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Integrate CRISPR/Cas9 for protein expression of HLA-B*38:68Q via precise gene editing.
The determination of null- or low-expressed HLA alleles is clinically relevant in both hematopoietic stem cell transplantation and solid organ transplantation. We studied the expression level of a questionable (Q) HLA-B*38:68Q allele, which carries a 9-nucleotide (nt) deletion at codon 230-232 in exon 4 of HLA-B*38:01:01:01 using CRISPR/Cas9 gene editing technology. CRISPR/Cas9 gene editing of HLA-B*38:01:01:01 homozygous EBV B cell line resulted in one HLA-B*38:68Q/B*38:01:01:01 heterozygous and one HLA-B*38:68Q homozygous clone. Flow cytometric analysis of monoclonal anti-Bw4 antibody showed the protein expression of HLA-B*38:01:01:01 in homozygous cells was 2.2 fold higher than HLA-B*38:68Q/B*38:01:01:01 heterozygous cells, and the expression of HLA-B*38:68Q/B*38:01:01:01 heterozygous cells was over 2.0 fold higher than HLA-B*38:68Q homozygous cells. The HLA-B*38:68Q expression was further confirmed using anti-B38 polyclonal antibody. Similarly, the expression of the HLA-B*38:01:01:01 homozygous cells was 1.5 fold higher than that of HLA-B*38:68Q/B*38:01:01:01 heterozygous cells, and the HLA-B*38:68Q/B*38:01:01:01 heterozygous cells was over 1.6 fold higher than that of HLA-B*38:68Q homozygous cells. The treatment of HLA-B*38:68Q homozygous cells with IFN-γ significantly increased its expression. In conclusion, we demonstrate that HLA-B*38:68Q is a low-expressing HLA allele. The CRISPR/Cas9 technology is a useful tool to induce precise gene editing in HLA genes to enable the characterization of HLA gene variants on expression and function.
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Transient cotransformation of CRISPR/Cas9 and oligonucleotide templates enables efficient editing of target loci in Physcomitrella patens.
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A New RNA-Dependent Cas12g Nuclease.
The development of RNA-targeting CRISPR-Cas systems represents a major step forward in the field of gene editing and regulation. RNA editing presents a viable alternative to genome editing in certain scenarios as it offers a reversible and manageable approach, reducing the likelihood of runaway mutant variants. One of the most promising applications is in the treatment of genetic disorders caused by mutations in RNA molecules. In this study, we investigate a previously undescribed Cas12g nuclease which was found in metagenomes from promising thermophilic microbial communities during the expedition to the Republic of North Ossetia-Alania in 2020. The method outlined in this study can be applied to other Cas orthologs and variants, leading to a better understanding of the CRISPR-Cas system and its enzymatic activities. The cis-cleavage activity of the new type V-G Cas effector was indicated by in vitro RNA cleavage experiments. While CRISPR-Cas systems are known for their high specificity, there is still a risk of unintended cleavage of nontargeted RNA molecules. Ultimately, the search for new genome editing tools and the study of their properties will remove barriers to research in this area. With continued research and development, we may be able to unlock their full potential.
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Social Inequality and Human Genome Editing: A Nuanced Analysis of the Ubuntuan Ethical Prism.
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Toward Responsible Human Genome Editing.
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Genome Editing, Transcriptional Regulation, and Forward Genetic Screening Using CRISPR-Cas12a Systems in Yarrowia lipolytica.
Class II Type V endonucleases have increasingly been adapted to develop sophisticated and easily accessible synthetic biology tools for genome editing, transcriptional regulation, and functional genomic screening in a wide range of organisms. One such endonuclease, Cas12a, presents itself as an attractive alternative to Cas9-based systems. The ability to mature its own guide RNAs (gRNAs) from a single transcript has been leveraged for easy multiplexing, and its lack of requirement of a tracrRNA element, also allows for short gRNA expression cassettes. To extend these functionalities into the industrially relevant oleaginous yeast Yarrowia lipolytica, we developed a set of CRISPR-Cas12a vectors for easy multiplexed gene knockout, repression, and activation. We further extended the utility of this CRISPR-Cas12a system to functional genomic screening by constructing a genome-wide guide library targeting every gene with an eightfold coverage. Pooled CRISPR screens conducted with this library were used to profile Cas12a guide activities and develop a machine learning algorithm that could accurately predict highly efficient Cas12a gRNA. In this protocols chapter, we first present a method by which protein coding genes may be functionally disrupted via indel formation with CRISPR-Cas12a systems. Further, we describe how Cas12a fused to a transcriptional regulator can be used in conjunction with shortened gRNA to achieve transcriptional repression or activation. Finally, we describe the design, cloning, and validation of a genome-wide library as well as a protocol for the execution of a pooled CRISPR screen, to determine guide activity profiles in a genome-wide context in Y. lipolytica. The tools and strategies discussed here expand the list of available synthetic biology tools for facile genome engineering in this industrially important host.
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CRISPR-Cas9-Mediated Genome Editing in Paenibacillus polymyxa.
In recent years, the clustered regularly interspaced palindromic repeats-Cas (CRISPR-Cas) technology has become the method of choice for precision genome editing in many organisms due to its simplicity and efficacy. Multiplex genome editing, point mutations, and large genomic modifications are attractive features of the CRISPR-Cas9 system. These applications facilitate both the ease and velocity of genetic manipulations and the discovery of novel functions. In this protocol chapter, we describe the use of a CRISPR-Cas9 system for multiplex integration and deletion modifications, and deletions of large genomic regions by the use of a single guide RNA (sgRNA), and, finally, targeted point mutation modifications in Paenibacillus polymyxa.
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CRISPR/Cas9-Mediated Genome Editing in Zebrafish.
The CRISPR/Cas9 system is a powerful tool for genome editing in zebrafish. This workflow takes advantage of the genetic tractability of zebrafish and will allow users to edit genomic sites and produce mutant lines using selective breeding. Established lines may then be employed by researchers for downstream genetic and phenotypic analyses.
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Tweaking genes with CRISPR or viruses fixes blood disorders.
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Cloning-Free Genome Editing by CRISPR/T7RNAP/Cas9 in Trypanosoma cruzi.
The genetic manipulation of the human parasite Trypanosoma cruzi has been significantly improved since the implementation of the CRISPR/Cas9 technology for genome editing in this organism. Initially, the system was successfully used for gene knockout and endogenous C-terminal tagging in T. cruzi. Recently, an updated version of this technology has been used for gene complementation, site-directed mutagenesis, and N-terminal tagging in trypanosomatids. This cloning-free strategy, called CRISPR/T7RNAP/Cas9, is extremely useful for identifying essential genes when null mutants are not viable. Mutant cell lines obtained by this new system have been used for the functional characterization of proteins in different developmental stages of this parasite's life cycle, including infective trypomastigotes and intracellular amastigotes. In this chapter, we describe the methodology to achieve genome editing by CRISPR/T7RNAP/Cas9 in T. cruzi. Our method involves the generation of T. cruzi epimastigotes that constitutively express the T7 RNA polymerase (T7RNAP) and SpCas9, and their co-transfection with an sgRNA template and donor DNA(s) as polymerase chain reaction (PCR) products. Using this strategy, we have generated genetically modified parasites in 2-3 weeks without the need for gene cloning, cell sorting, or having to perform several transfection attempts to verify the sgRNA efficiency for targeting the gene of interest. The methodology has been organized according to three main genetic purposes: gene knockout, gene complementation of knockout cell lines, and endogenous (N- or C-terminal) tagging in T. cruzi.
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