Precise and efficient insertion of A673T mutation in APP gene using MSYM.
Ontology highlight
ABSTRACT:
SUBMITTER: Xia Q
PROVIDER: S-EPMC11320440 | biostudies-literature | 2024 Nov
REPOSITORIES: biostudies-literature
Publications
Precise and efficient insertion of A673T mutation in APP gene using MSYM.
Xia Qing Q Liu Zijie Z Yang Xinyu X Xiao Jiaying J Zhao Xue X Zhao Yu Y Zheng Feifei F Ge Fangliang F Ye Ke K Liu Lulu L Wang Dayong D Gao Xu X
Genes & diseases 20231027 6
Similar Datasets
Project description:The amyloid precursor protein (APP) is a transmembrane protein mostly found in neurons. Cleavage of this protein by β-secretase can lead to the formation of amyloid-β (Aβ) peptide plaque, which leads to Alzheimer's disease. Genomic analysis of an Icelandic population that did not show symptoms of Alzheimer's at an advanced age led to the discovery of the A673T mutation. This mutation can reduce β-secretase cleavage by 40%. We hypothesized that the insertion of this mutation in patients' neurons could be an effective and sustainable method of slowing down or even stopping the progression of Alzheimer's disease. We modified the APP gene in HEK293T cells and in SH-SY5Y neuroblastoma using a Cas9 nickase (Cas9n)-deaminase enzyme to convert the alanine codon to a threonine. Several Cas9n-deaminase variants were tested to compare their efficiency of conversion. The results were characterized and quantified by deep sequencing. We successfully introduced the A673T mutation in 53% of HEK293T cells alongside a new mutation (E674K), which seemed to further reduce Aβ peptide accumulation. Our approach aimed to provide a new strategy for the treatment of Alzheimer's and in so doing, demonstrate the capacity of base editing techniques for treating genetic diseases.
| S-EPMC7985668 | biostudies-literature
Project description:A coding gene variant A673T (rs63750847) in the APP gene has recently been recognized as a protective variant of late-onset Alzheimer's Disease in a large Icelandic population and has been observed recurrently in populations from Nordic countries. The variant also was related to longevity in the Icelandic population. However, because of the extreme rarity of A673T in non-Nordic populations, the association with Alzheimer's disease has not yet been formally replicated. Because the variant has not been reported among the Danes, we aimed to study its frequency among healthy middle-age twins and oldest-old singletons and explore the possible effects on longevity and cognitive abilities. Surprisingly, only 1 of 3487 unrelated Danes carried the A673T variant, (0.014% [95% CI 0.000-0.080]), which was significantly lower than in the other Nordic countries averaging to 0.43% (95% CI 0.40-0.46). In conclusion, the A673T variant is rarer in Danes than other Nordic countries, thus precluding assessment of association with longevity or cognitive functioning.
| S-EPMC4562869 | biostudies-literature
Project description:Alzheimer's disease (AD) is the result of abnormal processing of the amyloid precursor protein (APP) by β-secretase and γ-secretase, which leads to the formation of toxic β-amyloid peptides. The toxic β-amyloid peptides induce neuron death, memory problems, and AD development. Several APP mutations increase the risk of developing early-onset AD. However, the A673T mutation identified in the Icelandic population prevents AD development by reducing the cleavage of APP by β-secretase. In this study, we inserted the A673T mutation in human cells using the CRISPR prime editing (PE) technique. Repeated PE treatments resulted in the insertion of the A673T mutation in up to 49.2% of APP genes when a second nick was induced in the other DNA strand. When the protospacer adjacent motif used for PE was also mutated, up to 68.9% of the APP genes contained the protective A673T mutation. PE is a promising approach to introduce the A673T mutation precisely without mutating nearby nucleotides.
| S-EPMC9009594 | biostudies-literature
Project description:ImportanceRecently, a rare variant in the amyloid precursor protein gene (APP) was described in a population from Iceland. This variant, in which alanine is replaced by threonine at position 673 (A673T), appears to protect against late-onset Alzheimer disease (AD). We evaluated the frequency of this variant in AD cases and cognitively normal controls to determine whether this variant will significantly contribute to risk assessment in individuals in the United States.ObjectiveTo determine the frequency of the APP A673T variant in a large group of elderly cognitively normal controls and AD cases from the United States and in 2 case-control cohorts from Sweden.Design, setting, and participantsCase-control association analysis of variant APP A673T in US and Swedish white individuals comparing AD cases with cognitively intact elderly controls. Participants were ascertained at multiple university-associated medical centers and clinics across the United States and Sweden by study-specific sampling methods. They were from case-control studies, community-based prospective cohort studies, and studies that ascertained multiplex families from multiple sources.Main outcomes and measuresGenotypes for the APP A673T variant were determined using the Infinium HumanExome V1 Beadchip (Illumina, Inc) and by TaqMan genotyping (Life Technologies).ResultsThe A673T variant genotypes were evaluated in 8943 US AD cases, 10 480 US cognitively normal controls, 862 Swedish AD cases, and 707 Swedish cognitively normal controls. We identified 3 US individuals heterozygous for A673T, including 1 AD case (age at onset, 89 years) and 2 controls (age at last examination, 82 and 77 years). The remaining US samples were homozygous for the alanine (A673) allele. In the Swedish samples, 3 controls were heterozygous for A673T and all AD cases were homozygous for the A673 allele. We also genotyped a US family previously reported to harbor the A673T variant and found a mother-daughter pair, both cognitively normal at ages 72 and 84 years, respectively, who were both heterozygous for A673T; however, all individuals with AD in the family were homozygous for A673.Conclusions and relevanceThe A673T variant is extremely rare in US cohorts and does not play a substantial role in risk for AD in this population. This variant may be primarily restricted to Icelandic and Scandinavian populations.
| S-EPMC4324097 | biostudies-literature
Project description:Precise genome editing of plants has the potential to reshape global agriculture through the targeted engineering of endogenous pathways or the introduction of new traits. To develop a CRISPR nuclease-based platform that would enable higher efficiencies of precise gene insertion or replacement, we screened the Cpf1 nucleases from Francisella novicida and Lachnospiraceae bacterium ND2006 for their capability to induce targeted gene insertion via homology directed repair. Both nucleases, in the presence of a guide RNA and repairing DNA template flanked by homology DNA fragments to the target site, were demonstrated to generate precise gene insertions as well as indel mutations at the target site in the rice genome. The frequency of targeted insertion for these Cpf1 nucleases, up to 8%, is higher than most other genome editing nucleases, indicative of its effective enzymatic chemistry. Further refinements and broad adoption of the Cpf1 genome editing technology have the potential to make a dramatic impact on plant biotechnology.
| S-EPMC5599503 | biostudies-literature
Project description:Efficient and precise targeted insertion holds great promise but remains challenging in plant genome editing. An efficient nonhomologous end-joining-mediated targeted insertion method was recently developed by combining clustered regularly interspaced short palindromic repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated nuclease 9 (SpCas9) gene editing with phosphorothioate modified double-stranded oligodeoxynucleotides (dsODNs). Yet, this approach often leads to imprecise insertions with no control over the insertion direction. Here, we compared the influence of chemical protection of dsODNs on efficiency of targeted insertion. We observed that CRISPR/SpCas9 frequently induced staggered cleavages with 1-nucleotide 5' overhangs; we also evaluated the effect of donor end structures on the direction and precision of targeted insertions. We demonstrate that chemically protected dsODNs with 1-nucleotide 5' overhangs significantly improved the precision and direction control of target insertions in all tested CRISPR targeted sites. We applied this method to endogenous gene tagging in green foxtail (Setaria viridis) and engineering of cis-regulatory elements for disease resistance in rice (Oryza sativa). We directionally inserted 2 distinct transcription activator-like effector binding elements into the promoter region of a recessive rice bacterial blight resistance gene with up to 24.4% efficiency. The resulting rice lines harboring heritable insertions exhibited strong resistance to infection by the pathogen Xanthomonas oryzae pv. oryzae in an inducible and strain-specific manner.
| S-EPMC10396358 | biostudies-literature
Project description: Not available
| S-EPMC11224055 | biostudies-literature
Project description:Several mutations conferring protection against Alzheimer's disease (AD) have been described, none as profound as the A673T mutation, where carriers are four times less likely to get AD compared to noncarriers. This mutation results in reduced amyloid beta (Aβ) protein production in vitro and lower lifetime Aβ concentration in carriers. Better understanding of the protective mechanisms of the mutation may provide important insights into AD pathophysiology and identify productive therapeutic intervention strategies for disease modification. Aβ(1-42) protein forms oligomers that bind saturably to a single receptor site on neuronal synapses, initiating the downstream toxicities observed in AD. Decreased formation, toxicity, or stability of soluble Aβ oligomers, or reduction of synaptic binding of these oligomers, may combine with overall lower Aβ concentration to underlie A673T's disease protecting mechanism. To investigate these possibilities, we compared the formation rate of soluble oligomers made from Icelandic A673T mutant and wild type (wt) Aβ(1-42) synthetic protein, the amount and intensity of oligomer bound to mature primary rat hippocampal/cortical neuronal synapses, and the potency of bound oligomers to impact trafficking rate in neurons in vitro using a physiologically relevant oligomer preparation method. At equal protein concentrations, mutant protein forms approximately 50% or fewer oligomers of high molecular weight (>50 kDa) compared to wt protein. Mutant oligomers are twice as potent at altering the cellular vesicle trafficking rate as wt at equivalent concentrations, however, mutant oligomers have a >4-fold lower binding affinity to synaptic receptors (Kd = 1,950 vs. 442 nM). The net effect of these differences is a lower overall toxicity at a given concentration. This study demonstrates for the first time that mutant A673T Aβ oligomers prepared with this method have fundamentally different assembly characteristics and biological impact from wt protein and indicates that its disease protecting mechanism may result primarily from the mutant protein's much lower binding affinity to synaptic receptors. This suggests that therapeutics that effectively reduce oligomer binding to synapses in the brain may be beneficial in AD.
| S-EPMC8246829 | biostudies-literature
Project description:The introduction of engineered site-specific DNA endonucleases has brought precise genome editing in many model organisms and human cells into the realm of possibility. In zebrafish, loss-of-function alleles have been successfully produced; however, germ line transmission of functional targeted knock-ins of protein tags or of SNP exchanges have not been reported. Here we show by phenotypic rescue that the CRISPR/Cas system can be used to target and repair a premature stop codon at the albino (alb) locus in zebrafish with high efficiency and precision. Using circular donor DNA containing CRISPR target sites we obtain close to 50% of larvae with precise homology-directed repair of the alb(b4) mutation, a small fraction of which transmitted the repaired allele in the germ line to the next generation (3/28 adult fish). The in vivo demonstration of germ line transmission of a precise SNP exchange in zebrafish underscores its suitability as a model for genetic research.
| S-EPMC4299274 | biostudies-literature
Project description: Not available
| S-EPMC6557807 | biostudies-literature