Compositions and methods for site-specific modification
OwnerSongyuan Li, Marcello Maresca +1inventors
InventorsSONGYUAN LI, MARCELLO MARESCA, SASA SVIKOVIC
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Abstract
The present disclosure provides a polynucleotide comprising an RNA guide sequence, a Cas-binding region, and a DNA template sequence. The disclosure also provides compositions comprising a Cas nuclease or a Cas nickase and one or more polynucleotides comprising a guide sequence, a Cas binding region, and a DNA template sequence. The disclosure further provides a fusion protein comprising a Cas nuclease or a Cas nickase and a DNA polymerase recruitment moiety. Also provided are methods for providing a targeted insertion in a target DNA of a cell.
Claims144
A polynucleotide comprising (i) an RNA guide sequence;
- (ii) a Cas-binding region;
- and (iii) a DNA template sequence, wherein the DNA template sequence is at a 3′ end of the polynucleotide.
A polynucleotide comprising:
- (i) an RNA guide sequence;
- (ii) a Cas-binding region;
- and (iii) a DNA template sequence, wherein the DNA template sequence is at a 3′ end of the polynucleotide, and wherein the DNA template sequence comprises a phosphorothioate bond.
A composition comprising:
- a Cas nuclease or a Cas nickase;
- and
- a polynucleotide comprising:
- (i) a guide sequence;
- (ii) a Cas-binding region;
- and (iii) a DNA template sequence, wherein the DNA template sequence is at a 3′ end of the polynucleotide.
A composition comprising:
- a Cas nuclease or a Cas nickase;
- a first polynucleotide comprising:
- (i) a guide sequence;
- (ii) a Cas-binding region;
- and (iii) a first hybridization region, wherein the first hybridization region is at a 3′ end of the first polynucleotide;
- and
- a second polynucleotide comprising:
- (i) a second hybridization region that is complementary to the first hybridization region and (ii) a DNA template sequence.
A composition comprising:
- a Cas nuclease or a Cas nickase;
- a first polynucleotide comprising:
- (i) a guide sequence;
- and (ii) a first hybridization region, wherein the first hybridization region is at a 3′ end of the first polynucleotide;
- and
- a second polynucleotide comprising:
- (i) a second hybridization region that is complementary to the first hybridization region;
- (ii) a Cas-binding region;
- and (iii) a DNA template sequence.
A composition comprising:
- a Cas nuclease or a Cas nickase;
- a first polynucleotide comprising:
- (i) a guide sequence;
- (ii) a Cas-binding region;
- (iii) a first hybridization region;
- and (iv) a primer-binding sequence, wherein the primer-binding sequence is at a 3′ end of the first polynucleotide;
- and
- a second polynucleotide comprising:
- (i) a second hybridization region that is complementary to the first hybridization region;
- and (ii) a sequence of interest (SOI).
A composition comprising:
- a Cas nuclease or a Cas nickase;
- a first polynucleotide comprising:
- (i) a guide sequence;
- (ii) a Cas-binding region;
- and (iii) a first hybridization region, wherein the first hybridization region is at a 3′ end of the first polynucleotide;
- a second polynucleotide comprising:
- (i) a second hybridization region that is complementary to the first hybridization region;
- (ii) a third hybridization region;
- and (iii) a primer-binding sequence, wherein the primer-binding sequence is at a 3′ end of the second polynucleotide;
- and
- a third polynucleotide comprising:
- (i) a fourth hybridization region that is complementary to the third hybridization region;
- and (ii) a sequence of interest (SOI).
A fusion protein comprising (i) a Cas nuclease or a Cas nickase;
- and (ii) a DNA polymerase recruitment protein, a DNA ligase, a DNA ligase recruitment moiety, a DNA binding protein, a DNA repair protein, or combination thereof.
The method of any one of claims 140 to 143, further comprising contacting the target DNA with a DNA polymerase, a protein in a DNA repair pathway, or combination thereof.
A composition comprising:
- a Cas nuclease or a Cas nickase;
- a first polynucleotide comprising:
- (i) a guide sequence;
- (ii) a Cas-binding region;
- (iii) a first hybridization region;
- and (iv) a primer-binding sequence, wherein the primer-binding sequence is at a 3′ end of the first polynucleotide;
- and
- a second polynucleotide comprising:
- (i) a second hybridization region that is complementary to the first hybridization region;
- and (ii) a sequence of interest (SOI).
- a third polynucleotide comprising:
- (i) a third hybridization region that is partially complementary to the second polynucleotide;
- and (ii) a sequence of interest (SOI).
Dependencies are inferred from the claim text.