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Long-read Individual-molecule Sequencing Reveals CRISPR-induced Genetic Heterogeneity in Human ESCs


Understanding the genetic heterogeneity of a cell population is extremely important for studies of biological systems. However, it remains challenging to analyze various types of genetic variants, because current methods are inadequate for detecting rare and/or complex variants. To address these issues, we develop a universal method to label individual DNA molecules for analyzing diverse types of rare genetic variants, with frequency as low as 4x10-5, using short- or long-read sequencing. It enables base-resolution haplotype-resolved quantitative characterization of rare variants. We showed that Cas9 cleavage induced SVs in ~4% of edited hESCs. Surprisingly, a significant number of SVs (up to 87%) were reoccurring deletions and insertions. These data provide the first quantitative evidence of nonrandom repair outcomes of Cas9 cutting and hotspots for Cas9-induced large indels.

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