Journal: Nucleic Acids Research

Article Title: Indel pattern-guided repair mapping reveals genome-wide DNA repair networks in CRISPR/Cas9 editing

doi: 10.1093/nar/gkag260

Figure Lengend Snippet: IPGRM as a genome-wide platform for systematic dissection of DSB repair mechanisms. ( A ) Schematic of vectors encoding the GeCKOv2 CRISPR knockout library with secondary Cas9 cleavage sites (sg3 or sg81) positioned adjacent to library-encoded sgRNAs. ( B ) Composition of the GeCKOv2 library, including coding genes, pre-miRNA loci, and non-targeting controls. ( C ) Workflow of IPGRM. HEK293T cells were transduced with the GeCKOv2 library and subsequently edited at sg3 or sg81 loci. Repair outcomes were captured by PCR amplicon sequencing and classified into seven indel patterns, allowing each sgRNA-mediated knockout to be directly linked to a corresponding repair outcome profile. Insertion length distributions in the sg3 ( D ) and sg81 ( E ) libraries. Deletion length distributions in the sg3 ( F ) and sg81 ( G ) libraries. Sequence profiles of the most frequent deletions: 3 bp in the sg3 library ( H ) and 5 bp in the sg81 library ( I ). Microhomologous bases are indicated by underlining. ( J ) Proportional distributions of the seven indel patterns in the sg3 and sg81 libraries.

Article Snippet: The human GeCKOv2 lentiviral library (GENEWIZ, Suzhou), containing 122 756 sgRNAs targeting 19 050 genes (6 sgRNAs per gene), 1864 miRNAs (4 sgRNAs per miRNA), and 1000 NonTarget controls, was transduced into 3 × 108 HEK293T cells at an MOI of 0.3.

Techniques: Genome Wide, Dissection, CRISPR, Knock-Out, Transduction, Amplification, Sequencing