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Recent progress in CRISPR/Cas9 system for eye disorders.
Ocular disorders encompass a broad spectrum of phenotypic and clinical symptoms resulting from several genetic variants and environmental factors. The unique anatomy and physiology of the eye facilitate validation of cutting-edge gene editing treatments. Genome editing developments have allowed researchers to treat a variety of diseases, including ocular disorders. The clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) system holds considerable promise for therapeutic applications in the field of ophthalmology, including repair of aberrant genes and treatment of retinal illnesses related to the genome or epigenome. Application of CRISPR/Cas9 systems to the study of ocular disease and visual sciences have yielded innovations including correction of harmful mutations in patient-derived cells and gene modifications in several mammalian models of eye development and disease. In this study, we discuss the generation of several ocular disease models in mammalian cell lines and in vivo systems using a CRISPR/Cas9 system. We also provide an overview of current uses of CRISPR/Cas9 technologies for the treatment of ocular pathologies, as well as future challenges.
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Synthetic AAV/CRISPR vectors for blocking HIV-1 expression in persistently infected astrocytes.
Astrocytes, the most abundant cells in the mammalian brain, perform key functions and are involved in several neurodegenerative diseases. The human immunodeficiency virus (HIV) can persist in astrocytes, contributing to the HIV burden and neurological dysfunctions in infected individuals. While a comprehensive approach to HIV cure must include the targeting of HIV-1 in astrocytes, dedicated tools for this purpose are still lacking. Here we report a novel Adeno-associated virus-based vector (AAV9P1) with a synthetic surface peptide for transduction of astrocytes. Analysis of AAV9P1 transduction efficiencies with single brain cell populations, including primary human brain cells, as well as human brain organoids demonstrated that AAV9P1 targeted terminally differentiated human astrocytes much more efficiently than neurons. We then investigated whether AAV9P1 can be used to deliver HIV-inhibitory genes to astrocytes. To this end we generated AAV9P1 vectors containing genes for HIV-1 proviral editing by CRISPR/Cas9. Latently HIV-1 infected astrocytes transduced with these vectors showed significantly diminished reactivation of proviruses, compared with untransduced cultures. Sequence analysis identified mutations/deletions in key HIV-1 transcriptional control regions. We conclude that AAV9P1 is a promising tool for gene delivery to astrocytes and may facilitate inactivation/destruction of persisting HIV-1 proviruses in astrocyte reservoirs.
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Gene Manipulation Using Fusion Guide RNAs for Cas9 and Cas12a.
The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) protein has emerged as a genome engineering tool for various organisms. Known as the CRISPR-Cas system, Cas endonucleases such as Cas9 and Cas12a (also known as Cpf1) and guide RNA (gRNA) complexes recognize and cleave the target DNA, allowing for targeted gene manipulation. Along with the Cas protein engineering, gRNA engineering has broadened the applications of the CRISPR-Cas system. Recently, we have developed fusion guide RNAs (fgRNAs) for orthogonal gene manipulation using Cas9 and Cas12a. Here, we describe the methods for designing and generating fgRNAs-expression constructs to achieve multiplex genome editing and gene manipulation in human cells.
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Evolving Therapeutic Options for the Treatment of Duchenne Muscular Dystrophy.
Duchenne muscular dystrophy (DMD) is the most common childhood form of muscular dystrophy. It is caused by mutations in the DMD gene, leading to reduced or absent expression of the dystrophin protein. Clinically, this results in loss of ambulation, cardiomyopathy, respiratory failure, and eventually death. In the past decades, the use of corticosteroids has slowed down the disease progression. More recently, the development of genetically mediated therapies has emerged as the most promising treatment for DMD. These strategies include exon skipping with antisense oligonucleotides, gene replacement therapy with adeno-associated virus, and gene editing with CRISPR (clustered regularly interspaced short palindromic repeats) technology. In this review, we highlight the most up-to-date therapeutic progresses in the field, with emphasis on past and recent experiences, as well as the latest clinical results of DMD micro-dystrophin gene therapy. Additionally, we discuss the lessons learned along the way and the challenges encountered, all of which have helped advance the field, with the potential to finally alleviate such a devastating disease.
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CRISPR/Cas9 gene editing strategy for cancer therapy: non-viral nanocarrier-mediated delivery of plasmids, RNA and ribonucleoprotein complexes.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) gene editing systems are among the most prevalent tools in modern genetic research and have been extensively studied in the context of cancer therapy. CRISPR/Cas9 systems can be categorized into three main delivery forms: plasmid DNA (pDNA) containing both Cas9 and guide RNA (sgRNA), messenger RNA (mRNA) of Cas9 along with sgRNA, and ribonucleoprotein (RNP) complexes consisting of Cas9 protein bound to sgRNA. This article will review the various CRISPR/Cas9 systems, focusing on the non-viral especially nanocarrier delivery strategies and common protocols used for their delivery and the challenges encountered during this process. This review will offer guidance on the potential applications of CRISPR/Cas9 gene editing technology for the treatment of malignant tumors in the future.
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Genome Editing in Human Pluripotent Stem Cells.
Pluripotent stem cells (PSCs), defined by their capacity for self-renewal and differentiation into all cell types, are an integral tool for basic biological research and disease modeling. However, full use of PSCs for research and regenerative medicine requires the ability to precisely edit their DNA to correct disease-causing mutations and for functional analysis of genetic variations. Recent advances in DNA editing of human stem cells (including PSCs) have benefited from the use of designer nucleases capable of making double-strand breaks (DSBs) at specific sequences that stimulate endogenous DNA repair. The clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas9 system has become the preferred designer nuclease for genome editing in human PSCs and other cell types. Here we describe the principles for designing a single guide RNA to uniquely target a gene of interest and describe strategies for disrupting, inserting, or replacing a specific DNA sequence in human PSCs. The improvements in efficiency and ease provided by these techniques allow individuals to precisely engineer PSCs in a way previously limited to large institutes and core facilities.
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Application of CRISPR/Cas System in the Metabolic Engineering of Small Molecules.
Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated Cas protein technology area is rapidly growing technique for genome editing and modulation of transcription of several microbes. Successful engineering in microbes requires an emphasis on the aspect of efficiency and targeted aiming, which can be employed using CRISPR/Cas system. Hence, this type of system is used to modify the genome of several microbes such as yeast and bacteria. In recent years, CRISPR/Cas systems have been chosen for metabolic engineering in microbes due to their specificity, orthogonality, and efficacy. Therefore, we need to review the scheme which was acquired for the execution of the CRISPR/Cas system for the modification and metabolic engineering in yeast and bacteria. In this review, we highlighted the application of the CRISPR/Cas system which has been used for the production of small molecules in the microbial system that is chemically and biologically important.
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Functional analysis of Bombyx Wnt1 during embryogenesis using the CRISPR/Cas9 system.
Recently established, custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system provide attractive genome editing tools. Targeted gene mutagenesis using the CRISPR/Cas9 system has been achieved in several orders of insects. However, outside of studies on Drosophila melanogaster and the lepidopteron model insect Bombyx mori, little success has been reported, which is largely due to a lack of effective genetic manipulation tools that can be used in other insect orders. To create a simple and effective method of gene knockout analysis, especially for dissecting gene functioning during insect embryogenesis, we performed a functional analysis of the Bombyx Wnt1 (BmWnt1) gene using Cas9/sgRNA-mediated gene mutagenesis. The Wnt1 gene is required for embryonic patterning in various organisms, and its crucial roles during embryogenesis have been demonstrated in several insect orders. Direct injection of Cas9 mRNA and BmWnt1-specific sgRNA into Bombyx embryos induced a typical Wnt-deficient phenotype: injected embryos could not hatch and exhibited severe defects in body segmentation and pigmentation in a dose-dependent manner. Quantitative real-time PCR (qRT-PCR) analysis revealed that Hox genes were down-regulated after BmWnt1 depletion. Furthermore, large deletion, up to 18Kb, ware generated. The current study demonstrates that using the CRISPR/Cas9 system is a promising approach to achieve targeted gene mutagenesis during insect embryogenesis.
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Precision genome editing in plants: state-of-the-art in CRISPR/Cas9-based genome engineering.
Traditionally, generation of new plants with improved or desirable features has relied on laborious and time-consuming breeding techniques. Genome-editing technologies have led to a new era of genome engineering, enabling an effective, precise, and rapid engineering of the plant genomes. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (CRISPR/Cas9) has emerged as a new genome-editing tool, extensively applied in various organisms, including plants. The use of CRISPR/Cas9 allows generating transgene-free genome-edited plants ("null segregants") in a short period of time. In this review, we provide a critical overview of the recent advances in CRISPR/Cas9 derived technologies for inducing mutations at target sites in the genome and controlling the expression of target genes. We highlight the major breakthroughs in applying CRISPR/Cas9 to plant engineering, and challenges toward the production of null segregants. We also provide an update on the efforts of engineering Cas9 proteins, newly discovered Cas9 variants, and novel CRISPR/Cas systems for use in plants. The application of CRISPR/Cas9 and related technologies in plant engineering will not only facilitate molecular breeding of crop plants but also accelerate progress in basic research.
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Palindromic target site identification in SARS-CoV-2, MERS-CoV and SARS-CoV-1 by adopting CRISPR-Cas technique.
Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated Cas protein (CRISPR-Cas) has turned out to be a very important tool for the rapid detection of viruses. This can be used for the identification of the target site in a virus by identifying a 3-6 nt length Protospacer Adjacent Motif (PAM) adjacent to the potential target site, thus motivating us to adopt CRISPR-Cas technique to identify SARS-CoV-2 as well as other members of Coronaviridae family. In this regard, we have developed a fast and effective method using k-mer technique in order to identify the PAM by scanning the whole genome of the respective virus. Subsequently, palindromic sequences adjacent to the PAM locations are identified as the potential target sites. Palindromes are considered in this work as they are known to identify viruses. Once all the palindrome-PAM combinations are identified, PAMs specific for the RNA-guided DNA Cas9/Cas12 endonuclease are identified to bind and cut the target sites. In this regard, PAMs such as 5'-TGG-3' and 5'-TTTA-3' in NSP3 and Exon for SARS-CoV-2, 5'-GGG-3' and 5'-TGG-3' in Exon and NSP2 for MERS-CoV and 5'-AGG-3' and 5'-TTTG-3' in Helicase and NSP3 respectively for SARS-CoV-1 are identified corresponding to SpCas9 and FnCas12a endonucleases. Finally, to recognise the target sites of Coronaviridae family as cleaved by SpCas9 and FnCas12a, complements of the palindromic target regions are designed as primers or guide RNA (gRNA). Therefore, such complementary gRNAs along with respective Cas proteins can be considered in assays for the identification of SARS-CoV-2, MERS-CoV and SARS-CoV-1.
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