Search

KR-20260062957-A - Class 2 Type V CRISPR-Cas Prime Edit

KR20260062957AKR 20260062957 AKR20260062957 AKR 20260062957AKR-20260062957-A

Abstract

The present invention relates to the field of gene genome editing. In particular, it relates to a CRISPR-Cas class 2 type V prime editing system comprising a prime editor, a prime editor complex, and a prime editing guide RNA system, as well as means and methods for modifying a nucleic acid sequence of interest using the prime editing system of the present invention.

Inventors

  • 드 블레스샤우어, 다비트
  • 묄레바터르, 프랑크
  • 쿠이이페르스, 아네-마리
  • 스탈스, 라이몬트 휘버르트 요세퍼
  • 반 데르 오스트, 존
  • 드'할루인, 카텔린
  • 스바르톄스, 토마스
  • 페르하겐, 한
  • 비예가스 와렌, 리카르도

Assignees

  • 바스프 아그리컬쳐럴 솔루션즈 유에스 엘엘씨

Dates

Publication Date
20260507
Application Date
20240827
Priority Date
20230831

Claims (20)

  1. Prime Editor Complex including the following: (a) at least one prime edit guide RNA (pegRNA) system as defined in any one of claims 11 to 18; and (b) At least one Cas12a Prime editor including the following: (i) at least one Cas12a enzyme, preferably having nikase activity (nCas12a), more preferably having non-target strand (NTS) nikase activity, or its active fragment; (ii) at least one reverse transcribtase, or its active fragment; (iii) Optionally: at least one linker that co-co-links at least one Cas12a enzyme, or its active fragment, and at least one reverse transcribtase, or its active fragment; and (iv) Optionally: at least one organelle localization signal.
  2. A prime editor complex according to claim 1, wherein at least one organelle localization signal is located at the N-terminus and/or C-terminus of at least one Cas12a enzyme, or its active fragment, and/or at least one reverse transcriptase, or its active fragment.
  3. The prime editor complex of claim 1 or 2, wherein at least one Cas12a enzyme, or an RNase dead Cas12a enzyme having an active fragment containing an H759A mutation, optionally containing a mutation at position H759, or its active fragment.
  4. A prime editor complex according to any one of claims 1 to 3, wherein the Cas12a prime editor comprises at least one ssDNA-binding and/or ssDNA-stabilizing protein, its domain or active fragment, preferably Brex27, RPA70-A, RPA70-B, RPA70-C, RPA32-D, BRCA2-OB2, BRCA2-OB3, HNRNPK KH, PUF60RRM, or Rad51DBD.
  5. A prime editor complex according to any one of claims 1 to 4, wherein (ii) at least one reverse transcriptase, or its active fragment, is connected to (iii) at least one Cas12a enzyme, or its active fragment, at the N-terminus or C-terminus, preferably at the N-terminus, through at least one linker.
  6. A prime editor complex according to any one of claims 1 to 5, wherein (c) preferably further comprises a reverse transcriptase protein comprising at least one organelle localization signal and preferably an MS2 coat protein (MCP).
  7. A prime editor complex according to any one of claims 1 to 6, wherein at least one Cas12a enzyme, or its active fragment, further comprises the N-terminal domain of an RNA binding factor, preferably La or MCP.
  8. In claim 7, comprising a reverse transcription protein as defined in claim 6 comprising an N-terminal domain of La and optionally an MCP, and a pegRNA system as defined in claim 13, and optionally a kking crRNA as defined in claim 18, wherein a) the crRNA comprises, in the 5' to 3' direction, (i) a CRISPR-Cas class 2 type V scaffold sequence, (ii) a spacer sequence; and optionally (vi) optionally a second scaffold sequence identical to the first scaffold sequence and/or; wherein b) The prime edit template RNA (petRNA) comprises an MS2 stem loop in the 5' to 3' direction, optionally a linker sequence, a reverse transcriptase template having a length of 9 to 150 nucleotides; a primer binding site having a length of 5 to 50 nucleotides; optionally a linker sequence; and an MS2 stem loop. Prime Editor Complex.
  9. In any one of claims 1 to 8, at least one Cas12a enzyme is an nCas12a comprising at least one mutation in a core read domain conferring nikase activity, optionally wherein at least one nCas12a comprises or is composed of an amino acid sequence selected from sequence identification numbers: 14 to 29, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to the same, and/or wherein at least one nCas12a is a sequence identification number: 14 starting at position 927 to A prime editor complex comprising a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with any one of 29 core read domains, or a corresponding core read domain.
  10. A prime editor complex according to any one of claims 1 to 9, wherein the Cas12a prime editor comprises or is composed of an amino acid sequence selected from sequence identification numbers: 65 to 69, 213, or 268 to 277, or a sequence having at least 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% sequence identity with respect to the same.
  11. As a prime edit guide RNA (pegRNA) system comprising the following parts: (i) CRISPR-Cas class 2 type V scaffold sequence; (ii) Spacer sequence; (iii) Random linker sequence; (iv) a reverse transcriptase template having a length of 9 to 150 nucleotides; (v) a primer binding site having a length of 5 to 50 nucleotides; and (vi) arbitrarily, arbitrarily a second scaffold sequence identical to the first scaffold sequence; Here, the primer binding site is complementary to the non-target strand. pegRNA system.
  12. In Paragraph 11, including the following: a) pegRNA comprising parts (i), (ii), optionally (iii), (iv), (v), and optionally (vi); or b) crisper RNA (crRNA) and prime edit template RNA (petRNA), where 1) The above crRNA comprises parts (i), (ii) and optionally (vi); 2) The above petRNA optionally comprises (iii), (iv) and (v). pegRNA system.
  13. In paragraph 12, the system comprises crRNA and petRNA, wherein the petRNA is a linear pegRNA system.
  14. A pegRNA system according to any one of claims 11 to 13, wherein the pegRNA or petRNA further comprises the following: (vii) optionally at least one 3' linker sequence; and (viii) at least one structured motif comprising at least one MS2 stem loop, preferably at least one hairpin comprising at least two MS2 stem loops, and/or at least one pseudonote sequence.
  15. A pegRNA system according to any one of claims 11 to 14, wherein (iv) the reverse transcriptase template has a length of 25 to 140 nucleotides, or 30 to 120 nucleotides, or 40 to 100 nucleotides, or 50 to 90 nucleotides, or 60 to 90 nucleotides.
  16. A pegRNA system according to any one of claims 11 to 15, wherein (v) the primer binding site has a length of 6 to 40 nucleotides, or 6 to 30 nucleotides, or 7 to 20 nucleotides, or 7 to 15 nucleotides, or 9 to 12 nucleotides.
  17. In any one of paragraphs 11 through 16, a) pegRNA comprises a portion (i), (ii), optionally (iii), (iv), (v), optionally (vi), and where present, optionally (vii) and (viii) in the 5' to 3' direction in the following order: 1) (i), (ii), optionally (iii), (iv), (v) and optionally (vi); or 2) (iv), (v), (iii), (i), (ii), optionally (vi); or 3) (i), (ii), optionally (iii), (iv), (v), optionally (vii), (viii) and optionally (vi); or b) the crRNA comprises a portion (i), (ii) as defined in any one of claims 11 to 16 in the 5' to 3' direction and optionally (vi); and the petRNA comprises a portion (viii) as defined in any one of claims 11 to 16 in the 5' to 3' direction, optionally (iii), (iv), (v), optionally (iii), and (viii). pegRNA system.
  18. A pegRNA system according to any one of claims 11 to 17, further comprising a kneading crRNA, wherein the kneading crRNA comprises a CRISPR-Cas class 2 type V scaffold sequence; a spacer sequence; and optionally a second scaffold sequence, wherein the target site of the spacer sequence of the kneading crRNA is located near the target site of the spacer sequence of the pegRNA or petRNA and on the opposite strand of the target site.
  19. As a Cas12a prime editor including the following: (i) at least one Cas12a enzyme having nikase activity (nCas12a), preferably having non-target strand nikase activity, or its active fragment; (ii) at least one reverse transcribtase, or its active fragment; (iii) at least one linker that co-links at least one nCas12a, or its active fragment, and at least one reverse transcribtase, or its active fragment, or at least one non-co-linked linker; (iv) at least one organelle localization signal at any time; Herein (i) at least one nCas12a is fused to (ii) at least one reverse transcriptase, and at least one organelle localization signal is located at the N-terminus of the Cas12a prime editor, and at least one organelle localization signal is located at the C-terminus of the Cas12a prime editor and/or; Herein, at least one nCas12a is an RNase dead nCas12a including the H759A mutation, optionally including a mutation at position H759, and/or; Herein, the Cas12a prime editor comprises at least one ssDNA-binding and/or ssDNA-stabilizing protein, its domain, or active fragment, preferably Brex27, RPA70-A, RPA70 B, RPA70-C, RPA32-D, BRCA2-OB2, BRCA2-OB3, HNRNPK KH, PUF60RRM, or Rad51DBD; Here, the Cas12a prime editor preferably comprises at least one organelle localization signal and preferably comprises a reverse transcriptase protein in trans that comprises an MS2 coat protein (MCP). Cas12a Prime Editor.
  20. In claim 19, the Cas12a prime editor comprises a reverse transcriptase protein in trans, and further comprises an RNA binding factor, preferably an N-terminal domain of La or MCP, more preferably wherein the Cas12a prime editor comprises an N-terminal domain of La, optionally a reverse transcriptase protein as defined in claim 6 comprising MCP, a pegRNA system as defined in claim 13, and optionally a nicking crRNA as defined in claim 18, wherein a) the crRNA comprises, in the 5' to 3' direction, (i) a CRISPR-Cas class 2 type V scaffold sequence, (ii) a spacer sequence, and optionally (vi) an optional second scaffold sequence identical to the first scaffold sequence; b) The prime edit template RNA (petRNA) comprises an MS2 stem loop in the 5' to 3' direction, optionally a linker sequence, a reverse transcriptase template having a length of 9 to 150 nucleotides; a primer binding site having a length of 5 to 50 nucleotides; optionally a linker sequence; and an MS2 stem loop. Cas12a Prime Editor.

Description

Class 2 Type V CRISPR-Cas Prime Edit The present invention relates to the field of gene genome editing. In particular, it relates to a CRISPR-Cas class 2 type V prime editing system comprising a prime editor, a prime editor complex, and a prime editing guide RNA system, as well as means and methods for modifying a nucleic acid sequence of interest using the prime editing system of the present invention. Prime editing is a "search-and-replace" genome editing tool that mediates targeted insertions, deletions, all 12 possible base-to-base swaps, and combinations thereof without requiring double-strand breaks (DSBs) or distinct donor DNA templates (ss/ds) (Anzalone et al., Nature. 2019 Dec; 576(7785):149-157). For prime editing, so-called prime editing guide RNAs (pegRNAs) are used. PegRNAs typically include a single guide RNA (sgRNA) with a target-specific sequence (spacer), and an extension containing a primer binding site (PBS) and a reverse transcriptase template (RTT) sequence to be introduced into the targeted DNA sequence. The PBS region is complementary to the non-target strand, and after base pairing, the non-target strand will generate primers for a reverse transcriptase (RT) that combine (via physical covalent linkage or as a standalone) with a guide RNA that binds to a Cas protein (typically a dsDNA nuclease or ssDNA nuclease (nicase), which generates specific DSBs or SSBs of the target DNA, respectively). Subsequently, the sequence of the RTT sequence is reverse transcribed from the pegRNA into the edited target DNA sequence. Three generations of prime editors were used in different target cells: PE1, PE2, and PE3 (Anzalone et al. cited above). PE1 is based on Molony murine leukemia virus reverse transcriptase (M-MLV RT). PE2 (referred to as pPE2 in plants) is based on the M-MLV RT D200N/L603W/T330P/T306K/W313F variant. PE3 (referred to as pPE3 in plants) uses an additional guide RNA that specifically targets non-edited strand DNA adjacent to the edited sequence (Marzec et al., Trends Cell Biol. 2020 Apr;30(4):257-259. doi: 10.1016/j.tcb.2020.01.004; Xu et al. Plant Commun. 2020 Apr 8;1(3):100043. doi: 10.1016/j.xplc.2020.100043; Lin et al. Nat Biotechnol. 2020 May;38(5):582-585. doi: 10.1038/s41587-020-0455-x.). It has been revealed that M-MLV RT can be exchanged for different RTs, such as cauliflower mosaic virus (CaMV) RT or retron-derived RT (Lin et al. cited above). PegRNA can be designed and optimized for different target sequences, cells, or constructs. For example, prime editing in plants is described in the literature [Sretenovic and Qi (Nat Plants. 2022 Jan;8(1):20-22. doi: 10.1038/s41477-021-01047-0)], and optimized prime editing in monocotyledonous plants is described in the literature [Jin et al. (Science. 2012 Aug 17;337(6096):816-21)]. Classic prime editors, as known in the relevant technical field, rely on the CRISPR-Cas9 (Class 2, Type II) Cas system. In this approach, Cas9 H840A nicase is used to generate an SSB that nicks a non-target strand, thereby exposing a 3'-hydroxyl group, which directly primes the reverse transcription of an edit-coding extension on pegRNA to the target site. However, despite the high level of interest in Class 2 Type V CRISPR-Cas systems, particularly Cas12a (Cpf1) systems, there is still a great need for functional Class 2 Type V CRISPR-Cas prime editing systems. One problem with Class 2 Type V CRISPR-Cas systems is that specific nikases equivalent to Cas9 H840A nikases are not available. Unlike Cas9, Cas12a uses a single catalytic site located in the RuvC domain to sequentially cleave both DNA strands, whereas the Nuc domain plays a role in substrate DNA regulation (Swarts et al., 2017 Molecular Cell. 2017 Apr 20;66(2):221-233.e4.doi: 10.1016/j.molcel.2017.03.016.; Swarts and Jinek, Molecular Cell. 2019 Feb 7;73(3):589-600.e4. doi: 10.1016/j.molcel.2018.11.021). These differences in structural organization hinder the true nikase design of Cas12a compared to Cas9, a CRISPR nuclease having two distinct domains, HNH and RuvC, which catalyze the cleavage of the target strand and non-target strand, respectively. Recently, a system referred to as CRISPR-based RNA-coded DNA substitution of alleles ("REDRAW") has been proposed (Kim et al. https://doi.org/10.1101/2022.12.13.520319). This system aims to overcome the lack of a specific Cas12a nikase by using a modified architecture in which spacer sequences and primer binding sites anneal to the same strand (target strand). However, the use of nucleases is associated with increased off-target effects and the generation of unwanted indels, which significantly reduces the efficiency of precise editing and necessitates complex and time-consuming steps (i.e., backcrossing in plants, or NHEJ inhibition in human cells) to remove these unwanted byproducts in subsequent stages. Accordingly, the object of the present invention is to provide a functional and efficient Class 2 Type V CRISPR-Cas prime editing system, prefera