Journal: Molecular Therapy. Nucleic Acids

Article Title: Comprehensive assessment of on- and off-target mutagenesis via lipid nanoparticle delivery of CRISPR-Cas9 genome editing

doi: 10.1016/j.omtn.2026.102958

Figure Lengend Snippet: Whole-genome sequencing analysis of human iPSCs treated with LNP-CRISPR (A) Schematic of the experimental design. (B) On-target indel frequencies measured by TIDE assay for each gRNA sample. (C) The genome editing efficiency by the dual gRNA sample was assessed as DNA copy number loss at the exon 45 region by droplet digital PCR (ddPCR) analysis. (D) TapeStation DNA electrophoresis to assess the DNA cleavage at the dual gRNAs. The arrowhead indicates the DNA band cleaved by gRNA #1 and gRNA #23. (E) When indels are detected from whole genome sequences, spontaneous indels (i.e., heterozygous indels) are detected as identical indel patterns in multiple sequence reads (middle). Therefore, they can be distinguished from noise, such as randomly occurring sequence errors (left). On the other hand, indels induced by genome editing typically induce different indel patterns across multiple cells. Therefore, overlapping but not identical indel patterns can be detected in one place as an “indel cluster.” (F) Number of overlapped indels (small, large, or total) obtained by the “ bedtools merge ” function from the WGS analysis. Regions with three or more indel patterns were defined as indel clusters and shown in the far-right column. (G) A scatterplot of allele fractions versus indel pattern counts for all the indels. The on-target sites are shown as diamonds, and the off-target sites are shown as circles.

Article Snippet: WGS libraries were prepared by Macrogen Japan Corp. using the Illumina TruSeq DNA PCR-Free kit (350 bp insert).

Techniques: Sequencing, CRISPR, Digital PCR, Nucleic Acid Electrophoresis

Journal: Molecular Therapy. Nucleic Acids

Article Title: Comprehensive assessment of on- and off-target mutagenesis via lipid nanoparticle delivery of CRISPR-Cas9 genome editing

doi: 10.1016/j.omtn.2026.102958

Figure Lengend Snippet: Integrated analysis of off-target sites from the in silico , in vitro , and in cellulo datasets (A) Genome editing with CRISPR-Cas9 involves DNA binding, cleavage, and mutagenesis, which may alter gene function and, in some cases, lead to phenotypic effects. In silico off-target analysis tools typically predict potential binding sites. CIRCLE-seq identifies in vitro cleavage sites. WGS detects mutagenesis in cells or tissues. (B) Venn diagrams of integrated off-target analyses. For CIRCLE-seq and Cas-OFFinder, both gRNA #1 and gRNA #23 are integrated. For the WGS, a total of six datasets, comprising three conditions, gRNA #1 alone, gRNA #23 alone, and gRNA #1 + #23 tested in two iPSC lines (1383D2 and 1383D6), are merged. (C) Summary of the off-target sites detected by integrated analysis. The two on-target sites are shown in the top two rows highlighted in light beige. MM: the number of mismatched base pairs. Of note, the GPHN gene is listed in COSMIC as a fusion partner of MLL . (D) Indel frequencies at the candidate sites identified in C were quantified by amplicon sequencing. Values represent the fraction of reads containing indels at each putative Cas9 cutting site. Color intensity reflects indel frequency, with darker red indicating higher values (see color scale on the right). The two on-target sites shown in C are presented as a single combined row.

Article Snippet: WGS libraries were prepared by Macrogen Japan Corp. using the Illumina TruSeq DNA PCR-Free kit (350 bp insert).

Techniques: In Silico, In Vitro, CRISPR, Binding Assay, Mutagenesis, Amplification, Sequencing