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Found: 26 147
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Identification of pathogenic variants in cancer genes using base editing screens with editing efficiency correction.
BACKGROUND: Millions of nucleotide variants are identified through cancer genome sequencing and it is clinically important to identify the pathogenic variants among them. By introducing base substitutions at guide RNA target regions in the genome, CRISPR-Cas9-based base editors provide the possibility for evaluating a large number of variants in their genomic context. However, the variability in editing efficiency and the complexity of outcome mapping are two existing problems for assigning guide RNA effects to variants in base editing screens. RESULTS: To improve the identification of pathogenic variants, we develop a framework to combine base editing screens with sgRNA efficiency and outcome mapping. We apply the method to evaluate more than 9000 variants across all the exons of BRCA1 and BRCA2 genes. Our efficiency-corrected scoring model identifies 910 loss-of-function variants for BRCA1/2, including 151 variants in the noncoding part of the genes such as the 5' untranslated regions. Many of them are identified in cancer patients and are reported as "benign/likely benign" or "variants of uncertain significance" by clinicians. Our data suggest a need to re-evaluate their clinical significance, which may be helpful for risk assessment and treatment of breast and ovarian cancer. CONCLUSIONS: Our results suggest that base editing screens with efficiency correction is a powerful strategy to identify pathogenic variants in a high-throughput manner. Applying this strategy to assess variants in both coding and noncoding regions of the genome could have a direct impact on the interpretation of cancer variants.
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PhieABEs: a PAM-less/free high-efficiency adenine base editor toolbox with wide target scope in plants.
Adenine base editors (ABEs), which are generally engineered adenosine deaminases and Cas variants, introduce site-specific A-to-G mutations for agronomic trait improvement. However, notably varying editing efficiencies, restrictive requirements for protospacer-adjacent motifs (PAMs) and a narrow editing window greatly limit their application. Here, we developed a robust high-efficiency ABE (PhieABE) toolbox for plants by fusing an evolved, highly active form of the adenosine deaminase TadA8e and a single-stranded DNA-binding domain (DBD), based on PAM-less/free Streptococcus pyogenes Cas9 (SpCas9) nickase variants that recognize the PAM NGN (for SpCas9n-NG and SpGn) or NNN (for SpRYn). By targeting 29 representative targets in rice and assessing the results, we demonstrate that PhieABEs have significantly improved base-editing activity, expanded target range and broader editing windows compared to the ABE7.10 and general ABE8e systems. Among these PhieABEs, hyper ABE8e-DBD-SpRYn (hyABE8e-SpRY) showed nearly 100% editing efficiency at some tested sites, with a high proportion of homozygous base substitutions in the editing windows and no single guide RNA (sgRNA)-dependent off-target changes. The original sgRNA was more compatible with PhieABEs than the evolved sgRNA. In conclusion, the DBD fusion effectively promotes base-editing efficiency, and this novel PhieABE toolbox should have wide applications in plant functional genomics and crop improvement.
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Functional correction and genome integrity with duplex base editing of β-thalassemic hematopoietic stem cells.
BACKGROUND: Beta-thalassemia is among the most common monogenic disorders, posing a major global health challenge. Editing of genetic modifiers, such as BCL11A erythroid enhancer and HBG promoters, enhances fetal hemoglobin expression and confers major therapeutic potential. Double-strand-break (DSB)-independent genome editing tools, such as base editors (BE), are potentially safer and better suited for multiplexed application than DSB-dependent CRISPR/Cas technology. However, harmful on- and off-target events remain a concern and must be excluded before clinical application, including chromosomal rearrangements invisible to standard detection technologies. RESULTS: Using primary patient-derived CD34+ cells from three donors, we investigate simplex and duplex BE-based disruption of the BCL11A erythroid enhancer and the BCL11A binding site (-115 bp) on the HBG promoter for DNA-level and functional studies at the RNA, protein, and morphological level. Analyses include direct comparison to DSB-based editing, the current clinically applied standard, and CAST-seq to assess recombination events, allowing wider inferences on relative safety. RNA-seq analyses for clones of primary CD34+ cells across all treatments confirm peak HBG induction for duplex BE and comparable effects on apoptotic and immune response signatures. Overall, duplex BE produces robust γ-globin and fetal hemoglobin induction, improves functional correction over simplex editing and results in low incidence of genomic alterations in both target loci. CONCLUSIONS: Duplex BE targeting both BCL11A erythroid enhancer and HBG promoter enables functional correction and genome integrity. Our study highlights the efficacy, safety, and therapeutic potential of the present duplex BE approach.
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Prime Editing for Precision Genetic Medicine: A Systematic Review of Technologies, Delivery, and Therapeutic Applications.
BACKGROUND: Prime editing has rapidly evolved from a CRISPR-based "search-and-replace" approach for precise sequence modification into a diverse family of genome editing technologies. This systematic review maps the technological evolution of prime editing, with emphasis on editor architecture, guide RNA engineering, delivery, therapeutic applications, computational approaches, and emerging capabilities. METHODS: PubMed and Web of Science were systematically searched for studies in which prime editing constituted a substantive experimental, technological, computational, delivery, or therapeutic component. After deduplication and screening, candidate studies underwent manual re-screening against prespecified eligibility criteria. Reviews, corrections, plant and bacterial studies, conventional CRISPR or base editing studies without a substantive prime editing component, and other non-relevant records were excluded. A total of 294 studies were included in the final systematic evidence synthesis. Because of substantial heterogeneity in editor architectures, targets, experimental models, outcomes, and reporting, the literature was synthesized using systematic mapping and qualitative thematic analysis rather than meta-analysis. RESULTS: The evidence demonstrates rapid diversification from the original Cas9 nickase-reverse transcriptase-prime editing guide RNA architecture through improvements in pegRNA design, Cas and reverse transcriptase engineering, DNA repair modulation, delivery, computational design, and increasingly complex sequence modification. Therapeutic studies span disease modeling, correction of pathogenic variants, ex vivo applications, and direct in vivo editing; however, high editing efficiency does not necessarily translate into functional or therapeutic rescue. Large-sequence insertion and replacement strategies further extend the capabilities of prime editing, although these approaches remain less mature than small-sequence correction and face substantial challenges in efficiency, fidelity, cargo delivery, and genomic safety. CONCLUSIONS: Prime editing has developed into a versatile precision genome editing platform, but the evidence base remains heterogeneous and predominantly preclinical. Translation to genetic medicine will require improvements in reproducibility across targets and cell types, delivery to clinically relevant tissues, product purity, genomic safety, and demonstration of meaningful functional benefit. Emerging large-sequence editing approaches broaden the potential scope of prime editing but should be distinguished from technologies with established experimental and therapeutic evidence.
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In vivo photoreceptor base editing ameliorates rhodopsin-E150K autosomal-recessive retinitis pigmentosa in mice.
Rhodopsin, the prototypical class-A G-protein coupled receptor, is a highly sensitive receptor for light that enables phototransduction in rod photoreceptors. Rhodopsin plays not only a sensory role but also a structural role as a major component of the rod outer segment disc, comprising over 90% of the protein content of the disc membrane. Mutations in RHO which lead to structural or functional abnormalities, including the autosomal recessive E150K mutation, result in rod dysfunction and death. Therefore, correction of deleterious rhodopsin mutations could rescue inherited retinal degeneration, as demonstrated for other visual genes such as RPE65 and PDE6B. In this study, we describe a CRISPR/Cas9 adenine base editing strategy to correct the E150K mutation and demonstrate precise in vivo editing in a Rho-E150K mouse model of autosomal recessive retinitis pigmentosa (RP). Using ultraviolet-visible spectroscopy, mass spectrometry, and the G-protein activation assay, we characterized wild-type rhodopsin and rhodopsin variants containing bystander base edits. Subretinal injection of dual-adeno-associated viruses delivering our base editing strategy yielded up to 44% Rho correction in homozygous Rho-E150K mice. Injection at postnatal day 15, but not later time points, restored rhodopsin expression, partially rescued retinal function, and partially preserved retinal structure. These findings demonstrate that in vivo base editing can restore the function of mutated structural and functional proteins in animal models of disease, including rhodopsin-associated RP and suggest that the timing of gene-editing is a crucial determinant of successful treatment outcomes for degenerative genetic diseases.
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Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases.
Mitochondrial DNA (mtDNA) editing paves the way for disease modeling of mitochondrial genetic disorders in cell lines and animals and also for the treatment of these diseases in the future. Bacterial cytidine deaminase DddA-derived cytosine base editors (DdCBEs) enabling mtDNA editing, however, are largely limited to C-to-T conversions in the 5'-TC context (e.g., TC-to-TT conversions), suitable for generating merely 1/8 of all possible transition (purine-to-purine and pyrimidine-to-pyrimidine) mutations. Here, we present transcription-activator-like effector (TALE)-linked deaminases (TALEDs), composed of custom-designed TALE DNA-binding arrays, a catalytically impaired, full-length DddA variant or split DddA originated from Burkholderia cenocepacia, and an engineered deoxyadenosine deaminase derived from the E. coli TadA protein, which induce targeted A-to-G editing in human mitochondria. Custom-designed TALEDs were highly efficient in human cells, catalyzing A-to-G conversions at a total of 17 target sites in various mitochondrial genes with editing frequencies of up to 49%.
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Innate programmable DNA binding by CRISPR-Cas12m effectors enable efficient base editing.
Cas9 and Cas12 nucleases of class 2 CRISPR-Cas systems provide immunity in prokaryotes through RNA-guided cleavage of foreign DNA. Here we characterize a set of compact CRISPR-Cas12m (subtype V-M) effector proteins and show that they provide protection against bacteriophages and plasmids through the targeted DNA binding rather than DNA cleavage. Biochemical assays suggest that Cas12m effectors can act as roadblocks inhibiting DNA transcription and/or replication, thereby triggering interference against invaders. Cryo-EM structure of Gordonia otitidis (Go) Cas12m ternary complex provided here reveals the structural mechanism of DNA binding ensuring interference. Harnessing GoCas12m innate ability to bind DNA target we fused it with adenine deaminase TadA-8e and showed an efficient A-to-G editing in Escherichia coli and human cells. Overall, this study expands our understanding of the functionally diverse Cas12 protein family, revealing DNA-binding dependent interference mechanism of Cas12m effectors that could be harnessed for engineering of compact base-editing tools.
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Gene editing technologies and applications for insects.
Initially discovered in bacteria, CRISPR-based genome editing endonucleases have proven remarkably amenable for adaptation to insects. To date, these endonucleases have been utilized in a plethora of both model and non-model insects including diverse flies, bees, beetles, butterflies, moths, and grasshoppers, to name a few, thereby revolutionizing functional genomics of insects. In addition to basic genome editing, they have also been invaluable for advanced genome engineering and synthetic biology applications. Here we explore the recent genome editing advancements in insects for generating site-specific genomic mutations, insertions, deletions, as well as more advanced applications such as Homology Assisted Genome Knock-in (HACK), potential to utilize DNA base editing, generating predictable reciprocal chromosomal translocations, and development gene drives to control the fate of wild populations.
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[Improvement of Crops Using the CRISPR/Cas System: New Target Genes].
The success of genome editing of crops using the CRISPR/Cas system largely depends on the correct choice of target genes, for which directed changes will increase yield and improve the quality of plant raw materials and resistance to biotic and abiotic stress factors. This work systematizes and catalogs data on target genes used to improve cultivated plants. The latest systematic review examined articles indexed in the Scopus database and published before August 17, 2019. Our work covers the period from August 18, 2019 to March 15, 2022. A search according to the given algorithm allowed us to identify 2090 articles, among which only 685 contain the results of gene editing of 28 species of cultivated plants (the search was carried out for 56 crops). A significant part of these papers considered either editing of target genes, which was previously carried out in similar works, or studies related to the field of reverse genetics, and only 136 articles contain data on editing of new target genes, whose modification is aimed at improving plant traits important for breeding. In total, 287 target genes of cultivated plants were subjected to editing in order to improve properties significant for breeding over the entire period of the CRISPR/Cas system application. This review presents a detailed analysis of the editing of new target genes. The studies were most often aimed at increasing productivity and disease resistance, as well as improving the properties of plant materials. It was noted whether it was possible to obtain stable transformants at the time of publication and whether editing was applied to non-model cultivars. The range of modified cultivars of a number of crops has been significantly expanded, in particular, for wheat, rice, soybean, tomato, potato, rapeseed, grape, and maize. In the vast majority of cases, editing constructs were delivered using agrobacterium-mediated transformation, less commonly, using biolistics, protoplast transfection, and haploinducers. The desired change in traits was most often achieved by gene knockout. In some cases, knockdown and nucleotide substitutions in the target gene were carried out. To obtain nucleotide substitutions in the genes of cultivated plants, base-editing and prime-editing technologies are increasingly used. The emergence of a convenient CRISPR/Cas editing system has contributed to the development of specific molecular genetics of many crop species.
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Precise mutagenesis in zebrafish using cytosine base editors.
Base editing is a powerful CRISPR-based technology for introducing precise substitutions into the genome. This technology greatly advances mutagenesis possibilities in vivo, particularly in zebrafish, for which the generation of precise point mutations is still challenging. Zebrafish have emerged as an important model for genetic studies and in vivo disease modeling. With the development of different base editor variants that recognize protospacer-adjacent motifs (PAMs) other than the classical 5'-NGG-3' PAM, it is now possible to design and test several guide RNAs to find the most efficient way to precisely introduce the desired substitution. Here, we describe the experimental design strategies and protocols for cytosine base editing in zebrafish, from guide RNA design and selection of base editor variants to generation of the zebrafish mutant line carrying the substitution of interest. By using co-selection by introducing a loss-of-function mutation in genes necessary for the formation of pigments, injected embryos with highly efficient base editing can be directly analyzed to determine the phenotypic impact of the targeted substitution. The generation of mutant embryos after base editor injections in zebrafish can be completed within 2 weeks.
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