Biology subjects

Lamothe, G.

Publications and source records attributed to Lamothe, G..

3 recordsLinked to original sources

bioRxiv2025

Split Cas12a protospacer engineering enables ultra-specific, PAM-free detection

ABSTRACTCRISPR-Cas12a is a programmable, RNA-guided endonuclease that has revolutionized biotechnology, with applications in genome engineering and diagnostics. To induce nuclease activity, Cas12a must first interact with the target dsDNA duplex by associating with a short protospacer adjacent motif (PAM) in the sequence. In this study we have split this target duplex to create PAM-proximal and PAM-distal duplex regions, which has allowed us to regulate trans-cleavage activity when these regions are included in combination or separately. These observations on Cas12a activity led to hypotheses into the related functional mechanisms, which we have tested and that have highlighted DNA/protein interactions during Cas12a complex assembly that were not otherwise apparent. Selective destabilization of the nucleic acid complexes appears to drive greater reliance on the Cas12a protein for complex stability. We have exploited this to provide significant improvements in both structural selectivity and nucleotide specificity in PAM-proximal and PAM-distal duplex regions, respectively. The result is an architecture that shows promise as a PAM-free ultra-specific platform to resolve single nucleotide polymorphisms. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=102 SRC="FIGDIR/small/667643v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@b1771dorg.highwire.dtl.DTLVardef@103197aorg.highwire.dtl.DTLVardef@7dd393org.highwire.dtl.DTLVardef@cd812d_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2020

Base editing strategy allows insertion of the A673T mutation in APP gene to prevent the development of Alzheimer's disease

Amyloid precursor protein (APP), a membrane protein mostly found in neurons, is preferentially cut by the -secretase enzyme, however, abnormal cleavage by {beta}-secretase leads to the formation of {beta}-amyloid peptide plaque in the brains of Alzheimers patients. Genome analysis of an Icelandic population that did not appear to show symptoms of Alzheimers at advanced age led to the discovery of the A673T mutation, reducing {beta}-secretase cleavage by 40%. We hypothesized that the insertion of this mutation in a patients genome could be an effective and sustainable method to slow down or prevent the progression of familial and sporadic forms of Alzheimers disease. We have thus modified the APP gene in HEK293T cells and in SH-SY5Y neuroblastoma using a Cas9n-deaminase enzyme, which changes a cytosine into a thymine, thus converting 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 modified the APP gene in up to 56.7% of the HEK293T cells. Our approach aimed to attest to the efficiency of base editing in the development of treatments against genetic diseases as well as provide a new strategy for the treatment of Alzheimers.

bioengineering↗

bioRxiv2020

Protective mutation A673T as a potential gene therapy for most forms of APP Familial Alzheimer's Disease

The accumulation of plaque in the brain leads to the onset and development of Alzheimers disease. The Amyloid precursor protein (APP) is usually cut by -secretase, however an abnormal cleavage profile by {beta}-secretase (BACE1) leads to the accumulation of A{beta} peptides, which forms these plaques. Numerous APP gene mutations favor plaque accumulation, causing Familial Alzheimer Disease (FAD). However, a variant of the APP gene (A673T) in Icelanders reduces BACE1 cleavage by 40 %. A library of plasmids containing APP genes with 29 FAD mutations with or without the additional A673T mutation was generated and transfected in neuroblastomas to assess the effect of this mutation on A{beta} peptide production. In most cases the production of A{beta} peptides was decreased by the co-dominant A673T mutation. The reduction of A{beta} peptide concentrations for the London mutation (V717I) even reached the same level as A673T carriers. These results suggest that the insertion of A673T in the APP gene of genetically susceptible FAD patients may prevent the onset of, slow down, or stop the progression of the disease.

neuroscience↗