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Enhancing gene editing and gene targeting efficiencies in Arabidopsis thaliana by using an intron-containing version of ttLbCas12a.
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Can CRISPR help in the fight against parasitic worms?
The first reports of CRISPR/Cas9 genome editing in flatworms could usher in a new era of research on these dangerous human parasites.
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Co-Treatment of Erythroid Cells from β-Thalassemia Patients with CRISPR-Cas9-Based β039-Globin Gene Editing and Induction of Fetal Hemoglobin.
Gene editing (GE) is an efficient strategy for correcting genetic mutations in monogenic hereditary diseases, including β-thalassemia. We have elsewhere reported that CRISPR-Cas9-based gene editing can be employed for the efficient correction of the β039-thalassemia mutation. On the other hand, robust evidence demonstrates that the increased production of fetal hemoglobin (HbF) can be beneficial for patients with β-thalassemia. The aim of our study was to verify whether the de novo production of adult hemoglobin (HbA) using CRISPR-Cas9 gene editing can be combined with HbF induction protocols. The gene editing of the β039-globin mutation was obtained using a CRISPR-Cas9-based experimental strategy; the correction of the gene sequence and the transcription of the corrected gene were analyzed by allele-specific droplet digital PCR and RT-qPCR, respectively; the relative content of HbA and HbF was studied by high-performance liquid chromatography (HPLC) and Western blotting. For HbF induction, the repurposed drug rapamycin was used. The data obtained conclusively demonstrate that the maximal production of HbA and HbF is obtained in GE-corrected, rapamycin-induced erythroid progenitors isolated from β039-thalassemia patients. In conclusion, GE and HbF induction might be used in combination in order to achieve the de novo production of HbA together with an increase in induced HbF.
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CRISPR-Cas9 gene editing induced complex on-target outcomes in human cells.
CRISPR-Cas9 is a powerful tool for editing the genome and holds great promise for gene therapy applications. Initial concerns of gene engineering focus on off-target effects. However, in addition to short indel mutations (often <50 bp), an increasing number of studies have revealed complex on-target results after double-strand break repair by CRISPR-Cas9, such as large deletions, gene rearrangement, and loss of heterozygosity. These unintended mutations are potential safety concerns in clinical gene editing. Here, in this review, we summarize the significant findings of CRISPR-Cas9-induced on-target deleterious outcomes and discuss putative ways to achieve safe gene therapy.
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CRISPR-Cas12a (Cpf1): A Versatile Tool in the Plant Genome Editing Tool Box for Agricultural Advancement.
Global population is predicted to approach 10 billion by 2050, an increase of over 2 billion from today. To meet the demands of growing, geographically and socio-economically diversified nations, we need to diversity and expand agricultural production. This expansion of agricultural productivity will need to occur under increasing biotic, and environmental constraints driven by climate change. Clustered regularly interspaced short palindromic repeats-site directed nucleases (CRISPR-SDN) and similar genome editing technologies will likely be key enablers to meet future agricultural needs. While the application of CRISPR-Cas9 mediated genome editing has led the way, the use of CRISPR-Cas12a is also increasing significantly for genome engineering of plants. The popularity of the CRISPR-Cas12a, the type V (class-II) system, is gaining momentum because of its versatility and simplified features. These include the use of a small guide RNA devoid of trans-activating crispr RNA, targeting of T-rich regions of the genome where Cas9 is not suitable for use, RNA processing capability facilitating simpler multiplexing, and its ability to generate double strand breaks (DSB) with staggered ends. Many monocot and dicot species have been successfully edited using this Cas12a system and further research is ongoing to improve its efficiency in plants, including improving the temperature stability of the Cas12a enzyme, identifying new variants of Cas12a or synthetically producing Cas12a with flexible PAM sequences. In this review we provide a comparative survey of CRISPR-Cas12a and Cas9, and provide a perspective on applications of CRISPR-Cas12 in agriculture.
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Plasmid- or Ribonucleoprotein-Mediated CRISPR/Cas Gene Editing in Primary Murine T Cells.
The CRISPR/Cas technology allows for genome editing in primary T cells. We herein describe the activation of primary murine CD4+ or CD8+ T cells, followed by electroporation with plasmid or ribonucleoproteins (RNP) for gene modification. Gene edited T cells can subsequently be transferred to host mice for in vivo studies or cultured in vitro for further characterization. This protocol enables sophisticated genetic analysis of T cells using commonly available virus-free reagents.
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Dibenzocyclooctyne-Branched Primer Assembled Gene Nanovector and Its Potential Applications in Genome Editing.
The CRISPR/Cas9 system has been widely used as an efficient genome editing toolkit for gene therapy. The delivery of vectors encoding the full CRISPR/Cas9 components including Cas9 gene and gRNA expression element into cells is the crucial step to effective genome editing. However, the cargo gene sequence for genome editing is usually large, which reduces the cargo encapsulation efficiency and affects the vector size. To obtain a nanovector with high cargo gene loading capacity and biocompatible size, we report the construction of a gene nanovector from branch-PCR with a dibenzocyclooctyne (DBCO)-branched primer and establish the correlation mapping between gene length and nanovector size. The results show that the size of nanovectors can be tuned according to the gene length. According to the findings, we constructed nanovectors carrying the full CRISPR/Cas9 components in 100-200 nm and validated their application in genome editing. The results show that this kind of nanovector exhibits higher serum stability than plasmids and can reach comparable genome editing efficiency with plasmids. Hence, this type of gene nanovector obtained through branch-PCR can carry large gene cargos and maintain a biocompatible nanoscale size, which we envisage will expand its medical applications in gene therapy.
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Engineering the Delivery System for CRISPR-Based Genome Editing.
Clustered regularly interspaced short palindromic repeat-CRISPR-associated protein (CRISPR-Cas) systems, found in nature as microbial adaptive immune systems, have been repurposed into an important tool in biological engineering and genome editing, providing a programmable platform for precision gene targeting. These tools have immense promise as therapeutics that could potentially correct disease-causing mutations. However, CRISPR-Cas gene editing components must be transported directly to the nucleus of targeted cells to exert a therapeutic effect. Thus, efficient methods of delivery will be critical to the success of therapeutic genome editing applications. Here, we review current strategies available for in vivo delivery of CRISPR-Cas gene editing components and outline challenges that need to be addressed before this powerful tool can be deployed in the clinic.
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Visualization analysis of CRISPR/Cas9 gene editing technology studies.
The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is an adaptive immune defense system that resists the invasion of viruses and plasmids heterologous genetic material in bacteria and archaea. Taking the literature related to gene editing technology of CRISPR/Cas9 from the Web of Science database from 2002 to 2015, we use the software CiteSpaceV to analyze co-cited literature in order to establish the research hotspots and fronts recently in this field by knowledge mapping.
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Inhibition of CRISPR-Cas9 ribonucleoprotein complex assembly by anti-CRISPR AcrIIC2.
CRISPR-Cas adaptive immune systems function to protect bacteria from invasion by foreign genetic elements. The CRISPR-Cas9 system has been widely adopted as a powerful genome-editing tool, and phage-encoded inhibitors, known as anti-CRISPRs, offer a means of regulating its activity. Here, we report the crystal structures of anti-CRISPR protein AcrIIC2Nme alone and in complex with Nme1Cas9. We demonstrate that AcrIIC2Nme inhibits Cas9 through interactions with the positively charged bridge helix, thereby preventing sgRNA loading. In vivo phage plaque assays and in vitro DNA cleavage assays show that AcrIIC2Nme mediates its activity through a large electronegative surface. This work shows that anti-CRISPR activity can be mediated through the inhibition of Cas9 complex assembly.
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