US 2026/0049352 A1 · ADAPTERED-TAG BLOCKING OLIGONUCLEOTIDES

Abstract

Described herein are compositions and methods for reducing adaptered-tag sequencing reads during the identification and nomination of on- and off-target CRISPR edited sites. One embodiment is a method for reducing adaptered-tag sequencing reads during the identification and nomination of on- and off-target CRISPR edited sites, the method comprising: contacting in an amplification reaction one or more adaptered-tag blocking oligonucleotides with an isolated genomic DNA having one or more tag sequences and adapter sequences; wherein the adaptered-tag blocking oligonucleotides comprise one or more blocking moieties and hybridize to adaptered-tag sequences at a junction region between the adapter and tag sequences to reduce amplification of the adaptered-tag sequences.

Claims20

Dependent claims

A method for reducing adaptered-tag sequencing reads during the identification and nomination of on- and off-target CRISPR edited sites, the method comprising:

contacting in an amplification reaction one or more adaptered-tag blocking oligonucleotides with an isolated genomic DNA having one or more tag sequences and adapter sequences;
wherein the adaptered-tag blocking oligonucleotides comprise one or more blocking moieties and hybridize to adaptered-tag sequences at a junction region between the adapter and tag sequences to reduce amplification of the adaptered-tag sequences.

A method for identifying and nominating on- and off-target CRISPR edited sites with improved accuracy and sensitivity, the method comprising:

(a) performing a multiplex PCR reaction comprising:
(i) one or more tag-specific oligonucleotide primers, each having a cleavage region comprising a ribonucleotide (rN) positioned 5′ of a blocking group and a complementary region flanking one or more tag sequences, wherein the blocking group prevents primer extension and/or inhibits the oligonucleotide primer from serving as a template for DNA synthesis;
(ii) one or more adapter-specific oligonucleotide primers, each having a cleavage region comprising a rN positioned 5′ of a blocking group and a complementary region flanking the 5′ end of a universal adapter sequence;
(iii) one or more adaptered-tag blocking oligonucleotides corresponding to each strand of the tag sequences and comprising one or more blocking moieties, wherein the adaptered-tag blocking oligonucleotides hybridize to top and bottom strands of adaptered-tag sequences at a junction region between the universal adapter and tag sequences and inhibit annealing of the tag-specific oligonucleotide primers to the top and bottom strands of the adaptered-tag sequences, thereby reducing amplification of the adaptered-tag sequences;
and
(iv) a cleaving enzyme;
(b) hybridizing the tag-specific oligonucleotide primers to one or more incorporated tag sequences to form a tag sequence double stranded substrate and hybridizing one or more adapter-specific oligonucleotide primers to the 5′ end of the universal adapter sequence;
(c) cleaving at a point within or adjacent to the cleavage regions with the cleaving enzyme to remove the blocking groups from the one or more tag-specific oligonucleotide primers and the one or more adapter-specific oligonucleotide primers;
(d) amplifying a portion of isolated genomic DNA comprising the one or more incorporated tag sequences and the universal adapter sequence;
and
(e) sequencing the amplified portion of the isolated genomic DNA, thereby identifying on- and off-target CRISPR edited sites.

8 more elements

Dependencies are inferred from the claim text.

Adaptered-tag blocking oligonucleotides

Life of this patent

Adaptered-tag blocking oligonucleotides

Life of this patent

Abstract

Claims20