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CRISPR-Cas9n-mediated ELANE promoter editing for gene therapy of severe congenital neutropenia.
Severe congenital neutropenia (CN) is an inherited pre-leukemia bone marrow failure syndrome commonly caused by autosomal-dominant ELANE mutations (ELANE-CN). ELANE-CN patients are treated with daily injections of recombinant human granulocyte colony-stimulating factor (rhG-CSF). However, some patients do not respond to rhG-CSF, and approximately 15% of ELANE-CN patients develop myelodysplasia or acute myeloid leukemia. Here, we report the development of a curative therapy for ELANE-CN through inhibition of ELANE mRNA expression by introducing two single-strand DNA breaks at the opposing DNA strands of the ELANE promoter TATA box using CRISPR-Cas9D10A nickases-termed MILESTONE. This editing effectively restored defective neutrophil differentiation of ELANE-CN CD34+ hematopoietic stem and progenitor cells (HSPCs) in vitro and in vivo, without affecting the functions of the edited neutrophils. CRISPResso analysis of the edited ELANE-CN CD34+ HSPCs revealed on-target efficiencies of over 90%. Simultaneously, GUIDE-seq, CAST-Seq, and rhAmpSeq indicated a safe off-target profile with no off-target sites or chromosomal translocations. Taken together, ex vivo gene editing of ELANE-CN HSPCs using MILESTONE in the setting of autologous stem cell transplantation could be a universal, safe, and efficient gene therapy approach for ELANE-CN patients.
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An introduction and use of the CRISPR-Cas systems.
Clusters of regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins (Cas) system (CRISPR-Cas) is a rapidly evolving field of targeted genome engineering. The type II CRISPR-Cas9 is used for genome editing of many organisms. Single guide RNA (sgRNA) can bind to Cas9 protein that can target desired sequences in presence of protospacer adjacent motif (PAM) sequences. This complex binds and generate a DSB that is repaired by NHEJ or HDR pathways, subsequently gene insertion/deletion (Indels) is generated that leads to change in the organism's genotype followed by its phenotype. In this chapter, CRISPR-mediated targeted genome editing in different lower organisms has been highlighted to promote its basic understanding to be applied for biotechnological, biomedical and therapeutic applications.
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Protocol for efficient CRISPR-Cas9-mediated fluorescent tag knockin in hard-to-transfect erythroid cell lines.
Precise insertion of fluorescent tags by CRISPR-Cas9-mediated homologous recombination (HR) in mammalian genes is a powerful tool allowing to study gene function and protein gene products. Here, we present a protocol for efficient HR-mediated targeted insertion of fluorescent markers in the genome of hard-to-transfect erythroid cell lines MEL (mouse erythroleukemic) and MEDEP (mouse ES cell-derived erythroid progenitor line). We describe steps for plasmid construction, electroporation, amplification, and verification of genome editing. We then detail procedures for isolating positive clones and validating knockin clones. For complete details on the use and execution of this protocol, please refer to Deleuze et al.1.
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Amelioration of Alpha-1 Antitrypsin Deficiency Diseases with Genome Editing in Transgenic Mice.
Alpha-1 antitrypsin deficiency (AATD) is a hereditary liver disease caused by mutations in the SERPINA1 serine protease inhibitor gene. Most severe patients are homozygous for PiZ alleles (PiZZ; amino acid E324K), which lead to protein aggregates in hepatocytes and reduced circulating levels of AAT. The liver aggregates typically lead to fibrosis, cirrhosis, and hepatocellular carcinoma, and the reduced circulating AAT levels can lead to emphysema and chronic obstructive pulmonary diseases. In this study, two CRISPR/Cas9 gene editing approaches were used to decrease liver aggregates and increase systemic AAT-M levels in the PiZ transgenic mouse. In the first approach, AAT expression in hepatocytes was reduced more than 98% following the systemic delivery of AAV8-CRISPR targeting exon 2 of hSERPINA1, leading to reduced aggregates in hepatocytes. In the second approach, a second adeno-associated virus, which provided the donor template to correct the Z mutation, was also administered. These treated mice had reduced AAT expression (> 98%) and a low level (5%) of wildtype AAT-M mRNA. Taken together, this study shows that CRISPR gene editing can efficiently reduce liver expression of AAT-Z and restore modest levels of wildtype AAT-M in a mouse model of AATD, raising the possibility of CRISPR gene editing therapeutic for AATD.
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RNA-guided genome engineering: paradigm shift towards transposons.
CRISPR-Cas systems revolutionized the genome engineering field but need to induce double-strand breaks (DSBs) and may be difficult to deliver due to their large protein size. Tn7-like transposons such as CRISPR-associated transposons (CASTs) can be repurposed for RNA-guided DSB-free integration, and obligate mobile element guided activity (OMEGA) proteins of the IS200/IS605 transposon family have been developed as hypercompact RNA-guided genome editing tools. CASTs and OMEGA are exciting, innovative genome engineering tools that can improve the precision and efficiency of editing. This review explores the recent developments and uses of CASTs and OMEGA in genome editing across prokaryotic and eukaryotic cells. The pros and cons of these transposon-based systems are deliberated in comparison to other CRISPR systems.
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CRISPR-Cas9 mediated gene deletions in lager yeast Saccharomyces pastorianus.
BACKGROUND: The ease of use of CRISPR-Cas9 reprogramming, its high efficacy, and its multiplexing capabilities have brought this technology at the forefront of genome editing techniques. Saccharomyces pastorianus is an aneuploid interspecific hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus that has been domesticated for centuries and is used for the industrial fermentation of lager beer. For yet uncharacterised reasons, this hybrid yeast is far more resilient to genetic alteration than its ancestor S. cerevisiae. RESULTS: This study reports a new CRISPR-Cas9 method for accurate gene deletion in S. pastorianus. This method combined the Streptococcus pyogenes cas9 gene expressed from either a chromosomal locus or from a mobile genetic element in combination with a plasmid-borne gRNA expression cassette. While the well-established gRNA expression system using the RNA polymerase III dependent SNR52 promoter failed, expression of a gRNA flanked with Hammerhead and Hepatitis Delta Virus ribozymes using the RNA polymerase II dependent TDH3 promoter successfully led to accurate deletion of all four alleles of the SeILV6 gene in strain CBS1483. Furthermore the expression of two ribozyme-flanked gRNAs separated by a 10-bp linker in a polycistronic array successfully led to the simultaneous deletion of SeATF1 and SeATF2, genes located on two separate chromosomes. The expression of this array resulted in the precise deletion of all five and four alleles mediated by homologous recombination in the strains CBS1483 and Weihenstephan 34/70 respectively, demonstrating the multiplexing abilities of this gRNA expression design. CONCLUSIONS: These results firmly established that CRISPR-Cas9 significantly facilitates and accelerates genome editing in S. pastorianus.
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Optimizing genome editing efficiency in Streptomyces fradiae via a CRISPR/Cas9n-mediated editing system.
Streptomyces fradiae is an important bioresource to produce various antibacterial natural products, however, the time-consuming and labor-intensive genome editing toolkits hindered the construction and application of engineered strains, and this study aimed to establish an efficient CRISPR/Cas9n genome editing system in S. fradiae. Initially, the CRISPR/Cas9-mediated editing tool was employed to replace those awkward genome editing tools that relied on homologous recombination, while the off-target Cas9 exhibited high toxicity to S. fradiae Sf01. Therefore, the nickase mutation D10A, high-fidelity mutations including N497A, R661A, Q695A, and Q926A, and thiostrepton-induced promotor PtipA were incorporated into the Cas9 expression cassette, which reduced its toxicity. The deletion of single gene neoI and long fragment sequence (13.3 kb) were achieved with efficiencies of 77.8% and 44%, respectively. Additionally, the established tool was applied to facilitate the rapid deletion of nagB, replacement of Pfrr with PermE*, and integration of exogenous vgbS, with respective efficiencies of 77.8%, 100%, and 67.8%, and all of the above modification strategies benefited neomycin synthesis in S. fradiae. Taken together, this research established an efficient CRISPR/Cas9n-mediated genome editing toolkit in S. fradiae, paving the way for developing high-performance neomycin-producing strains and facilitating the genetic modification of Streptomyces.IMPORTANCEThis study describes the development and application of a genome editing system mediated by CRISPR/Cas9n in Streptomyces fradiae for the first time, which overcomes the challenges associated with genome editing caused by high GC content (74.5%) coupling with complex genome structure, and reduces the negative impact of "off-target effect." Our work not only provides a facile editing tool for constructing S. fradiae strains of high-yield neomycin but also offers the technical guidance for the design of a CRISPR/Cas9n mediated genome editing tool in those creatures with high GC content genomes.
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Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing.
Herpesvirus of turkeys (HVT) is an ideal viral vector for the generation of recombinant vaccines against a number of avian diseases, such as avian influenza (AI), Newcastle disease (ND), and infectious bursal disease (IBD), using bacterial artificial chromosome (BAC) mutagenesis or conventional recombination methods. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system has been successfully used in many settings for gene editing, including the manipulation of several large DNA virus genomes. We have developed a rapid and efficient CRISPR/Cas9-mediated genome editing pipeline to generate recombinant HVT. To maximize the potential use of this method, we present here detailed information about the methodology of generating recombinant HVT expressing the VP2 protein of IBDV. The VP2 expression cassette is inserted into the HVT genome via an NHEJ (nonhomologous end-joining)-dependent repair pathway. A green fluorescence protein (GFP) expression cassette is first attached to the insert for easy visualization and then removed via the Cre-LoxP system. This approach offers an efficient way to introduce other viral antigens into the HVT genome for the rapid development of recombinant vaccines.
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Introducing precise genetic modifications into human 3PN embryos by CRISPR/Cas-mediated genome editing.
PURPOSE: As a powerful technology for genome engineering, the CRISPR/Cas system has been successfully applied to modify the genomes of various species. The purpose of this study was to evaluate the technology and establish principles for the introduction of precise genetic modifications in early human embryos. METHODS: 3PN zygotes were injected with Cas9 messenger RNA (mRNA) (100 ng/μl) and guide RNA (gRNA) (50 ng/μl). For oligo-injections, donor oligo-1 (99 bp) or oligo-2 (99 bp) (100 ng/μl) or dsDonor (1 kb) was mixed with Cas9 mRNA (100 ng/μl) and gRNA (50 ng/μl) and injected into the embryos. RESULTS: By co-injecting Cas9 mRNA, gRNAs, and donor DNA, we successfully introduced the naturally occurring CCR5Δ32 allele into early human 3PN embryos. In the embryos containing the engineered CCR5Δ32 allele, however, the other alleles at the same locus could not be fully controlled because they either remained wild type or contained indel mutations. CONCLUSIONS: This work has implications for the development of therapeutic treatments of genetic disorders, and it demonstrates that significant technical issues remain to be addressed. We advocate preventing any application of genome editing on the human germline until after a rigorous and thorough evaluation and discussion are undertaken by the global research and ethics communities.
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Multiplexed orthogonal genome editing and transcriptional activation by Cas12a.
CRISPR-Cas9-based combinatorial perturbation approaches for orthogonal knockout and gene activation have been impeded by complex vector designs and co-delivery of multiple constructs. Here, we demonstrate that catalytically active CRISPR-Cas12a fused to a transcriptional-activator domain enables flexible switching between genome editing and transcriptional activation by altering guide length. By leveraging Cas12a-mediated CRISPR-RNA array processing, we illustrate that Cas12a-VPR enables simplified multiplexed knockout and transcriptional activation in vitro and in vivo.
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