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Editing an α-globin enhancer in primary human hematopoietic stem cells as a treatment for β-thalassemia.
β-Thalassemia is one of the most common inherited anemias, with no effective cure for most patients. The pathophysiology reflects an imbalance between α- and β-globin chains with an excess of free α-globin chains causing ineffective erythropoiesis and hemolysis. When α-thalassemia is co-inherited with β-thalassemia, excess free α-globin chains are reduced significantly ameliorating the clinical severity. Here we demonstrate the use of CRISPR/Cas9 genome editing of primary human hematopoietic stem/progenitor (CD34+) cells to emulate a natural mutation, which deletes the MCS-R2 α-globin enhancer and causes α-thalassemia. When edited CD34+ cells are differentiated into erythroid cells, we observe the expected reduction in α-globin expression and a correction of the pathologic globin chain imbalance in cells from patients with β-thalassemia. Xenograft assays show that a proportion of the edited CD34+ cells are long-term repopulating hematopoietic stem cells, demonstrating the potential of this approach for translation into a therapy for β-thalassemia.β-thalassemia is characterised by the presence of an excess of α-globin chains, which contribute to erythrocyte pathology. Here the authors use CRISP/Cas9 to reduce α-globin expression in hematopoietic precursors, and show effectiveness in xenograft assays in mice.
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CRISPR-Cas-mediated gene editing in lactic acid bacteria.
The high efficiency, convenience and diversity of clustered regular interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems are driving a technological revolution in the gene editing of lactic acid bacteria (LAB). Cas-RNA cassettes have been adopted as tools to perform gene deletion, insertion and point mutation in several species of LAB. In this article, we describe the basic mechanisms of the CRISPR-Cas system, and the current gene editing methods available, focusing on the CRISPR-Cas models developed for LAB. We also compare the different types of CRISPR-Cas-based genomic manipulations classified according to the different Cas proteins and the type of recombineering, and discuss the rapidly evolving landscape of CRISPR-Cas application in LAB.
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Creating a patient carried Men1 gene point mutation on wild type iPSCs locus mediated by CRISPR/Cas9 and ssODN.
A patient specific point mutation (c.1288G>T) of Men1 gene was introduced into wide type iPSC line with CRISPR/Cas9 and single-stranded donor oligonucleotides carrying the mutation. The mutated iPSC line has a heterozygous c.1288G>T mutation on exon-9 of Men1 that was confirmed by sequencing analysis. The karyotype of this line was normal and the pluripotency was demonstrated by its ability to differentiate into three germ layers. These artificially created Men1 mutation in wild type iPSC line will help to dissect out the molecular basis of two patients carried the same mutation from one family who were differentially represented hypoglycemia.
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Counterpoint: The Potential Harms of Human Gene Editing Using CRISPR-Cas9.
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CRISPR/Cas9-Based Gene Editing in Soybean.
CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR associated Cas9)-based gene editing is a robust tool for functional genomics research and breeding programs in various crops. In soybean, a number of laboratories have obtained mutants by CRISPR/Cas9 system; however, there has been not yet a detailed method for the CRISPR/Cas9-based gene editing in soybean. Here, we describe the procedures for constructing the CRISPR/Cas9 plasmid suitable for soybean gene editing and the modified protocols for Agrobacterium-mediated soybean transformation and regeneration from cotyledonary node explants containing the Cas9/sgRNA (single guide RNA) transgenes.
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CRISPR/Cpf1-Mediated Multiplex and Large-Fragment Gene Editing in Staphylococcus aureus.
Staphylococcus aureus is a major human pathogen that causes a variety of infections, including life-threatening diseases. Research on S. aureus is constrained by complex and limited genetic manipulation methods. Here, we report a CRISPR/Cpf1-mediated system, pCpfSA, for rapid and versatile genome editing in S. aureus. In direct comparison with the existing CRISPR/Cas9-mediated genome-editing system, the pCpfSA system exhibits enhanced colony-forming units (CFUs) after editing and an expanded targetable range with comparable editing efficiency. Given the precursor crRNA (pre-crRNA) processing activity of Cpf1, the pCpfSA system also allows multiplex gene editing and large-fragment DNA knockout simply by introducing two crRNAs and the corresponding donor templates, which is difficult to achieve using the CRISPR/Cas9 system, thereby greatly expanding the genome editor toolbox for S. aureus.
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Methods for detecting off-target effects of CRISPR/Cas9.
The CRISPR/Cas9 system has emerged as a revolutionary tool for gene editing, widely used in the biomedical field due to its simplicity, efficiency, and cost-effectiveness. However, evidence suggests that CRISPR/Cas9 can induce off-target effects, leading to unintended mutations that may compromise the precision of gene modifications. Consequently, predicting，detecting and evaluating these off-target effects is crucial for optimizing the accuracy and reliability of CRISPR/Cas9 system. This paper provides an overview of the various methodologies and strategies, used or to be used for identifying off-target effects in CRISPR/Cas9-based genome editing, offering insights to improve the precision and safety of CRISPR applications in research and therapeutics.
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[The Washington summit: orange light for genome editing?].
The summit organised in early December 2015 considered in depth the various issues (technical, scientific, societal and ethical) raised by the prospect of genome editing using the extremely effective CRISPR system. Germline editing (for therapeutic or "enhancement" purposes) was stated to be irresponsible under current conditions, but the possibility that this could be considered in the future was not excluded; a mechanism for monitoring progress and possibly revising recommendations was proposed.
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Kinetics of dCas9 target search in Escherichia coli.
How fast can a cell locate a specific chromosomal DNA sequence specified by a single-stranded oligonucleotide? To address this question, we investigate the intracellular search processes of the Cas9 protein, which can be programmed by a guide RNA to bind essentially any DNA sequence. This targeting flexibility requires Cas9 to unwind the DNA double helix to test for correct base pairing to the guide RNA. Here we study the search mechanisms of the catalytically inactive Cas9 (dCas9) in living Escherichia coli by combining single-molecule fluorescence microscopy and bulk restriction-protection assays. We find that it takes a single fluorescently labeled dCas9 6 hours to find the correct target sequence, which implies that each potential target is bound for less than 30 milliseconds. Once bound, dCas9 remains associated until replication. To achieve fast targeting, both Cas9 and its guide RNA have to be present at high concentrations.
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CRISPR plants now subject to tough GM laws in European Union.
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