Systems and methods for modifying a polynucleotide
AssigneeChildren's Medical Center Corporation
InventorsDaniel E. Bauer, Sébastien Levesque
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Abstract
The present disclosure features systems and methods for modifying a target polynucleotide.
Claims20
A method for enhancing prime editing in a hematopoietic stem and progenitor cell (HSPC), the method comprising contacting the HSPC with the following components under conditions permissive for genome editing, the components comprising
- a programmable DNA binding protein;
- a DNA nickase;
- a polymerase;
- a prime editing guide RNA comprising a gRNA target sequence that binds target DNA, a primer binding sequence that primes DNA synthesis, and a template containing an edited RNA sequence, wherein the guide RNA is capable of directing the fusion protein to a target sequence in a polynucleotide or a primer binding sequence that binds the nicked single stranded DNA and a template sequence to specify the edit to be introduced by the polymerase;
- and
- a VPX polypeptide or a polynucleotide encoding a VPX polypeptide, and/or exogenous deoxynucleosides or deoxynucleotides.
A method of modifying an HSPC genome, the method comprising contacting the HSPC with the following components under conditions permissive for genome editing, the components comprising
- a programmable DNA binding protein;
- a DNA nickase;
- a polymerase;
- a prime editing guide RNA comprising a gRNA target sequence that binds target DNA, a primer binding sequence that primes DNA synthesis, and a template containing an edited RNA sequence, wherein the guide RNA is capable of directing the fusion protein to a target sequence in a polynucleotide;
- and
- a VPX polypeptide or a polynucleotide encoding a VPX polypeptide and/or exogenous deoxynucleosides or deoxynucleotides.
An HSPC comprising:
- a programmable DNA binding protein;
- a DNA nickase;
- a polymerase;
- a prime editing guide RNA comprising a gRNA target sequence that binds target DNA, a primer binding sequence that primes DNA synthesis, and a template containing an edited RNA sequence, wherein the guide RNA is capable of directing the fusion protein to a target sequence in a polynucleotide;
- and
- a VPX polypeptide or a polynucleotide encoding a VPX polypeptide.
A method for enhancing prime editing in a hematopoietic stem and progenitor cell (HSPC), the method comprising contacting the HSPC with the following components under conditions permissive for genome editing, the components comprising
- a programmable DNA binding protein;
- a DNA nickase;
- a DNA polymerase;
- a prime editing guide RNA comprising a gRNA target sequence that binds target DNA, a primer binding sequence that primes DNA synthesis, and a template containing an edited RNA sequence, wherein the guide RNA is capable of directing the fusion protein to a target sequence in a polynucleotide;
- and
- a VPX polypeptide or a polynucleotide encoding a VPX polypeptide,
- wherein the template is designed to evade mismatch repair.
A method for enhancing prime editing in a hematopoietic stem and progenitor cell (HSPC), the method comprising contacting the HSPC with the following components under conditions permissive for genome editing, the components comprising
- a programmable DNA binding protein;
- a DNA nickase;
- a DNA polymerase;
- a prime editing guide RNA comprising a gRNA target sequence that binds target DNA, a primer binding sequence that primes DNA synthesis, and a template containing an edited RNA sequence, wherein the guide RNA is capable of directing the fusion protein to a target sequence in a polynucleotide;
- a VPX polypeptide or a polynucleotide encoding a VPX polypeptide;
- and
- a mismatch repair inhibitor.
A method for enhancing prime editing in a human hematopoietic stem and progenitor cell (HSPC), the method comprising contacting the HSPC with the following components under conditions permissive for genome editing, the components comprising
- a programmable DNA binding protein;
- a DNA nickase;
- a reverse transcriptase;
- a pair of prime editing guide RNAs each comprising a gRNA target sequence that binds target DNA, a primer binding sequence that primes DNA synthesis, and a template containing an edited RNA sequence, wherein the guide RNA is capable of directing the fusion protein to a target sequence in a polynucleotide;
- a VPX polypeptide or a polynucleotide encoding a VPX polypeptide,
- wherein the template in each prime editing guide RNA is complementary to the template in the other prime editing guide RNA.
Dependencies are inferred from the claim text.