scholarly journals Using CRISPR-Cas9 for Therapeutic Protein Production (Review Article)

2021 ◽  
Vol 9 (1) ◽  
Author(s):  
Ashraf Ullah Khan ◽  
Zabi Ullah
Keyword(s):  
Nucleic Acids ◽  
Protein Production ◽  
Gene Editing ◽  
Ex Vivo ◽  
Somatic Cells ◽  
Review Article ◽  
Crispr Locus ◽  
Target Effects ◽  
Subsequent Integration

Existence of CRISPR/Cas9 systems in bacteria and archaea has been noted to be the reason for these organisms’ ability to disarm invading nucleic acids. Such immunity is noted to arise from the targeting of the invading nucleic acids by guiding RNAs (sgRNAs), their cleavage by Cas9 (an endonuclease), and their subsequent integration into CRISPR locus. Recent studies have shown that the CRISPR/Cas9 tool can be adopted for gene editing in eukaryotic cells and thus offering potential for its use to treat genetic conditions. In this review, CRISPR/Cas9 has been shown to be an effective genome-editing tool with studies showing efficacy in zygote editing, in-vivo editing of somatic cells and ex-vivo editing of somatic cells. Occurrence of off-target effects however make zygote editing in human cells ethically questionable due to possibility of introducing unwanted mutations that may be passed on to the progeny. Nevertheless, observations that such off-target effects arise mainly from the promiscuity of sgRNAs rather that errors in CRISPR/Cas9 system show promise for increased specificity by developing better sgRNAs.  Such increased specificity will facilitate the adoption of CRISPR/Cas9 for clinical use in treatment of conditions such as β-thalassemia, cystic fibrosis, Duchenne muscular dystrophy and HIV.

scholarly journals Peptide Nucleic Acids and Gene Editing: Perspectives on Structure and Repair

Molecules ◽  
2020 ◽  
Vol 25 (3) ◽  
pp. 735 ◽  
Author(s):  
Nicholas G. Economos ◽  
Stanley Oyaghire ◽  
Elias Quijano ◽  
Adele S. Ricciardi ◽  
W. Mark Saltzman ◽  
...  
Keyword(s):  
Nucleic Acids ◽  
Gene Editing ◽  
Molecular Design ◽  
Peptide Nucleic Acids ◽  
Therapeutic Application ◽  
Monogenic Disorders ◽  
Structural Biochemistry ◽  
Nucleic Acid Structures ◽  
Target Effects

Unusual nucleic acid structures are salient triggers of endogenous repair and can occur in sequence-specific contexts. Peptide nucleic acids (PNAs) rely on these principles to achieve non-enzymatic gene editing. By forming high-affinity heterotriplex structures within the genome, PNAs have been used to correct multiple human disease-relevant mutations with low off-target effects. Advances in molecular design, chemical modification, and delivery have enabled systemic in vivo application of PNAs resulting in detectable editing in preclinical mouse models. In a model of β-thalassemia, treated animals demonstrated clinically relevant protein restoration and disease phenotype amelioration, suggesting a potential for curative therapeutic application of PNAs to monogenic disorders. This review discusses the rationale and advances of PNA technologies and their application to gene editing with an emphasis on structural biochemistry and repair.

scholarly journals CRISPR/Cas-based Diagnostics and Gene Therapy

2021 ◽  
Author(s):  
Qiu Meiyu ◽  
Li Pei
Keyword(s):  
Gene Therapy ◽  
Gene Editing ◽  
Ex Vivo ◽  
Point Of Care ◽  
Cost Effective ◽  
Blood Disorders ◽  
Initial Stage ◽  
Cas Gene ◽  
Cas Proteins

Clustered regularly interspaced short palindromic repeats (CRISPR) technology, an easy, rapid, cost-effective, and precise gene-editing technique, has revolutionized diagnostics and gene therapy. Fast and accurate diagnosis of diseases is essential for point-of-care-testing (POCT) and specialized medical institutes. The CRISPR-associated (Cas) proteins system shed light on the new diagnostics methods at point-of-care (POC) owning to its advantages. In addition, CRISPR/Cas-based gene-editing technology has led to various breakthroughs in gene therapy. It has been employed in clinical trials for a variety of untreatable diseases, including cancer, blood disorders, and other syndromes. Currently, the clinical application of CRISPR/Cas has been mainly focused on ex vivo therapies. Recently, tremendous efforts have been made in the development of ex vivo gene therapy based on CRISPR-Cas9. Despite these efforts, in vivo CRISPR/Cas gene therapy is only in its initial stage. Here, we review the milestones of CRISPR/Cas technologies that advanced the field of diagnostics and gene therapy. We also highlight the recent advances of diagnostics and gene therapy based on CRISPR/Cas technology. In the last section, we discuss the strength and significant challenges of the CRISPR/Cas technology for its future clinical usage in diagnosis and gene therapy.

scholarly journals CRISPR Therapeutics: New Emerging Developments and Clinical Applications

2021 ◽  
Vol 11 (5) ◽  
pp. 193-195
Author(s):  
Kaiser Jay Aziz-Andersen
Keyword(s):  
Stem Cells ◽  
Gene Editing ◽  
Ex Vivo ◽  
Human Subjects ◽  
Clinical Applications ◽  
Approval Process ◽  
Hematopoietic Stem ◽  
Immune System Diseases ◽  
Wide Range

CRISPR gene editing is a genetic engineering technique applied in clinical applications in which the genomes of living organisms may be modified. It is based on the principles of the CRISPR-Cas9 antiviral defense system. It is based on delivering the Cas9 nuclease complexed with a synthetic guide RNA into a living organism cell and that organisms’s genome can be “cut” and –“paste” at a desired location, allowing existing genes to be modified for desired outcome (i.e., CRISPR for Precision Medicine). CRISPR gene editing harnesses the natural defense mechanisms of some bacteria to cut human DNA strands. Then the DNA strand either heals itself or injects a new piece of DNA to mend the gap. Studies have been reported in Lung Cancer diagnosis and treatments. CRISPR-based engineering techniques have been developed for T Cells and Stem cells applications (i.e. Gene Corrections in Hematopoietic Stem Cells for the Treatment of Blood and Immune System Diseases). Even though earlier CRISPR methodologies were used for performing simple DNA edits, recent applications include the ability to delete genes or insert genes, and edit regulatory regions in a wide range of cell types. The role of CRISPR in human therapeutics is currently focused on utilizing CRISPR techniques to perform either in vivo editing of human cells–everything from the head, eye all the way to neurons and liver cells--or performing ex vivo therapies. The FDA’s new genomic CRISPR technology based products approval process begins with review and evaluation of preclinical studies in order to establish and characterize the proposed product’s safety profile. New genomic products must be shown to be safe and effective for the FDA approval process. The sponsor of the new genomic product must show that the product is safe and effective in human subjects.1

scholarly journals In vivo CRISPR-Cas gene editing with no detectable genome-wide off-target mutations

2018 ◽  
Author(s):  
Pinar Akcakaya ◽  
Maggie L. Bobbin ◽  
Jimmy A. Guo ◽  
Jose M. Lopez ◽  
M. Kendell Clement ◽  
...  
Keyword(s):  
Genome Editing ◽  
Ex Vivo ◽  
Strong Support ◽  
Guide Rna ◽  
Genome Wide ◽  
Highly Sensitive ◽  
Further Development ◽  
Target Effects ◽  
Cas Gene

CRISPR-Cas genome-editing nucleases hold substantial promise for human therapeutics1–5 but identifying unwanted off-target mutations remains an important requirement for clinical translation6, 7. For ex vivo therapeutic applications, previously published cell-based genome-wide methods provide potentially useful strategies to identify and quantify these off-target mutation sites8–12. However, a well-validated method that can reliably identify off-targets in vivo has not been described to date, leaving the question of whether and how frequently these types of mutations occur. Here we describe Verification of In Vivo Off-targets (VIVO), a highly sensitive, unbiased, and generalizable strategy that we show can robustly identify genome-wide CRISPR-Cas nuclease off-target effects in vivo. To our knowledge, these studies provide the first demonstration that CRISPR-Cas nucleases can induce substantial off-target mutations in vivo, a result we obtained using a deliberately promiscuous guide RNA (gRNA). More importantly, we used VIVO to show that appropriately designed gRNAs can direct efficient in vivo editing without inducing detectable off-target mutations. Our findings provide strong support for and should encourage further development of in vivo genome editing therapeutic strategies.

scholarly journals crisprSQL: a novel database platform for CRISPR/Cas off-target cleavage assays

2020 ◽  
Vol 49 (D1) ◽  
pp. D855-D861
Author(s):  
Florian Störtz ◽  
Peter Minary
Keyword(s):  
Gene Editing ◽  
Target Prediction ◽  
Current Version ◽  
Therapeutic Gene ◽  
Guide Rnas ◽  
Rodent Cell ◽  
Ongoing Development ◽  
Database Platform ◽  
Target Effects

Abstract With ongoing development of the CRISPR/Cas programmable nuclease system, applications in the area of in vivo therapeutic gene editing are increasingly within reach. However, non-negligible off-target effects remain a major concern for clinical applications. Even though a multitude of off-target cleavage datasets have been published, a comprehensive, transparent overview tool has not yet been established. Here, we present crisprSQL (http://www.crisprsql.com), an interactive and bioinformatically enhanced collection of CRISPR/Cas9 off-target cleavage studies aimed at enriching the fields of cleavage profiling, gene editing safety analysis and transcriptomics. The current version of crisprSQL contains cleavage data from 144 guide RNAs on 25,632 guide-target pairs from human and rodent cell lines, with interaction-specific references to epigenetic markers and gene names. The first curated database of this standard, it promises to enhance safety quantification research, inform experiment design and fuel development of computational off-target prediction algorithms.

Structure of DNA-PAMAM dendrimer complexes studied using small-angle scattering techniques

2020 ◽  
Vol 27 ◽  
Author(s):  
Bradley W. Mansel ◽  
Hsin-Lung Chen
Keyword(s):  
Nucleic Acids ◽  
Small Angle ◽  
Ex Vivo ◽  
Pamam Dendrimer ◽  
Modern Medicine ◽  
Small Angle Scattering ◽  
In Vivo Studies ◽  
Angle Scattering ◽  
Low Cytotoxicity

: Gene therapy is one of the most important developments for modern medicine. As such methods for the compaction and delivery of nucleic acids bearing therapeutic sequences is essential. The quest for non-viral carriers of nucleic acids has produced a number of possible candidates with dendrimer being among the most promising. Their hyper-branched structure and well-defined size together with low cytotoxicity has found success in both ex-vivo and in-vivo studies. The compaction of DNA with dendrimer has produced a rich array of different structures depending on the physiochemical conditions. Mechanisms that drive the compaction have been shown to be a number of physical interactions that reduce the large polymeric entity from 100s of nanometers to some tens of nanometers to fit into the cell nucleus. The mechanisms driving the compaction of DNA will be discussed in detail while the focus will be directed to tuning the structural properties of the complexes and their structural characterization using small-angle scattering techniques.

scholarly journals A versatile toolkit for CRISPR-Cas13-based RNA manipulation in Drosophila

2020 ◽  
Vol 21 (1) ◽  
Author(s):  
Nhan Huynh ◽  
Noah Depner ◽  
Raegan Larson ◽  
Kirst King-Jones
Keyword(s):  
Gene Expression ◽  
Nucleic Acids ◽  
High Efficiency ◽  
Ex Vivo ◽  
Transcriptional Level ◽  
Rna Targeting ◽  
Versatile Tool ◽  
Mitochondrial Transcripts ◽  
Overall Performance

AbstractAdvances in CRISPR technology have immensely improved our ability to manipulate nucleic acids, and the recent discovery of the RNA-targeting endonuclease Cas13 adds even further functionality. Here, we show that Cas13 works efficiently in Drosophila, both ex vivo and in vivo. We test 44 different Cas13 variants to identify enzymes with the best overall performance and show that Cas13 could target endogenous Drosophila transcripts in vivo with high efficiency and specificity. We also develop Cas13 applications to edit mRNAs and target mitochondrial transcripts. Our vector collection represents a versatile tool collection to manipulate gene expression at the post-transcriptional level.

scholarly journals Chemical Synthesis of Stimuli-Responsive Guide RNA for Conditional Control of CRISPR-Cas9 Gene Editing

2021 ◽  
Author(s):  
Chunmei Gu ◽  
Lu Xiao ◽  
Jiachen Shang ◽  
Xiao Xu ◽  
Luo He ◽  
...  
Keyword(s):  
Chemical Synthesis ◽  
Nucleic Acids ◽  
Gene Editing ◽  
Live Cells ◽  
Stimuli Responsive ◽  
Guide Rna ◽  
Conditional Control ◽  
Target Effects

CRISPR-Cas9 promotes changes in identity or abundance of nucleic acids in live cells and is a programmable modality of broad biotechnological and therapeutic interest. To reduce off-target effects, tools for...

scholarly journals Gene editing and CRISPR in the clinic: current and future perspectives

2020 ◽  
Vol 40 (4) ◽  
Author(s):  
Matthew P. Hirakawa ◽  
Raga Krishnakumar ◽  
Jerilyn A. Timlin ◽  
James P. Carney ◽  
Kimberly S. Butler
Keyword(s):  
Clinical Trials ◽  
Genome Editing ◽  
Dna Sequences ◽  
Gene Editing ◽  
Ex Vivo ◽  
Scientific Basis ◽  
Current Status ◽  
Zinc Finger Nucleases ◽  
Transcription Activator

Abstract Genome editing technologies, particularly those based on zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR (clustered regularly interspaced short palindromic repeat DNA sequences)/Cas9 are rapidly progressing into clinical trials. Most clinical use of CRISPR to date has focused on ex vivo gene editing of cells followed by their re-introduction back into the patient. The ex vivo editing approach is highly effective for many disease states, including cancers and sickle cell disease, but ideally genome editing would also be applied to diseases which require cell modification in vivo. However, in vivo use of CRISPR technologies can be confounded by problems such as off-target editing, inefficient or off-target delivery, and stimulation of counterproductive immune responses. Current research addressing these issues may provide new opportunities for use of CRISPR in the clinical space. In this review, we examine the current status and scientific basis of clinical trials featuring ZFNs, TALENs, and CRISPR-based genome editing, the known limitations of CRISPR use in humans, and the rapidly developing CRISPR engineering space that should lay the groundwork for further translation to clinical application.

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