查看更多>>摘要：Bacteriophages have been used across various fields,and the utilization of CRISPR/Cas-based genome editing technology can accelerate the research and applications of bacteriophages.However,some bacteriophages can escape from the cleavage of Cas protein,such as Cas9,and decrease the efficiency of genome editing.This study focuses on the bacteriophage T7,which is widely utilized but whose mechanism of evading the cleavage of CRISPR/Cas9 has not been elucidated.First,we test the escape rates of T7 phage at different cleavage sites,ranging from 10-2 to 10-5.The sequencing results show that DNA point mutations and microhomology-mediated end joining(MMEJ)at the target sites are the main causes.Next,we indicate the existence of the hotspot DNA region of MMEJ and successfully reduce MMEJ events by designing targeted sites that bypass the hotspot DNA region.Moreover,we also knock out the ATP-dependent DNA ligase 1.3 gene,which may be involved in the MMEJ event,and the frequency of MMEJ at 4.3 is reduced from 83％to 18％.Finally,the genome editing efficiency in T7 △1.3 increases from 20％to 100％.This study reveals the mechanism of T7 phage evasion from the cleavage of CRISPR/Cas9 and demonstrates that the special design of editing sites or the deletion of key gene 1.3 can reduce MMEJ events and enhance gene editing efficiency.These findings will contribute to advancing CRISPR/Cas-based tools for efficient genome editing in phages and provide a theoretical foundation for the broader application of phages.
查看更多>>摘要：Hearing loss constitutes one of the most prevalent conditions within the field of otolaryngology.Recent in-vestigations have revealed that mutations in deafness-associated genes,including point mutations and variations in DNA sequences,can cause hearing impairments.With the ethology of deafness remaining unclear for a sub-stantial portion of the affected population,further screenings for pathogenic mutations are imperative to unveil the underlying mechanisms.On this study,by using next-generation sequencing,we examine 129 commonly im-plicated deafness-related genes in a Chinese family with hearing loss,revealing a novel heterozygous dominant mutation in the GJB2 gene(GJB2:c.65T>G:p.Lys22Thr).This mutation consistently occurs in affected family members but is not detected in unaffected individuals,strongly suggesting its causative role in hearing loss.Structural analysis indicates potential disruption to the Cx26 gap junction channel's hydrogen bond and electro-static interactions,aligning with predictions from the PolyPhen and SIFT algorithms.In conclusion,our study provides conclusive evidence that the identified heterozygous GJB2 mutation(GJB2:c.65T>G:p.Lys22Thr),specifically the K22T alteration,is the primary determinant of the family's deafness.This contribution enhances our understanding of the interplay between common deafness-associated genes and hearing loss,offering valuable insights for diagnostic guidance and the formulation of therapeutic strategies for this condition.
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