Methods of enriching targeted nucleic acid, identifying off-target and evaluating gene editing efficiency
AssigneeGenEditBio Limited
InventorsWenjing ZHOU, Bang WANG, Zongli ZHENG
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Abstract
The present disclosure relates to enriching nucleic acid from a sample. In some embodiments, the present disclosure provides methods for enriching at least one targeted nucleic acid, identifying genome-wide gene editing off-targets, and evaluating gene editing efficiency from a sample comprising a plurality of single-strand nucleic acid fragments. Others example embodiments are also described herein.
Claims30
A method of enriching at least one target nucleic acid from a sample comprising a plurality of single-strand nucleic acid fragments, the method comprising:
- (a) contacting a universal oligonucleotide adaptor with the sample to produce a ligation product, wherein the universal oligonucleotide adaptor is configured for ligating to a 5′ end of the single-strand nucleic acid fragments;
- (b) amplifying the ligation product by a first PCR with a first target-specific primer and optionally a first universal oligonucleotide adaptor primer to form a first PCR product;
- and
- (c) amplifying the first PCR product by a second PCR with a second target-specific primer and a second universal oligonucleotide adaptor primer to form a second PCR product, wherein the second target-specific primer is nested relative to the first target-specific primer.
A method of identifying genome-wide gene editing off-targets from a sample comprising a plurality of single-strand nucleic acid fragments, comprising:
- (a) contacting a universal oligonucleotide adaptor with the sample to produce a ligation product, wherein the universal oligonucleotide adaptor is configured for ligating to a 5′ end of the single-strand nucleic acid fragments;
- (b) amplifying the ligation product by performing a first PCR with a first target-specific primer to form a first PCR product;
- (c) amplifying the first PCR product by a second PCR with a sequencing specific adaptor primer and a second target-specific primer nested relative to the first target-specific primer, to form a sequencing library;
- (d) quantifying and reading the sequencing library to obtain sequencing results;
- and
- (e) mapping the sequencing results to a reference genome and evaluating gene editing off-targets.
A method of evaluating gene editing efficiency from a sample comprising a plurality of single-strand nucleic acid fragments, comprising:
- (a) contacting a universal oligonucleotide adaptor with the sample to produce a ligation product, wherein the universal oligonucleotide adaptor is configured for ligating to a 5′ end of the single-strand nucleic acid fragments;
- (b) amplifying the ligation product by performing a first PCR with a first target-specific primer to form a first PCR product, wherein the first target-specific primer is preferably configured for annealing to the single-strand nucleic acid fragments at an on-target, a predicted off-target, or a known off-targets;
- (c) amplifying the first PCR product by a second PCR with a sequencing specific adaptor primer and a second target-specific primer nested relative to the first target-specific primer, to form a sequencing library;
- (d) quantifying and reading the sequencing library to form sequencing results;
- and
- (e) mapping the sequencing results to a reference genome and evaluating gene editing efficiency.
A method of identifying genome-wide gene editing off-targets from a sample comprising a plurality of single-strand nucleic acid fragments, comprising:
- (a) contacting a universal oligonucleotide adaptor with the sample to produce a ligation product, wherein the universal oligonucleotide adaptor is configured for ligating to a 5′ end of the single-strand nucleic acid fragments;
- (b) amplifying the ligation product by a first PCR with a first set of target-specific primers, wherein the first set of target-specific primers are configured for annealing to the single-strand nucleic acid fragments 5′ of on-target and one or more predicted and/or known off-targets;
- (c) amplifying the first PCR product by a second PCR with a second set of target-specific primers and a universal oligonucleotide adaptor primer to form a sequencing library, wherein each of the second set of target-specific primers is nested relative to a corresponding primer of the first set of target-specific primers;
- and
- (d) sequencing the sequencing library to identify off-targets.
Dependencies are inferred from the claim text.