Methods and systems for identification of sequence adjacent motif, potency, and fidelity of a nuclease
AssigneeGenEditBio Limited
InventorsZongli Zheng, Miao Yu
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Abstract
Provided is a method for identification of sequence adjacent motif, potency, and fidelity of a nuclease.
Claims40
A method for identification of adjacent sequence motif (adjacent motif) of a nuclease using a sequence that occurs more than once in a genome, wherein the method comprises the steps of:
- (1) identifying a repeat sequence from a genome of interest, where the flanking sequences to the repeat sequence are diversified;
- (2) contacting a nuclease of interest with the genome to target the repeat sequence;
- and
- (3) analyzing the flanking sequences of modified or unmodified repeat sequence(s), so as to identify the adjacent sequence motif of the nuclease.
The method of any one of the preceding claims, wherein the genome of interest is a eukaryotic genome;
- optionally, the eukaryotic genome is from human, mouse, rat, pig, monkey, or other animals, plants, fungi, or protists.
The method of any one of the preceding claims, wherein the genome of interest is a prokaryotic genome;
- optionally, the prokaryotic genome is from bacterial or archaea.
A method for identification of editing efficiency of a nuclease using a sequence that occurs more than once in a genome, wherein the method comprises the steps of:
- (1) identifying a repeat sequence from a genome of interest, where the flanking sequences to the repeat sequence are diversified;
- (2) contacting a nuclease of interest with the genome to target the repeat sequence;
- and
- (3) measuring the editing efficiency of the target loci.
A method for identification of off-target of a nuclease using a sequence that occurs more than once in a genome, wherein the method comprises the steps of:
- (1) identifying a repeat sequence from a genome of interest, where the flanking sequences to the repeat sequence are diversified;
- (2) contacting a nuclease of interest with the genome to target the repeat sequence;
- and
- (3) quantifying the edited sequences with perfectly matched (on-targets) and with mismatched bases (off-targets).
A polynucleotide for a specific application, wherein the polynucleotide comprises a spacer sequence or a nucleic acid sequence encoding such spacer sequence, wherein the spacer sequence is capable of binding to a repeat sequence or a repeat's complementary sequence from a genome of interest and the flanking sequences to the repeat sequence are diversified;
- and wherein the specific application is identification of adjacent sequence motif of a nuclease, identification of editing efficiency of a nuclease, or identification of off-target of a nuclease.
Dependencies are inferred from the claim text.