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Improvement of wheat drought tolerance through editing of TaATX4 by CRISPR/Cas9.
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CRISPR/Cas9: a historical and chemical biology perspective of targeted genome engineering.
The CRISPR-Cas9 system has revolutionized the process of making changes to the DNA sequence of organisms. Relying on a simplistic model of RNA guided DNA binding and cleavage, this molecular toolbox has found application in nearly every branch of biological sciences. The story of CRISPR-Cas9 is one of discovery and development where a component of bacterial adaptive immunity has been harnessed to address important biological questions using significant inputs from physicochemical structure-function studies. In this review, we trace the evolution of CRISPR-Cas9 from its predecessor genome editing tools and document its current status with an emphasis on chemical biology aspects of modulating its activity to generate a potent tool for gene therapy applications.
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Large-scale genome editing based on high-capacity adenovectors and CRISPR-Cas9 nucleases rescues full-length dystrophin synthesis in DMD muscle cells.
Targeted chromosomal insertion of large genetic payloads in human cells leverages and broadens synthetic biology and genetic therapy efforts. Yet, obtaining large-scale gene knock-ins remains particularly challenging especially in hard-to-transfect stem and progenitor cells. Here, fully viral gene-deleted adenovector particles (AdVPs) are investigated as sources of optimized high-specificity CRISPR-Cas9 nucleases and donor DNA constructs tailored for targeted insertion of full-length dystrophin expression units (up to 14.8-kb) through homologous recombination (HR) or homology-mediated end joining (HMEJ). In muscle progenitor cells, donors prone to HMEJ yielded higher CRISPR-Cas9-dependent genome editing frequencies than HR donors, with values ranging between 6% and 34%. In contrast, AdVP transduction of HR and HMEJ substrates in induced pluripotent stem cells (iPSCs) resulted in similar CRISPR-Cas9-dependent genome editing levels. Notably, when compared to regular iPSCs, in p53 knockdown iPSCs, CRISPR-Cas9-dependent genome editing frequencies increased up to 6.7-fold specifically when transducing HMEJ donor constructs. Finally, single DNA molecule analysis by molecular combing confirmed that AdVP-based genome editing achieves long-term complementation of DMD-causing mutations through the site-specific insertion of full-length dystrophin expression units. In conclusion, AdVPs are a robust and flexible platform for installing large genomic edits in human cells and p53 inhibition fosters HMEJ-based genome editing in iPSCs.
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No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice.
CRISPR-Cas9 technologies have transformed genome-editing of experimental organisms and have immense therapeutic potential. Despite significant advances in our understanding of the CRISPR-Cas9 system, concerns remain over the potential for off-target effects. Recent studies have addressed these concerns using whole-genome sequencing (WGS) of gene-edited embryos or animals to search for de novo mutations (DNMs), which may represent candidate changes introduced by poor editing fidelity. Critically, these studies used strain-matched, but not pedigree-matched controls and thus were unable to reliably distinguish generational or colony-related differences from true DNMs. Here we used a trio design and whole genome sequenced 8 parents and 19 embryos, where 10 of the embryos were mutagenised with well-characterised gRNAs targeting the coat colour Tyrosinase (Tyr) locus. Detailed analyses of these whole genome data allowed us to conclude that if CRISPR mutagenesis were causing SNV or indel off-target mutations in treated embryos, then the number of these mutations is not statistically distinguishable from the background rate of DNMs occurring due to other processes.
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First-generation genome editing in potato using hairy root transformation.
Genome editing and cis-gene breeding have rapidly accelerated crop improvement efforts, but their impacts are limited by the number of species capable of being genetically transformed. Many dicot species, including some vital potato relatives being used to accelerate breeding and genetics efforts, remain recalcitrant to standard Agrobacterium tumefaciens-based transformation. Hairy root transformation using Agrobacterium rhizogenes (A. rhizogenes) provides an accelerated approach to generating transgenic material but has been limited to analysis of hairy root clones. In this study, strains of A. rhizogenes were tested in the wild diploid potato relative Solanum chacoense, which is recalcitrant to infection by Agrobacterium tumefaciens. One strain of A. rhizogenes MSU440 emerged as being capable of delivering a T-DNA carrying the GUS marker and generating transgenic hairy root clones capable of GUS expression and regeneration to whole plants. CRISPR/Cas9 reagents targeting the potato PHYTOENE DESATURASE (StPDS) gene were expressed in hairy root clones and regenerated. We found that 64%-98% of transgenic hairy root clones expressing CRISPR/Cas9 reagents carried targeted mutations, while only 14%-30% of mutations were chimeric. The mutations were maintained in regenerated lines as stable mutations at rates averaging at 38% and were capable of germ-line transmission to progeny. This novel approach broadens the numbers of genotypes amenable to Agrobacterium-mediated transformation while reducing chimerism in primary events and accelerating the generation of edited materials.
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[Advances in genome editing technologies in Komagataella phaffii and their applications in biomanufacturing].
Komagataella phaffii is widely recognized as a premier host for the production of recombinant proteins and value-added metabolites, owing to its low background secretion of endogenous proteins, strong capacity for heterologous protein secretion, and robust growth and metabolic performance under industrially relevant fermentation conditions. In recent years, rapid progress in genome editing technologies and synthetic biology toolkits has markedly improved the precision and efficiency of gene function interrogation, metabolic pathway reconstruction, and dynamic regulation in K. phaffii, thereby continuously strengthening its performance as a microbial cell factory. Consequently, beyond its established roles in producing recombinant proteins, industrial enzymes, and vaccine antigens, K. phaffii has also demonstrated substantial potential for the biosynthesis of natural products, biopharmaceutical molecules, and emerging biomaterials. This review systematically summarizes the evolution of genome engineering technologies in K. phaffii, spanning the transition from conventional recombination-based methods to next-generation precision editing tools. We highlight recent advances, optimization strategies, and engineering practices of CRISPR/Cas and related systems in this host. Moreover, in light of emerging research trends, we discuss key challenges and opportunities associated with improving editing efficiency, enabling rapid assembly of complex metabolic pathways, and accelerating industrial translation, thereby providing a reference for the rational engineering of Komagataella phaffii and its broader applications in synthetic biology and biomanufacturing.
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Modifications of fatty acid profile through targeted mutation at BnaFAD2 gene with CRISPR/Cas9-mediated gene editing in Brassica napus.
Genomic editing with CRISPR/Cas9 system can simultaneously modify multiple copies of theBnaFAD2 gene to develop novel variations in fatty acids profiles in polyploidy rapeseed. Fatty acid composition affects edible and processing quality of vegetable oil and has been one of the primary targets for genetic modification in oilseed crops including rapeseed (Brassica napus). Fatty acid desaturase 2 gene, FAD2, is a key player that affects three major fatty acids, namely oleic, linoleic and linolenic acid, in oilseed plants. Previously, we showed that there are four copies of BnaFAD2 in allotetraploid rapeseed. In this study, we further established spatiotemporal expression pattern of each copy of BnaFAD2 using published RNA-seq data. Genomic editing technology based on CRISPR/Cas9 system was used to mutate all the copies of BnaFAD2 to create novel allelic variations in oleic acid and other fatty acid levels. A number of mutants at two targeting sites were identified, and the phenotypic variation in the mutants was systematically evaluated. The oleic acid content in the seed of the mutants increased significantly with the highest exceeding 80% compared with wild type of 66.43%, while linoleic and linolenic acid contents decreased accordingly. Mutations on BnaFAD2.A5 caused more dramatic changes of fatty acid profile than the mutations on BnaFAD2.C5 alleles that were identified with gene editing technique for the first time. Moreover, combining different mutated alleles of BnaFAD2 can even broaden the variation more dramatically. It was found that effects of different mutation types at BnaFAD2 alleles on oleic levels varied, indicating a possibility to manipulate fatty acid levels by precise mutation at specific region of a gene.
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CRISPR-Cas9: From a bacterial immune system to genome-edited human cells in clinical trials.
The adaptive bacterial immune system CRISPR-Cas is revolutionizing all fields of life science and has opened up new frontiers toward personalised medicine. Since the elucidation of the molecular mechanism of Cas9 from Streptococcus pyogenes in 2012 and its development as a genomic engineering tool, genetic modifications in more than 40 species have been performed, over 290 patents have been filed worldwide and the first clinical trials using CRISPR-Cas-modified T-cells have recently been started in China and in the US. In this review we summarise current design developments, novel Cas systems and their antagonists, present and potential future applications as well as the ongoing debate on ethical issues, which has arisen through the CRISPR-Cas technology.
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Establishment of a CPAMD8-GFP reporter human embryonic stem cell line, IBBDe001-B, using CRISPR/Cas9 editing.
The human CPAMD8 gene encodes proteins in the A2M/C3 (alpha-2-macroglobulin/complement 3) family, predominantly expressed in the distal tips of the retinal neuroepithelium that form the iris and ciliary body. Mutations in CPAMD8 have been linked to anterior segment dysplasia and congenital glaucoma. Using CRISPR/Cas9 editing, we inserted a 3*EAAAK-EGFP fluorescent tag into the CPAMD8 gene, enabling real-time observation of its expression and providing insights into its biological functions. The resulting gene-edited cell line retained normal stem cell morphology and karyotype, expressed essential pluripotency markers, and exhibited differentiation potential.
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Generation of a FTO gene knockout human embryonic stem cell line using CRISPR/Cas9 editing.
Fat mass and obesity-associated protein (FTO) is the first protein found to have the activity of N6-methyladenosine (m6A) demethylation. It has been reported that FTO was involved in different physiological and pathological processes, including stem cell differentiation, sex determination, tumorigenesis, and progression. To further understand the exact role of FTO in these processes, we generated a FTO knockout human embryonic stem cell (hESC) line by CRISPR/Cas9 mediated gene editing method. This cell line maintained normal karyotype, pluripotency, and trilineage differentiation potential, which are considered as a model for function studies of the FTO protein in hESC self-renewal and differentiation.
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