Long-read target enrichment sequencing for rAAV integration site analysis in engineered clones with targeted viral insertion

Genome-Wide Characterization of Recombinant AAV6 Vector Integration in Human CD34＋ Cells.

The data support the genomic safety of rAAV6 and its applicability to hematological gene therapy and functional enrichment analysis indicated associations with general cellular and structural processes, without enrichment in oncogenic pathways.

Development and evaluation of a single-tube RT-ERA-CRISPR/Cas12a assay for simultaneous detection of enterovirus G and recombinant strains with papain-like cysteine protease gene insertion

Large-scale genome structure interrogation via recombinase-mediated rearrangements of multiplexed prime edits in repetitive elements.

This Protocol leverages prime editing to insert recombinase recognition sites into repetitive genomic regions, such as LINE-1 elements, thereby enabling extensive genetic modifications in human cells, and supports a wide range of studies, including genome-wide functional analyses and essentiality mapping.

CRISPR/Cas9-Mediated Site-Directed Mutagenesis of Genes in Klebsiella pneumoniae.

This protocol minimizes off-target editing, shortens experimental timelines, reduces screening workload, and provides a reliable platform for investigating resistance mechanisms, validating candidate genes, and supporting functional genomics studies in clinically relevant bacterial pathogens.

Efficient targeted gene knock-in in Ulva using Cas9 RNPs and long single-stranded DNA donors

This study establishes a streamlined and highly efficient framework for precise insertional mutagenesis and double-mutant generation in Ulva, thereby expanding the genetic engineering toolkit for this macroalga.

Lineage-associated small inversions disrupt dosT, dnaE2, and a promoter-adjacent region in some Mycobacterium tuberculosis isolates

ABSTRACT Large molecular inversions in the genome of Mycobacterium tuberculosis (Mtb) due to factors like the presence of insertion sequences and transposases are widely known. However, smaller inversions within coding sequences and non-coding control elements are rarely reported. The present study aims to identify inversions and their potential impact on Mtb biology in a lineage-specific manner. Structural variants (SVs) could only be detected by long reads. For this, we simulated long reads by de novo assembling the short-read sequencing data sets and subsequently aligned representative strains from each lineage using the Progressive Mauve algorithm. Independently, long-read sequencing from the Pacific Biosciences platform was acquired and analyzed using the structural variant identification method. Variants were merged, and Fisher’s exact test was carried out to identify the inversion association with lineages. To visualize deoxyribonucleic acid (DNA) features, the DNA-features-viewer tool was used. Simulated reads from short-read sequencing gave indications of lineage (L)-specific inversions. The long-read sequencing approach led to the identification of seven unique inversions: two positively associated with L1, one positively associated with L3, two negatively associated with L4, and two positively associated with L3 but negatively associated with L4 (P < 0.05). The inversions encompassed primarily non-essential genes like sdaA, dosT, Rv2026c, dnaE2, Rv1341, Rv1342, and lprD. An interesting inversion was observed in the upstream control element of purB and Rv0776c. The study sheds light on small inversions that may be causing alterations in expression, formation of fusion genes, and nonsense mutations that may have a role in lineage-specific phenotypic changes. IMPORTANCE The role of mutations like SNPs and INDELs and their association with drug resistance is well known in Mycobacterium tuberculosis (Mtb). However, structural variations, especially inversions, are largely overlooked and unreported. In this paper, publicly available whole-genome sequencing datasets from Illumina and Pacific Biosciences–Oxford Nanopore Technologies platform have been used to detect inversions and report seven unreported Mtb lineage-specific small inversions. The role of mutations like SNPs and INDELs and their association with drug resistance is well known in Mycobacterium tuberculosis (Mtb). However, structural variations, especially inversions, are largely overlooked and unreported. In this paper, publicly available whole-genome sequencing datasets from Illumina and Pacific Biosciences–Oxford Nanopore Technologies platform have been used to detect inversions and report seven unreported Mtb lineage-specific small inversions.