CRISPR nuclease off-target detection by sequencing (CROFT-seq)
Rapid, sensitive and cost-efficient detection and characterization method for in vitro double strand breaks produced by CRISPR-Cas9 nucleases from human genomic DNA samples.
1 . A method for detecting double strand breaks in DNA, wherein cleavage of the DNA is induced by programmable nucleases, the method comprises:
a) ligating an adapter to the cleaved DNA ends, wherein an adapter comprises a biotin for the purification of the ligated DNA;
b) incubating the ligated DNA under conditions sufficient to remove the unligated adapter with exonuclease I;
c) capturing ligated DNA on the streptavidin-coated magnetic beads;
d) adding an agent capable of removing non-biotinylated DNA strand unattached to the streptavidin-coated magnetic beads;
e) synthesizing complementary strands using non-biotinylated strand synthesis primers compatible for use in PCR library amplification and sequencing;
f) amplifying, pooling and purifying the PCR libraries; and
g) sequencing said PCR libraries.
2 . The method of claim 1 , wherein the DNA is one of dsDNA: synthetic genomic DNA, gDNA from a cell type of interest, or plasmid DNA.
3 . The method of claim 2 , wherein the DNA is isolated from a bacterial, mammalian, plant, yeast or fungal cell.
4 . The method of claim 1 , wherein the double strand break inducing agent is selected from the group consisting of: zinc-finger nucleases, meganucleases, transcription activator (TALEN), and CRISPR-Cas RNA-guided nucleases.
5 . The method of claim 1 , wherein the double strand break inducing agent is ribonucleoprotein (RNP) complex comprising a Cas9 endonuclease and a single-guide RNA (sgRNA).
6 . The method of claim 1 , wherein the double strand break inducing agent is ribonucleoprotein (RNP) complex comprising an engineered Cas9 variant and a single-guide RNA (sgRNA).
7 . The method of claim 1 , wherein the adapter comprises a next generation sequencing primer sequence or a randomized DNA barcode.
8 . The method of claim 1 , wherein the non-biotinylated strand synthesis primers comprise a next generation sequencing primer sequence, a randomized DNA barcode or unique molecular identifier (UMI).
9 . The method of claim 1 for detecting double strand breaks in DNA, wherein cleavage of the DNA is induced by programmable nucleases, wherein the method further comprises:
between steps b) and c) fragmenting the ligated DNA with DNase I under conditions required to fragment DNA to an average length of about 100-500 bp;
in step c) capturing the ligated and fragmented DNA on the streptavidin-coated magnetic beads; and
between steps d) and e) adding polyC tail to the 3′DNA end with terminal deoxynucleotidyl transferase (TdT) under conditions required to generate polyC tail of an average length of about 10-50 nt.
10 . The method of claim 9 , wherein the DNA is one of dsDNA: synthetic genomic DNA, gDNA from a cell type of interest, or plasmid DNA.
11 . The method of claim 10 , wherein the DNA is isolated from a bacterial, mammalian, plant, yeast or fungal cell.
12 . The method of claim 11 , wherein the double strand break inducing agent is selected from the group consisting of: zinc-finger nucleases, meganucleases, transcription activator (TALEN), and CRISPR-Cas RNA-guided nucleases.
13 . The method of claim 11 , wherein the double strand break inducing agent is ribonucleoprotein (RNP) complex comprising a Cas9 endonuclease and a single-guide RNA (sgRNA).
14 . The method of claim 11 , wherein the double strand break inducing agent is ribonucleoprotein (RNP) complex comprising an engineered Cas9 variant and a single-guide RNA (sgRNA).
15 . The method of claim 11 , wherein the adapter comprises a next generation sequencing primer sequence or a randomized DNA barcode.
16 . The method of claim 11 , wherein the non-biotinylated strand synthesis primers comprise a next generation sequencing primer sequence, a randomized DNA barcode or unique molecular identifier (UMI).
17 . The method of claim 1 , wherein the agent capable of removing non-biotinylated DNA strand is NaOH.