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US-12624354-B2 - Methods and compositions for prime editing nucleotide sequences

US12624354B2US 12624354 B2US12624354 B2US 12624354B2US-12624354-B2

Abstract

Compositions and methods are provided herein for conducting prime editing of a target DNA molecule (e.g., a genome) that enables the incorporation of a nucleotide change and/or targeted mutagenesis. The compositions include fusion proteins comprising nucleic acid programmable DNA binding proteins (napDNAbp) and a polymerase (e.g., reverse transcriptase), which is guided to a specific DNA sequence by a modified guide RNA, named an PEgRNA. The PEgRNA has been altered (relative to a standard guide RNA) to comprise an extended portion that provides a DNA synthesis template sequence which encodes a single strand DNA flap which is synthesized by the polymerase of the fusion protein and which becomes incorporated into the target DNA molecule.

Inventors

  • David R. Liu
  • Andrew Vito Anzalone
  • James William Nelson

Assignees

  • THE BROAD INSTITUTE, INC.
  • PRESIDENT AND FELLOWS OF HARVARD COLLEGE

Dates

Publication Date
20260512
Application Date
20240425

Claims (20)

  1. 1 . A prime editing system comprising: a) a prime editing guide RNA (PEgRNA) or one or more polynucleotides encoding the PEgRNA, wherein the PEgRNA comprises: i) a spacer sequence; ii) a gRNA core capable of complexing with a nucleic acid programmable DNA binding protein (napDNAbp) that is a nickase configured to cut a first strand of a double-stranded DNA sequence or a nuclease configured to cut both strands of the double-stranded DNA sequence; iii) an RNA extension arm comprising: (A) a DNA synthesis template comprising an edit template that encodes one or more edits compared a region downstream of the cut site in the first strand of the double-stranded DNA sequence, and (B) a primer binding site that is complementary to a region upstream of the cut site in the first strand of the double-stranded DNA sequence; and b) a prime editor, or one or more polynucleotides encoding the prime editor, the prime editor comprising: i) the nucleic acid programmable DNA binding protein (napDNAbp); and ii) a reverse transcriptase domain.
  2. 2 . The prime editing system of claim 1 , wherein the spacer sequence comprises a region of complementarity to a second strand of the double-stranded DNA sequence, and wherein the second strand is complementary to the first strand.
  3. 3 . The prime editing system of claim 1 , wherein the region upstream of the cut site to which the primer binding site is complementary is immediately 5′ of the cut site.
  4. 4 . The prime editing system of claim 1 , wherein the cut site is three nucleotides upstream of a protospacer adjacent motif (PAM).
  5. 5 . The prime editing system of claim 1 , wherein the primer binding site is from 7 to 17 nucleotides in length.
  6. 6 . The prime editing system of claim 1 , wherein the primer binding site is from 8 to 15 nucleotides in length.
  7. 7 . The prime editing system of claim 1 , wherein the spacer sequence is 20 nucleotides in length.
  8. 8 . The prime editing system of claim 7 , wherein the primer binding site comprises the reverse complement of nucleotides p to 17 of the spacer sequence, wherein p is an integer no greater than 13.
  9. 9 . The prime editing system of claim 1 , wherein the DNA synthesis template further comprises a homology arm that is complementary to a region downstream of the cut site in the first strand of the double-stranded DNA sequence.
  10. 10 . The prime editing system of claim 9 , wherein the homology arm is located 5′ of the edit template.
  11. 11 . The prime editing system of claim 10 , wherein the RNA extension arm comprises, from 5′ to 3′, the homology arm, the edit template, and the primer binding site.
  12. 12 . The prime editing system of claim 11 , wherein the edit template is directly adjacent to the primer binding site.
  13. 13 . The prime editing system of claim 1 , wherein the DNA synthesis template is from 10 to 16 nucleotides in length.
  14. 14 . The prime editing system of claim 1 , wherein the DNA synthesis template is from 12 to 17 nucleotides in length.
  15. 15 . The prime editing system of claim 1 , wherein the DNA synthesis template is less than 15 nucleotides in length.
  16. 16 . The prime editing system of claim 1 , wherein at least one of the edits encoded by the DNA synthesis template disrupts an endogenous PAM site associated with the spacer sequence.
  17. 17 . The prime editing system of claim 1 , wherein the PEgRNA is a single molecule comprising the spacer sequence, the gRNA core, and the RNA extension arm.
  18. 18 . The prime editing system of claim 17 , comprising in a 5′ to 3′ orientation: the spacer sequence, the gRNA core, and the RNA extension arm.
  19. 19 . The prime editing system of claim 1 , wherein the PEgRNA comprises at least one of: a modified nucleobase, a modified sugar, a modified phosphate group, or a nucleoside analog.
  20. 20 . The prime editing system of claim 1 , wherein the PEgRNA comprises one or more 3′ structures selected from the group consisting of linkers, stem loops, hairpins, toeloops, tetraloops, aptamers, and RNA-protein recruitment domains.

Description

RELATED APPLICATIONS This application is a continuation of and claims priority under 35 U.S.C. §§ 120 and 365(c) to U.S. patent application Ser. No. 18/064,738, filed Dec. 12, 2022, which is a continuation of and claims priority under 35 U.S.C. § 120 to U.S. patent application Ser. No. 17/219,635, filed Mar. 31, 2021, which claims priority under 35 U.S.C. §§ 120 and 365(c) to and is a continuation of International PCT Application PCT/US2020/023730, filed Mar. 19, 2020, which claims priority under 35 U.S.C. § 119 (c) to U.S. Provisional Application No. 62/820,813, filed Mar. 19, 2019, U.S. Provisional Application No. 62/858,958, filed Jun. 7, 2019, U.S. Provisional Application No. 62/889,996, filed Aug. 21, 2019, U.S. Provisional Application No. 62/922,654, filed Aug. 21, 2019, U.S. Provisional Application No. 62/913,553, filed Oct. 10, 2019, U.S. Provisional Application No. 62/973,558, filed Oct. 10, 2019, U.S. Provisional Application No. 62/931,195, filed Nov. 5, 2019, U.S. Provisional Application No. 62/944,231, filed Dec. 5, 2019, U.S. Provisional Application No. 62/974,537, filed Dec. 5, 2019, U.S. Provisional Application No. 62/991,069, filed Mar. 17, 2020, and U.S. Provisional Application No. 63/100,548, filed Mar. 17, 2020, the entire contents of each of which is incorporated herein by reference. GOVERNMENT SUPPORT This invention was made with government support under Grant Numbers AI142756, EB022376, GM007726, GM118062, GM954507, and HG009490 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING The contents of the electronic sequence listing (B119570096US06-SEQ-TNG.xml; Size: 5,406,784 bytes; and Date of Creation: Apr. 24, 2024) is herein incorporated by reference in its entirety. BACKGROUND OF THE INVENTION Pathogenic single nucleotide mutations contribute to approximately 50% of human diseases for which there is a genetic component,7 according to some estimates. Unfortunately, treatment options for patients with these genetic disorders remain extremely limited, despite decades of gene therapy exploration8. Perhaps the most parsimonious solution to this therapeutic challenge is direct correction of single nucleotide mutations in patient genomes, which would address the root cause of disease and would likely provide lasting benefit. Although such a strategy was previously unthinkable, recent improvements in genome editing capabilities brought about by the advent of the CRISPR/Cas system9 have now brought this therapeutic approach within reach. By straightforward design of a guide RNA (gRNA) sequence that contains ˜20 nucleotides complementary to the target DNA sequence, nearly any conceivable genomic site can be specifically accessed by CRISPR associated (Cas) nucleases1,2. To date, several monomeric bacterial Cas nuclease systems have been identified and adapted for genome editing applications10. This natural diversity of Cas nucleases, along with a growing collection of engineered variants11-14, offers fertile ground for developing new genome editing technologies. While gene disruption with CRISPR is now a mature technique, precision editing of single base pairs in the human genome remains a major challenge3. Homology directed repair (HDR) has long been used in human cells and other organisms to insert, correct, or exchange DNA sequences at sites of double strand breaks (DSBs) using donor DNA repair templates that encode the desired edits15. However, traditional HDR has very low efficiency in most human cell types, particularly in non-dividing cells, and competing non-homologous end joining (NHEJ) leads predominantly to insertion-deletion (indel) byproducts16. Other issues relate to the generation of DSBs, which can give rise to large chromosomal rearrangements and deletions at target loci17, or activate the p53 axis leading to growth arrest and apoptosis18,19. Several approaches have been explored to address these drawbacks of HDR. For example, repair of single-stranded DNA breaks (nicks) with oligonucleotide donors has been shown to reduce indel formation, but yields of desired repair products remain low20. Other strategies attempt to bias repair toward HDR over NHEJ using small molecule and biologic reagents21-23. However, the effectiveness of these methods is likely cell-type dependent, and perturbation of the normal cell state could lead to undesirable and unforeseeable effects. Recently, the inventors, led by Prof. David Liu et al., developed base editing as a technology that edits target nucleotides without creating DSBs or relying on HDR4-6,24-27. Direct modification of DNA bases by Cas-fused deaminase enzymes allows for C⋅G to T⋅A, or A⋅T to G⋅C, base pair conversions in a short target window (˜5-7 bases) with very high efficiency. As a result, base editors have been rapidly adopted by the scientific community. However, the following factors limit their generality for precision genome editing: (1) “by