviewed and discussed herein.
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