Project description:Cells were transfected with plasmids containing AsCas12f variants and sgRNAs for gene-editing. Off-target effect was determined using GUIDE-seq on an Illumina Nextseq platform.
2023-04-24 | GSE211600 | GEO
Project description:Gene-editing effciency of engineered AsCas12f variants
Project description:The spike S of SARS-CoV-2 recognizes ACE2 on the host cell membrane to initiate entry. Soluble decoy receptors, in which the ACE2 ectodomain is engineered to block S with high affinity, potently neutralize infection and, due to close similarity with the natural receptor, hold out the promise of being broadly active against virus variants without opportunity for escape. Here, we directly test this hypothesis. Using deep mutagenesis, we find that the ACE2-binding surface of the SARS-CoV-2 spike tolerates high mutational diversity, which may act as a source for resistance to therapeutics. However, saturation mutagenesis of the receptor-binding domain (RBD) followed by in vitro selection, with wild type ACE2 and the engineered decoy competing for binding sites, failed to find S mutants that discriminate in favor of the wild type receptor. We conclude that resistance to engineered decoys will be rare.
Project description:We used the whole exome sequencing to analyze the off-target effect of base editing system in mouse genomic DNA, which was extracted from haploid stem cells, mouse tails of semi-cloned embryos and mutant pups. The purpose of this sequencing is to find whether there exists off-target effect in the genome. By obtaining over 100 million reads of each sample from WES, we mapped the reads to the reference data base (mm10) and calculated the numbers of SNVs and indels. After filtering out naturally-occurring variants in the SNP database (dbSNP) and excluding SNPs also found in the wild-type genome, we next compared the DNA sequences at the remaining SNP sites with the on-target sequence. The results indicated that rare off-targets events happened in tested cell and embryos.