Abstract
Precisely measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality. Here we present Breaks Labeling In Situ and Sequencing (BLISS), featuring the following: (1) direct labelling of DSBs in fixed cells or tissue sections on a solid surface; (2) low-input requirement by linear amplification of tagged DSBs by in vitro transcription; (3) quantification of DSBs through unique molecular identifiers; and (4) easy scalability and multiplexing. We apply BLISS to profile endogenous and exogenous DSBs in low-input samples of cancer cells, embryonic stem cells and liver tissue. We demonstrate the sensitivity of BLISS by assessing the genome-wide off-target activity of two CRISPR-associated RNA-guided endonucleases, Cas9 and Cpf1, observing that Cpf1 has higher specificity than Cas9. Our results establish BLISS as a versatile, sensitive and efficient method for genome-wide DSB mapping in many applications.
Highlights
Measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality
We demonstrate the broad applicability of Breaks Labeling In Situ and Sequencing (BLISS) for genome-wide detection of both endogenous and exogenous DSBs in low-input samples of cells and tissues, as well as for genome-wide profiling of on- and off-target DSBs introduced by Cas9 and Cpf1 nucleases
DSBs are in situ blunted and ligated with a double-stranded DNA oligonucleotide adapter containing the T7 promoter sequence, the RA5 Illumina sequencing adapter, a random stretch of 8–12 nucleotides that serves as unique molecular identifier (UMI)19 and a sample barcode suitable for multiplexing (Supplementary Fig. 1a and Supplementary Data 1)
Summary
Measuring the location and frequency of DNA double-strand breaks (DSBs) along the genome is instrumental to understanding genomic fragility, but current methods are limited in versatility, sensitivity or practicality. In the past few years, several methods based on nextgeneration sequencing (NGS) have been developed to assess DSBs at genomic scale, including chromatin immunoprecipitation sequencing, direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing (BLESS), genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), in vitro Cas9-digested whole-genome sequencing (Digenome-seq), integrase-defective lentiviral vector (IDLV)-mediated DNA break capture, high-throughput, genome-wide, translocation sequencing and more recently End-Seq and DSBCapture. In the past few years, several methods based on nextgeneration sequencing (NGS) have been developed to assess DSBs at genomic scale, including chromatin immunoprecipitation sequencing, direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing (BLESS), genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), in vitro Cas9-digested whole-genome sequencing (Digenome-seq), integrase-defective lentiviral vector (IDLV)-mediated DNA break capture, high-throughput, genome-wide, translocation sequencing and more recently End-Seq and DSBCapture17 All of these methods represent important complementary tools to detect DSBs genome wide (Supplementary Table 1), they have important drawbacks. We demonstrate the broad applicability of BLISS for genome-wide detection of both endogenous and exogenous DSBs in low-input samples of cells and tissues, as well as for genome-wide profiling of on- and off-target DSBs introduced by Cas and Cpf nucleases
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