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Generation and immunofluorescent validation of gene knockouts in adult human colonic organoids using multi-guide RNA CRISPR-Cas9.
While readily achieved in cell lines, the application of CRISPR-Cas9 gene editing in human-derived organoids suffers from limited efficacy and complex protocols. Here, we describe a multi-guide RNA CRISPR-Cas9 gene-editing protocol which efficiently achieves complete gene knockout in adult human colonic organoids. This protocol also describes crucial steps including how to harvest patient tissue to maximize gene-editing efficacy and a technique to validate gene knockout following editing with immunofluorescent staining of the organoids against the target protein.
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CRISPR/Cas: An intriguing genomic editing tool with prospects in treating neurodegenerative diseases.
The CRISPR/Cas genome editing tool has led to a revolution in biological research. Its ability to target multiple genomic loci simultaneously allows its application in gene function and genomic manipulation studies. Its involvement in the sequence specific gene editing in different backgrounds has changed the scenario of treating genetic diseases. By unravelling the mysteries behind complex neuronal circuits, it not only paved way in understanding the pathogenesis of the disease but helped in the development of large animal models of different neuronal diseases; thereby opened the gateways of successfully treating different neuronal diseases. This review explored the possibility of using of CRISPR/Cas in engineering DNA at the embryonic stage, as well as during the functioning of different cell types in the brain, to delineate implications related to the use of this super-specialized genome editing tool to overcome various neurodegenerative diseases that arise as a result of genetic mutations.
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Non-Viral CRISPR/Cas Gene Editing In Vitro and In Vivo Enabled by Synthetic Nanoparticle Co-Delivery of Cas9 mRNA and sgRNA.
CRISPR/Cas is a revolutionary gene editing technology with wide-ranging utility. The safe, non-viral delivery of CRISPR/Cas components would greatly improve future therapeutic utility. We report the synthesis and development of zwitterionic amino lipids (ZALs) that are uniquely able to (co)deliver long RNAs including Cas9 mRNA and sgRNAs. ZAL nanoparticle (ZNP) delivery of low sgRNA doses (15 nm) reduces protein expression by >90 % in cells. In contrast to transient therapies (such as RNAi), we show that ZNP delivery of sgRNA enables permanent DNA editing with an indefinitely sustained 95 % decrease in protein expression. ZNP delivery of mRNA results in high protein expression at low doses in vitro (<600 pM) and in vivo (1 mg kg-1 ). Intravenous co-delivery of Cas9 mRNA and sgLoxP induced expression of floxed tdTomato in the liver, kidneys, and lungs of engineered mice. ZNPs provide a chemical guide for rational design of long RNA carriers, and represent a promising step towards improving the safety and utility of gene editing.
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Design and Assembly of CRISPR/Cas9 Lentiviral and rAAV Vectors for Targeted Genome Editing.
Clustered regularly interspaced short palindromic repeat (CRISPR/Cas) system has emerged as an extremely useful tool for biological research and as a potential technology for gene therapy approaches. CRISPR/Cas mediated genome editing can be used to easily and efficiently modify endogenous genes in a large variety of cells and organisms. Furthermore, a modified version of the Cas9 nuclease has been developed that can be used for regulation of endogenous gene expression and labeling of genomic loci, among other applications. This chapter provides an introduction to the basis of the technology and a detail protocol for the most classic application: gene inactivation by CRISPR/Cas9 nuclease system from Streptococcus pyogenes. This workflow can be easily adapted for other CRISPR systems and applications.
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Streamlining mouse genome editing by integrating AAV repair template delivery and CRISPR-Cas electroporation.
Recent genome editing techniques have substantially simplified the generation of genetically modified mice. A new study combines adeno-associated viruses (AAV) and electroporation to generate a robust pipeline to deliver CRISPR-Cas reagents into mouse embryos.
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Futuristic approach to cancer treatment.
Cancer is becoming one of the deadliest disease in both developed as well as developing countries and continuous effort is being made to find innovative therapies for myriad types of cancers that afflict the human body. Therapeutic options for cancer have grown exponentially over the time but we are quite a way off from finding a magic bullet that can help cure cancer and based on the current evidence we may never find a catch all cure ever and it becomes crucial that we keep on innovating and find multiple ways to attack the menace of this dreaded disease. Many patients suffer recurrence of disease and require second-line or in some cases more than two lines of treatment. In this review article we have discussed the available therapies along with the newer advancements that have been made in cancer therapy. Latest developments in treatment of various cancers that have been discussed include gene editing using CRISPR/Cas9, theranostics, viral mediated therapy, artificial intelligence, tumor infiltrating lymphocyte therapy, etc.
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CRISPR in livestock: From editing to printing.
Precise genome editing of large animals applied to livestock and biomedicine is nowadays possible since the CRISPR revolution. This review summarizes the latest advances and the main technical issues that determine the success of this technology. The pathway from editing to printing, from engineering the genome to achieving the desired animals, does not always imply an easy, fast and safe journey. When applied in large animals, CRISPR involves time- and cost-consuming projects, and it is mandatory not only to choose the best approach for genome editing, but also for embryo production, zygote microinjection or electroporation, cryopreservation and embryo transfer. The main technical refinements and most frequent questions to improve this disruptive biotechnology in large animals are presented. In addition, we discuss some CRISPR applications to enhance livestock production in the context of a growing global demand of food, in terms of increasing efficiency, reducing the impact of farming on the environment, enhancing pest control, animal welfare and health. The challenge is no longer technical. Controversies and consensus, opportunities and threats, benefits and risks, ethics and science should be reconsidered to enter into the CRISPR era.
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Utilizing codon degeneracy in the design of donor DNA for CRISPR/Cas9-mediated gene editing to streamline the screening process for single amino acid mutations.
Chlamydomonas reinhardtii, a unicellular green alga, has been widely used as a model organism for studies of algal, plant and ciliary biology. The generation of targeted amino acid mutations is often necessary, and this can be achieved using CRISPR/Cas9 induced homology-directed repair to install genomic modifications from exogenous donor DNA. Due to the low gene editing efficiency, the technical challenge lies in identifying the mutant cells. Direct sequencing is not practical, and pre-screening is required. Here, we report a strategy for generating and screening for amino acid point mutations using the CRISPR/Cas9 gene editing system. The strategy is based on designing donor DNA using codon degeneracy, which enables the design of specific primers to facilitate mutant screening by PCR. An in vitro assembled RNP complex, along with a dsDNA donor and an antibiotic resistance marker, was electroporated into wild-type cells, followed by PCR screening. To demonstrate this principle, we have generated the E102K mutation in centrin and the K40R mutation in α-tubulin. The editing efficiencies at the target sites for Centrin, TUA1, TUA2 were 4, 24 and 8% respectively, based on PCR screening. More than 80% of the mutants with the expected size of PCR products were precisely edited, as revealed by DNA sequencing. Subsequently, the precision-edited mutants were biochemically verified. The introduction of codon degeneracy did not affect the gene expression of centrin and α-tubulins. Thus, this approach can be used to facilitate the identification of point mutations, especially in genes with low editing rates.
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Discovery and engineering of AiEvo2, a novel Cas12a nuclease for human gene editing applications.
The precision of gene editing technology is critical to creating safe and effective therapies for treating human disease. While the programmability of CRISPR-Cas systems has allowed for rapid innovation of new gene editing techniques, the off-target activity of these enzymes has hampered clinical development for novel therapeutics. Here, we report the identification and characterization of a novel CRISPR-Cas12a enzyme from Acinetobacter indicus (AiCas12a). We engineer the nuclease (termed AiEvo2) for increased specificity, protospacer adjacent motif recognition, and efficacy on a variety of human clinical targets. AiEvo2 is highly precise and able to efficiently discriminate between normal and disease-causing alleles in Huntington's patient-derived cells by taking advantage of a single nucleotide polymorphism on the disease-associated allele. AiEvo2 efficiently edits several liver-associated target genes including PCSK9 and TTR when delivered to primary hepatocytes as mRNA encapsulated in a lipid nanoparticle. The enzyme also engineers an effective CD19 chimeric antigen receptor-T-cell therapy from primary human T cells using multiplexed simultaneous editing and chimeric antigen receptor insertion. To further ensure precise editing, we engineered an anti-CRISPR protein to selectively inhibit off-target gene editing while retaining therapeutic on-target editing. The engineered AiEvo2 nuclease coupled with a novel engineered anti-CRISPR protein represents a new way to control the fidelity of editing and improve the safety and efficacy of gene editing therapies.
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Clarifying the regulation of genome editing in Australia: situation for food.
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