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Fig. 1 | Genome Biology

Fig. 1

From: Effective control of large deletions after double-strand breaks by homology-directed repair and dsODN insertion

Fig. 1

CRISPR-Cas9 RNP cleavage leads to large deletions. a Experimental design. Three types of human cells were edited with Cas9-gRNA RNPs. Editing efficiencies of small indels were assessed by Illumina amplicon sequencing and CRISPResso2 analysis. Large deletions were determined by long PCR and nanopore sequencing. b A representative of coverage and alignment of nanopore sequencing reads of the BCL11A amplicon. “Mean depth” and “Read depth” were analyzed by Samtools and Seqkit, respectively. Deletions were calculated by the formula (Read depth − Mean depth)/(Read depth). The deletion index was defined as deletion (%) of edited cells minus deletion (%) of unedited wildtype cells (background noise). A white area indicates an apparent deletion around the gRNA targeting sites in the coverage of alleles from RNP-edited cells. c Reproducibility of the deletion index data. The edited samples were PCR amplified with primers carrying different barcode sequences, followed by nanopore sequencing. The correlation of replicates indicates the reproducibility of this study. d Large deletion levels in three cell types. We used the deletion indexes to quantitate large deletions. e Frequencies of indels determined by NGS and CRISPResso2 analysis. For d and e, error bars represent the mean ± SEM of 4 experiments. The data in d and e were statistically analyzed by a two-way ANOVA test. Adjusted p values were indicated. “ns” means no significance (p > 0.05)

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