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Genome editing using CRISPR/Cas9 to treat hereditary hematological disorders.
The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is a versatile and convenient genome-editing tool with prospects in gene therapy. This technique is based on customized site-specific nucleases with programmable guiding RNAs that cleave and introduce double-strand breaks (DSBs) at the target locus and achieve precise genome modification by triggering DNA repair mechanisms. Human hematopoietic stem/progenitor cells (HSPCs) are conventional cell targets for gene therapy in hematological diseases and have been widely used in most studies. Induced pluripotent stem cells (iPSCs) can be generated from a variety of somatic cells and hold great promise for personalized cell-based therapies. CRISPR/Cas9-mediated genome editing in autologous HSPCs and iPSCs is an ideal therapeutic solution for treating hereditary hematological disorders. Here, we review and summarize the latest studies about CRISPR/Cas9-mediated genome editing in patient-derived HSPCs and iPSCs to treat hereditary hematological disorders. Current challenges and prospects are also discussed.
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Gene editing technology for improving life quality: A dream coming true?
The fact that monogenic diseases are related to mutations in one specific gene, make gene correction one of the promising strategies in the future to treat genetic diseases or alleviate their symptoms. From this perspective, and along with recent advances in technology, genome editing tools have gained momentum and developed fast. In fact, clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9), transcription activator-like effector nucleases (TALENs), and zinc-finger nucleases (ZFNs) are regarded as novel technologies which are able to correct a number of genetic aberrations in vitro and in vivo. The number of ongoing clinical trials employing these tools has been increased showing the encouraging outcomes of these tools. However, there are still some major challenges with respect to the safety profile and directed delivery of them. In this paper, we provided updated information regarding the history, nature, methods of delivery, and application of the above-mentioned gene editing tools along with the meganucleases (an older similar tool) based on published in vitro and in vivo studies and introduced clinical trials which employed these technologies.
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A glance at genome editing with CRISPR-Cas9 technology.
In recent years, CRISPR-Cas9 technology is widely acknowledged for having major applications in the field of biotechnology for editing genome of any organism to treat a variety of complex diseases and for other purposes. The acronym 'CRISPR-Cas' stands for clustered regularly interspaced short palindromic repeats-CRISPR-associated genes. This genetic organization exists in prokaryotic organisms and aids in the development of adaptive immunity since a protein called Cas9 nuclease cleaves specific target nucleic acid sequences from foreign invaders and destroys them. This mode of action has gained interest of the researchers to understand the insights of CRISPR-Cas9 technology. Here, we review that CRISPR-Cas organization is restricted to two classes and possesses different protein effectors. We also review the architecture of CRISPR loci, mechanism involved in genome editing by CRISPR-Cas9 technology and pathways of repairing double-strand breaks (DSBs) generated during the process of genome editing. This review also presents the strategies to increase the Cas9 specificity and reduce off-target activity to achieve accurate genome editing. Further, this review provides information on CRISPR tools used for genome editing, databases that are required for storing data on loci, strategies for delivering CRISPR-Cas9 to cells under study and applications of CRISPR-Cas9 to various fields. Safety measures are implemented on this technology to avoid misuse or ethical issues. We also discuss about the future aspects and potential applications of CRISPR-Cas9 technology required mainly for the treatment of dreadful diseases, crop improvement as well as genetic improvement in human.
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Golden Gate Assembly of CRISPR gRNA expression array for simultaneously targeting multiple genes.
The engineered CRISPR/Cas9 technology has developed as the most efficient and broadly used genome editing tool. However, simultaneously targeting multiple genes (or genomic loci) in the same individual cells using CRISPR/Cas9 remain one technical challenge. In this article, we have developed a Golden Gate Assembly method for the generation of CRISPR gRNA expression arrays, thus enabling simultaneous gene targeting. Using this method, the generation of CRISPR gRNA expression array can be accomplished in 2 weeks, and contains up to 30 gRNA expression cassettes. We demonstrated in the study that simultaneously targeting 10 genomic loci or simultaneously inhibition of multiple endogenous genes could be achieved using the multiplexed gRNA expression array vector in human cells. The complete set of plasmids is available through the non-profit plasmid repository Addgene.
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Embryo Microinjection and Knockout Mutant Identification of CRISPR/Cas9 Genome-Edited Helicoverpa Armigera (Hübner).
The cotton bollworm, Helicoverpa armigera, is one of the most destructive pests in the world. A combination of molecular genetics, physiology, functional genomics, and behavioral studies has made H. armigera a model species in Lepidoptera Noctuidae. To study the in vivo functions of and interactions between different genes, clustered regularly interspaced short palindromic repeats (CRISPR)/ associated protein 9 (Cas9) genome editing technology is a convenient and effective method used for performing functional genomic studies. In this study, we provide a step-by-step systematic method to complete gene knockout in H. armigera using the CRISPR/Cas9 system. The design and synthesis of guide RNA (gRNA) are described in detail. Then, the subsequent steps consisting of gene-specific primer design for guide RNA (gRNA) creation, embryo collection, microinjection, insect rearing, and mutant detection are summarized. Finally, troubleshooting advice and notes are provided to improve the efficiency of gene editing. Our method will serve as a reference for the application of CRISPR/Cas9 genome editing in H. armigera as well as other Lepidopteran moths.
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Fast and global detection of periodic sequence repeats in large genomic resources.
Periodically repeating DNA and protein elements are involved in various important biological events including genomic evolution, gene regulation, protein complex formation, and immunity. Notably, the currently used genome editing tools such as ZFNs, TALENs, and CRISPRs are also all associated with periodically repeating biomolecules of natural organisms. Despite the biological importance of periodically repeating sequences and the expectation that new genome editing modules could be discovered from such periodical repeats, no software that globally detects such structured elements in large genomic resources in a high-throughput and unsupervised manner has been developed. We developed new software, SPADE (Search for Patterned DNA Elements), that exhaustively explores periodic DNA and protein repeats from large-scale genomic datasets based on k-mer periodicity evaluation. With a simple constraint, sequence periodicity, SPADE captured reported genome-editing-associated sequences and other protein families involving repeating domains such as tetratricopeptide, ankyrin and WD40 repeats with better performance than the other software designed for limited sets of repetitive biomolecular sequences, suggesting the high potential of this software to contribute to the discovery of new biological events and new genome editing modules.
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CRISPR/CAS9-based gene editing in cancer therapy: A systematic review and meta-analysis on current status and future directions.
BACKGROUND: The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has recently been discovered for gene editing and cancer therapy and its applications are expanding. This review and meta-analysis aim to assess the present and future of CRISPR/Cas9 based gene editing in cancer treatment and the way forward. METHODS: The search was conducted in PubMed from 2015 to 2025 and 89 relevant studies were identified. The study design, CRISPR/Cas9 targets, delivery methods, therapeutic efficacy and limitations were extracted from the studies. RESULTS: We reviewed the efficacy, challenges, and potential for translation of CRISPR/Cas9 in oncogene and tumor suppressor gene targeting and immune modulation. Several preclinical researches showed that CRISPR/Cas9 mediated disruption of oncogenes or restoration of tumor suppressor genes led to significant tumor regression. The evaluation was also extended to off target effects and integration with immunotherapy. CONCLUSION: From the findings of this work, it can be concluded that CRISPR/Cas9 is a promising tool, but there are several limitations including off target effects, delivery systems and ethical issues that need to be solved in order to improve the clinical significance.
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Genome Editing for Cardiovascular Diseases-A Brief Review for Cardiologists.
The recent technical advances in genome engineering have accelerated our understanding of the molecular mechanisms of human diseases and are leading to increased clinical applications of gene-targeting therapies. The field of cardiovascular medicine, rich in knowledge of molecular level disease mechanisms, is particularly well positioned to receive significant benefits from this technology. Specifically, a new generation of genome editing tools capable of introducing targeted sequence modifications at high frequencies initiated by induced DNA double-strand breaks has been developed. Of note is the RNA-guided genome editing system, clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9), which has provided researchers and clinicians a malleable gene-targeting platform with high specificity. Recent reports have robustly demonstrated proof-of-concept in using CRISPR-Cas9 based gene therapy for treating common cardiovascular diseases and are testaments that the new genome editing technology holds promise for treating patients with cardiovascular ailments in the clinic in the near future. In light of this trend, a basic understanding of genome editing technology is becoming more relevant to clinical cardiologists. To this end, a concise explanation of terms and the biological basis of genome editing, on-going research, and clinical trials highly relevant to clinical application are presented. In conclusion, the aim of this short review is to introduce clinicians to the core concepts of current genome editing technology.
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CETCh-seq: CRISPR epitope tagging ChIP-seq of DNA-binding proteins.
Chromatin immunoprecipitation followed by next-generation DNA sequencing (ChIP-seq) is a widely used technique for identifying transcription factor (TF) binding events throughout an entire genome. However, ChIP-seq is limited by the availability of suitable ChIP-seq grade antibodies, and the vast majority of commercially available antibodies fail to generate usable data sets. To ameliorate these technical obstacles, we present a robust methodological approach for performing ChIP-seq through epitope tagging of endogenous TFs. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing technology to develop CRISPR epitope tagging ChIP-seq (CETCh-seq) of DNA-binding proteins. We assessed the feasibility of CETCh-seq by tagging several DNA-binding proteins spanning a wide range of endogenous expression levels in the hepatocellular carcinoma cell line HepG2. Our data exhibit strong correlations between both replicate types as well as with standard ChIP-seq approaches that use TF antibodies. Notably, we also observed minimal changes to the cellular transcriptome and to the expression of the tagged TF. To examine the robustness of our technique, we further performed CETCh-seq in the breast adenocarcinoma cell line MCF7 as well as mouse embryonic stem cells and observed similarly high correlations. Collectively, these data highlight the applicability of CETCh-seq to accurately define the genome-wide binding profiles of DNA-binding proteins, allowing for a straightforward methodology to potentially assay the complete repertoire of TFs, including the large fraction for which ChIP-quality antibodies are not available.
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Split crRNA-Mediated Regulation of CRISPR-Cas12a Enables Construction of Biochemical Circuits for Sensitive Molecular Diagnostics.
The clustered regularly interspaced short palindromic repeat (CRISPR) system represents a fundamental tool for biotechnological applications. However, the ability to engineer intrinsic components of the CRISPR system to regulate its activity remains limited. Here, we present a strategy for the regulation of CRISPR-Cas12a by engineering an enhanced split CRISPR RNA (split en-crRNA) variant. Reassembly of this variant autonomously restores the full activity of Cas12a nuclease without the need for chemical modifications or exogenous activation stimuli. Leveraging this strategy, we developed CREST (CRISPR Reactions Elicited by Split en-crRNA Transcription) for molecular diagnostics. CREST integrates biochemical circuits that ingeniously harness isothermal amplification to trigger the on-demand synthesis and reassembly of a universal split en-crRNA, addressing longstanding challenges in CRISPR diagnostics, including the low efficiency of one-pot assays, protospacer adjacent motif restriction, the need for crRNA reoptimization, and limited reagent stability. The CREST assay enables robust detection of attomolar DNAs or RNAs within 15 to 20 min and demonstrates capability for identifying bacterial infections in patients. This study provides new insights into Cas12a regulation, which is expected to advance the development of controllable CRISPR technologies for diagnostics as well as broader applications such as genome editing and programmable gene expression.
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