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MEGA-CRISPR tool gives a power boost to cancer-fighting cells.
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Correction of β-thalassemia mutant by base editor in human embryos.
β-Thalassemia is a global health issue, caused by mutations in the HBB gene. Among these mutations, HBB -28 (A>G) mutations is one of the three most common mutations in China and Southeast Asia patients with β-thalassemia. Correcting this mutation in human embryos may prevent the disease being passed onto future generations and cure anemia. Here we report the first study using base editor (BE) system to correct disease mutant in human embryos. Firstly, we produced a 293T cell line with an exogenous HBB -28 (A>G) mutant fragment for gRNAs and targeting efficiency evaluation. Then we collected primary skin fibroblast cells from a β-thalassemia patient with HBB -28 (A>G) homozygous mutation. Data showed that base editor could precisely correct HBB -28 (A>G) mutation in the patient's primary cells. To model homozygous mutation disease embryos, we constructed nuclear transfer embryos by fusing the lymphocyte or skin fibroblast cells with enucleated in vitro matured (IVM) oocytes. Notably, the gene correction efficiency was over 23.0% in these embryos by base editor. Although these embryos were still mosaic, the percentage of repaired blastomeres was over 20.0%. In addition, we found that base editor variants, with narrowed deamination window, could promote G-to-A conversion at HBB -28 site precisely in human embryos. Collectively, this study demonstrated the feasibility of curing genetic disease in human somatic cells and embryos by base editor system.
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Prime Time for Genome Editing?
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Efficient genome editing in wheat using Cas9 and Cpf1 (AsCpf1 and LbCpf1) nucleases.
Genome editing can be used to create new wheat varieties with enhanced performance. Clustered regularly interspaced short palindromic repeat (CRISPR) is a powerful tool for knockout generation, precise modification, multiplex engineering, and the activation and repression of target genes. Targeted mutagenesis via RNA-guided genome editing using type II CRISPR-Cas9 is highly efficient in some plant species, but not in others. One possible solution is to use newly discovered variants of genome editing enzymes such as the class 2 system component Cpf1 (CRISPR from Prevotella and Francisella 1) in place of the more commonly used Cas9. We compared the editing efficiency of Cas9 and two Cpf1 orthologs, AsCpf1 (Acidaminococcus spp. BV3L6) and LbCpf1 (Lachnospiraceae bacterium ND2006) in wheat (Triticum aestivum). LbCpf1 had a higher editing efficiency for the target gene TaPDS than AsCpf1 and Cas9, and Cas9 induced more off-target mutations than AsCpf1 and LbCpf1, suggesting that CRISPR-LbCpf1 is a powerful genome editing tool for polyploid plants such as wheat.
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CRISPR/Cas9-mediated mutagenesis of phytoene desaturase in pigeonpea and groundnut.
The CRISPR/Cas9 technology, renowned for its ability to induce precise genetic alterations in various crop species, has encountered challenges in its application to grain legume crops such as pigeonpea and groundnut. Despite attempts at gene editing in groundnut, the low rates of transformation and editing have impeded its widespread adoption in producing genetically modified plants. This study seeks to establish an effective CRISPR/Cas9 system in pigeonpea and groundnut through Agrobacterium-mediated transformation, with a focus on targeting the phytoene desaturase (PDS) gene. The PDS gene is pivotal in carotenoid biosynthesis, and its disruption leads to albino phenotypes and dwarfism. Two constructs (one each for pigeonpea and groundnut) were developed for the PDS gene, and transformation was carried out using different explants (leaf petiolar tissue for pigeonpea and cotyledonary nodes for groundnut). By adjusting the composition of the growth media and refining Agrobacterium infection techniques, transformation efficiencies of 15.2% in pigeonpea and 20% in groundnut were achieved. Mutation in PDS resulted in albino phenotype, with editing efficiencies ranging from 4 to 6%. Sequence analysis uncovered a nucleotide deletion (A) in pigeonpea and an A insertion in groundnut, leading to a premature stop codon and, thereby, an albino phenotype. This research offers a significant foundation for the swift assessment and enhancement of CRISPR/Cas9-based genome editing technologies in legume crops.
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Next-generation CRISPR-Cas for genome editing: focusing on the Cas protein and PAM.
The emergence of the gene editing technology, especial CRISPR-Cas (clustered regularly intersected short palindromic repeats and CRISPR associated proteins), has greatly promoted the ability of human beings to transform natural species. It has been widely harnessed for the engineering in the medical, industrial, agricultural and other fields. The key component of the CRISPR-Cas system, the Cas protein, possesses its specific features, including self-activity, recognition site, cutting end and guide RNA. PAM (protein assistant motif) is a number of nucleotides adjacent to the target site, which is very important for the Cas protein to recognize the target sequence and is also the key characteristic of CRISPR-Cas. There are several reported methods for identification of PAM. In this review, we summarize the searching for the Cas protein, the identification of Cas mutants with desired traits and the mapping of the PAM (including the extending of PAM spectrum), in order to provide a reference for the development and optimization of next-generation gene editing tools.
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Applications of CRISPR/Cas gene-editing technology in yeast and fungi.
Genome editing technology has progressed rapidly in recent years. Although traditional gene-editing methods, including homologous recombination, zinc finger endonucleases, and transcription activator-like effector nucleases, have substantial implications for research in genetics and molecular biology, but they have remarkable limitations, including their low efficiency, high error rate, and complex design. A new gene-editing technology, the CRISPR/Cas system, was developed based on studies of archaeal and bacterial immune responses to viruses. Owing to its high target efficiency, simple primer design, and wide applications, the CRISPR/Cas system, whose developers were awarded the Nobel Prize in Chemistry in 2020, has become the dominant genomic editing technology in academia and the pharmaceutical industry. Here, we briefly introduce the CRISPR/Cas system and its main applications for genome engineering, metabolic engineering, and transcriptional regulation in yeast, filamentous fungi, and macrofungi. The polygene and polyploid editing, construction of yeast chromosomes, yeast library creation, regulation of metabolic pathways, and CRISPR activation/CRISPR interference systems are mainly summarized and discussed. The potential applications for the treatment of fungal infections and the further transformation and application of the CRISPR/Cas system in fungi are also proposed and discussed.
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The tsunami named CRISPR/Cas9.
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Erratic journey of CRISPR/Cas9 in oncology from bench-work to successful-clinical therapy.
CRISPR is a customized molecular scissor, comprising genetic guide made of RNA and an enzyme, Cas9 which snips DNA in simpler, cheaper and more precise way than any other gene editing tools. In recent years CRISPR/Cas has taken the research world by storm being go-to genome editor for potential gene therapy to fix cancer as well as several hereditary disorders. This review explores the literature around the mechanism of Nobel winning CRISPR/Cas9 and its journey from its discovery to various pre-clinical and clinical trials in oncology, focusing mostly on PD-1 knockout CAR-T cell therapy. It also discusses the hurdles and ethical dispute associated with CRISPR, such as unintended on-target and off-target cuts, embryonic germ-line editing. Despite the controversies regarding the safety of this technique, many studies reported promising results on targeting cancer and other diseases using CRISPR/Cas9. Outcomes from the first successful clinical trial showed the beneficial long term effect on genetically modified T-cells in targeting cancer cells which opens the door for CRISPR to be the most preferred technique to help treating cancer and other diseases in future. As far as germ-line editing is concerned, further studies are needed to support the safety of this technique in humans fixing genetic disorders and mutations. Therefore till date only somatic cell editing is ethically approved.
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Heritable Human Genome Editing: The International Commission Report.
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