WO2025153509A1 - Improved prime editing polypeptide - Google Patents

Abstract

A first aspect of the invention relates to a variant prime editing polypeptide composed of a SpCas9 nickase fused to an engineered Moloney Murine leukaemia virus (M-MLV) reverse transcriptase, characterized by a mutation, relative to a reference sequence SEQ ID NO 001, selected from the group consisting of A277D; K1865T; and S237A. Another aspect of the invention relates to a combination of a first and second split PE variant polypeptide comprising split intein component at its C- and N-terminus, respectively, which, when both are present within a target cell, are capable of forming a fusion polypeptide comprising a functional prime editing polypeptide according to the first aspect of the invention. Other aspects of the invention relate to a combination medicaments and nucleic acid sequences facilitating the PE variant of the invention.

Description

Improved Prime Editing Polypeptide

This application claims the right of priority of European application no. 24152221.8, filed 16 January 2024, which incorporated herein by reference.

Field

The present invention relates to improved variants of the prime editing enzyme comprised of a SpCasO nickase fused to an engineered reverse transcriptase (RT) derived from Moloney Murine leukaemia virus, as well as to systems employing and nucleic acids encoding improved variants.

Background

Prime editors (PEs) are capable of rewriting genetic information in the genome when administered together with a prime editing guide RNA (pegRNA). PEs comprise a SpCasO nickase (H840A - hereafter referred to as nCas9) fused to an engineered reverse transcriptase (RT) derived from Moloney Murine leukaemia virus (M-MLV). pegRNAs are based on a Cas9 guide RNA, fused at the 3’ end to a reverse transcriptase template (RTT) sequence and a primer binding site (PBS). When the PE ribonucleoprotein (RNP) complex binds the genomic target, the non-target strand is nicked, binds to the complementary PBS, and primes reverse transcription of the RTT segment of the pegRNA. Prime editing enables the installation of insertions, deletions and all 12 possible nucleotide conversions.

Despite their high versatility, PEs are comparably less efficient than classical Cas9 nucleases that operate by inducing DNA double strand breaks, or base editors (BEs) which modify DNA via deamination. Recent studies therefore employed rational design to increase the performance of prime editing, for example by protecting the pegRNA from exonucleases to generate enhanced pegRNAs (epegRNAs), or by altering cellular DNA mismatch repair. Similarly, optimization of the codon usage and domain architecture have resulted in an optimized PE protein (PEmax) (Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635-5652. e29 (2021 )). However, to our knowledge, directed protein evolution, which has previously been adapted to increase the targeting scope and efficiency of Cas9 nucleases and BEs8-13, has not yet been applied in the context of prime editing.

Based on the above-mentioned state of the art, the objective of the present invention is to provide means and methods to improve on current base editing technology. This objective is attained by the subject-matter of the independent claims of the present specification, with further advantageous embodiments described in the dependent claims, examples, figures and general description of this specification. Summary of the Invention

A first aspect of the invention relates to a variant polypeptide, which is derived from a prime editing (PE) polypeptide composed of a SpCas9 nickase (H840A - hereafter referred to as nCas9) fused to an engineered Moloney Murine leukaemia virus (M-MLV) reverse transcriptase (RT). The variant is characterized by an enhancing mutation, relative to a reference sequence SEQ ID NO 001 , selected from the group consisting of A277D; K1865T; and S237A.

Another aspect of the invention relates to combination of polypeptides that contain split intein elements which allow expressing and reconstituting the full prime editor polypeptide from the combination. This combination consists of a first split PE variant polypeptide comprising an N- terminal part of a split intein component at its C-terminus, and a second split PE variant polypeptide comprising a C-terminal part of a split intein component at its N-terminus. The first split PE variant polypeptide and the second split PE variant polypeptide, when both are present within a target cell, are capable of forming a fusion polypeptide comprising a functional prime editing polypeptide according to the first aspect of the invention.

Another aspect of the invention relates to a combination medicament, that comprises a first viral vector and a second viral vector encoding the PE polypeptide.

Terms and definitions

General

For purposes of interpreting this specification, the following definitions will apply and whenever appropriate, terms used in the singular will also include the plural and vice versa. In the event that any definition set forth below conflicts with any document incorporated herein by reference, the definition set forth shall control.

The terms “comprising”, “having”, “containing”, and “including”, and other similar forms, and grammatical equivalents thereof, as used herein, are intended to be equivalent in meaning and to be open-ended in that an item or items following any one of these words is not meant to be an exhaustive listing of such item or items, or meant to be limited to only the listed item or items. For example, an article “comprising” components A, B, and C can consist of (i.e., contain only) components A, B, and C, or can contain not only components A, B, and C but also one or more other components. As such, it is intended and understood that “comprises” and similar forms thereof, and grammatical equivalents thereof, include disclosure of embodiments of “consisting essentially of or “consisting of.” In particular embodiments relating to nucleic acid sequences, “consisting essentially of refers to a sequence comprising the referenced elements, and no more than 2% of excess nucleotides, in reference to the full sequence length, joining or framing the elements. In particular embodiments relating to amino acid sequences, “consisting essentially of refers to a sequence comprising the referenced elements, and no more than 2% of excess amino acid residues, particularly glycine, serine or alanine residues, in reference to the full sequence length, joining or framing the elements.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit, unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

Reference to “about” a value or parameter herein includes (and describes) variations that are directed to that value or parameter per se. For example, description referring to “about X” includes description of “X.”

As used herein, including in the appended claims, the singular forms “a”, “or” and “the” include plural referents unless the context clearly dictates otherwise.

"And/or" where used herein is to be taken as specific recitation of each of the two specified features or components with or without the other. Thus, the term "and/or" as used in a phrase such as "A and/or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and/or" as used in a phrase such as "A, B, and/or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques and biochemistry, organic synthesis). Standard techniques are used for molecular, genetic, and biochemical methods (see generally, Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed. (2012) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. and Ausubel et al., Short Protocols in Molecular Biology (2002) 5th Ed, John Wiley & Sons, Inc.) and chemical methods.

Any patent document cited herein shall be deemed incorporated by reference herein in its entirety.

Sequences

Sequences similar or homologous (e.g., at least about 85% sequence identity) to the sequences disclosed herein are also part of the invention. In some embodiments, the sequence identity at the amino acid level can be about 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. At the nucleic acid level, the sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively, substantial identity exists when the nucleic acid segments will hybridize under selective hybridization conditions (e.g., very high stringency hybridization conditions), to the complement of the strand. The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form. In the context of the present specification, the terms sequence identity and percentage of sequence identity refer to a single quantitative parameter representing the result of a sequence comparison determined by comparing two aligned sequences position by position. Methods for alignment of sequences for comparison are well-known in the art. Alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981 ), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988) or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analyses is publicly available, e.g., through the National Center for Biotechnology-Information (http://blast.ncbi.nlm.nih.gov/).

One example for comparison of amino acid sequences is the BLASTP algorithm that uses the default settings: Expect threshold: 10; Word size: 3; Max matches in a query range: 0; Matrix: BLOSUM62; Gap Costs: Existence 11 , Extension 1 ; Compositional adjustments: Conditional compositional score matrix adjustment. One such example for comparison of nucleic acid sequences is the BLASTN algorithm that uses the default settings: Expect threshold: 10; Word size: 28; Max matches in a query range: 0; Match/Mismatch Scores: 1 .-2; Gap costs: Linear. Unless stated otherwise, sequence identity values provided herein refer to the value obtained using the BLAST suite of programs (Altschul et al., J. Mol. Biol. 215:403-410 (1990)) using the above identified default parameters for protein and nucleic acid comparison, respectively.

Reference to identical sequences without specification of a percentage value implies 100% identical sequences (i.e. the same sequence).

The term having substantially the same biological activity in the context of the present invention relates to the prime editing function of the Y_17 polypeptide, i.e. Prime Editing activity as assayed according to the protocol included below as Example 7.

General Biochemistry: Peptides, Amino Acid Sequences

The term polypeptide in the context of the present specification relates to a molecule consisting of 50 or more amino acids that form a linear chain wherein the amino acids are connected by peptide bonds. The amino acid sequence of a polypeptide may represent the amino acid sequence of a whole (as found physiologically) protein or fragments thereof. The term "polypeptides" and "protein" are used interchangeably herein and include proteins and fragments thereof. Polypeptides are disclosed herein as amino acid residue sequences.

Amino acid residue sequences are given from amino to carboxyl terminus. Capital letters for sequence positions refer to L-amino acids in the one-letter code (Stryer, Biochemistry, 3^rd ed. p. 21 ). Lower case letters for amino acid sequence positions refer to the corresponding D- or (2R)- amino acids. Sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gin, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (lie, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (Vai, V).

The term variant refers to a polypeptide that differs from a reference polypeptide, but retains essential properties. A typical variant of a polypeptide differs in its primary amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical. A variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions). A substituted or inserted amino acid residue may or may not be one encoded by the genetic code. A variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.

In the context of the present specification, the term amino acid linker or peptide linker refers to a polypeptide of variable length that is used to connect two polypeptides in order to generate a single chain polypeptide. Exemplary embodiments of linkers useful for practicing the invention specified herein are oligopeptide chains consisting of 5, 10, 20, 30, 40 or 50 amino acids. The example shows a linker between the SpCasO and the M-MLV RT that consists of 34 amino acids and contains the sequence SGGSSGGSKR TADGSEFESP KKKRKVSGGS SGGS (SEQ ID NO 012).

There is no constraint on the amino acid composition of the linker. In certain embodiments, the linker consists of amino acids selected from the group of G S, A and D. An important characteristic of the conjugate peptide linkers as specified above are low immunogenicity, and a peptide length that allows the domains which are joined by the linker, to interact to form a functional entity as disclosed herein. In particular desirable embodiments of the domain peptide linkers specified above, the sequences are primarily made up of stretches of amino acids such as glycine (G) and serine (S).

In certain embodiments peptide linker is >15 amino acids in length, particularly 15 to 30 amino acids in length wherein the amino acids are selected from G S, A and D.

A non-limiting example of an amino acid linker is a monomer or di-, tri- or tetramer of a peptide motif composed of three or four glycine and one serine.

Any embodiments relating peptide linkers as disclosed herein, encompass structures in which amino acids with similar characteristics are exchanged, for example, the amino acids V, L, I, P, S, C, or M may replace G, S, or S, and D may be replaced by E.

The term nucleic acid expression vector in the context of the present specification relates to a plasmid, a viral genome or an RNA, which is used to transfect (in case of a plasmid or an RNA) or transduce (in case of a viral genome) a target cell with a certain gene of interest, or -in the case of an RNA construct being transfected- to translate the corresponding protein of interest from a transfected mRNA. For vectors operating on the level of transcription and subsequent translation, the gene of interest is under control of a promoter sequence and the promoter sequence is operational inside the target cell, thus, the gene of interest is transcribed either constitutively or in response to a stimulus or dependent on the cell’s status. In certain embodiments, the viral genome is packaged into a capsid to become a viral vector, which is able to transduce the target cell.

The term "pegRNA" in the context of the present specification relates to "prime editing guide RNA", which serves as a guide RNA molecule designed to direct the prime editing machinery to a specific target site in the genome. It contains two key regions:

Target Binding Region: This part of the pegRNA is complementary to the DNA sequence at the genomic site where the edit is intended. It helps the prime editing complex locate the correct spot for the edit.

Primer Binding Region: The primer binding region of the pegRNA serves as a template for the synthesis of the new DNA strand during the editing process. This region guides the insertion of the desired genetic information into the genome.

The terms capable of forming a hybrid or hybridizing sequence in the context of the present specification relate to sequences that under the conditions existing within the cytosol of a mammalian cell, are able to bind selectively to their target sequence. Such hybridizing sequences may be contiguously reverse-complementary to the target sequence, or may comprise gaps, mismatches or additional non-matching nucleotides. The minimal length for a sequence to be capable of forming a hybrid depends on its composition, with C or G nucleotides contributing more to the energy of binding than A or T/U nucleotides, and on the backbone chemistry.

Detailed Description of the Invention

The inventors employed Orthogonal DNA replication (OrthoRep; Ravikumar, A., Arrieta, A. & Liu, C. C. Nat Chem Biol AO, 175-177 (2014)), a semi-continuous protein evolution approach in yeast, to increase the activity of PEmax in a eukaryotic environment. Variants resulting from four rounds of evolution showed increased processivity in vitro in a cell lysate assay. Recombining the mutations of the two best-performing variants resulted in the superior variant PE_Y18, which contains amino acid substitutions A259D in nCas9 (the positions A259D and S219A are relative to the Cas9 protein (SEQ ID NO 007), not to SEQ ID NO 001 , where these positions are designated A277D and S237A, respectively) and K445T in the M-MLV RT (this position is relative to the M- MLV RT protein SEQ ID NO 008, not to SEQ ID No 001 , where this position is designated K1865T). PE_Y18 shows enhanced activity in cell lines when delivered as a plasmid, mRNA, or as ribonucleoprotein complex. Furthermore, PE_Y18 exhibited increased activities in the mouse brain and the liver upon AW-mediated delivery. A first aspect of the invention relates to a variant polypeptide, which is derived from a prime editing (PE) polypeptide composed of a SpCas9 nickase (H840A - hereafter referred to as nCas9) fused to an engineered Moloney Murine leukaemia virus (M-MLV) reverse transcriptase (RT). The variant is characterized by an enhancing mutation, relative to a reference sequence SEQ ID NO 001 , selected from the group consisting of A277D; K1865T; and S237A.

In certain embodiments, the variant lacks RNAse H activity. RNAseH activity is not strictly required for prime editing, and if the size that can be suited inside of a vector delivering the PE enzyme puts strict limits on enzyme size, the RNAseH domain can be deleted.

The RnaseH is present in SEQ ID NO 01-03 but is missing in SEQ ID NO 04-05. SEQ ID NO 04 and 05 have an RNAseH at the C-terminal position of the M-MLV RT, hence should not affect the counting.

In one embodiment, the variant according to the invention only comprises the enhancing mutations A277D and K1865T (construct Y_18). Of note, A277D is in relation again to the numeration of SEQ ID NO 001 , and will be A259D in relation to the numeration of the nCas9 domain (SEQ ID NO 007), and K1865T is also in relation again to the numeration of SEQ ID NO 001 , and will be K445T in relation to the numeration of the M-MLV domain (SEQ ID NO 008).

In certain embodiments, the variant that comprises the enhancing mutations A277D and K1865T is at least (>) 95% identical to SEQ ID NO 002. In particular embodiments, the variant is >98% identical to SEQ ID NO 002. In even more particular embodiments, the variant is >99% identical to SEQ ID NO 002, or is 100% identical to that sequence. Identity of <100% to SEQ ID NO 002 means that the variant may show small variations caused by neutral deletions, insertions or substitutions outside of the indicated positions 277 and 1865 relative to SEQ ID NO 002, while maintaining the prime editing activity of SEQ ID NO 002 as determined according to Example 7 of the present specification.

In one embodiment, the variant according to the invention only comprises the enhancing mutations S237A and K1865T (construct Y_17). Again, the position 237 is relative to SEQ ID NO 001 , whereas the position is designated S219A in relation to nCas9).

In certain embodiments, the variant that comprises the enhancing mutations enhancing mutations S237A and K1865T is at least (>) 95% identical to SEQ ID NO 003. In particular embodiments, the variant is >98% identical to SEQ ID NO 003. In even more particular embodiments, the variant is >99% identical to SEQ ID NO 003, or is 100% identical to that sequence. Identity of <100% to SEQ ID NO 003 means that the variant may show small variations caused by neutral deletions, insertions or substitutions outside of the indicated positions 277 and 1865 relative to SEQ ID NO 003, while maintaining the prime editing activity of SEQ ID NO 003 as determined according to Example 7 of the present specification. In one embodiment, the variant according to the invention comprises all three of the enhancing mutations S237A, A277D and K1865T. The variant may show small variations caused by neutral deletions, insertions or substitutions outside of the indicated positions 237, 277 and 1865 relative to SEQ ID NO 001 (SEQ ID NO 001 being mutated to S237A, A277D and K1865T as indicated) while maintaining the prime editing activity of the reference construct (SEQ ID NO 001 with S237A, A277D and K1865T) as determined according to Example 7 of the present specification.

In one embodiment, the variant according to the invention only comprises the enhancing mutation K1865T. The variant may show small variations caused by neutral deletions, insertions or substitutions outside of the indicated position 1865 relative to SEQ ID NO 001 (SEQ ID NO 001 being mutated to K1865T) while maintaining the prime editing activity of the reference construct (SEQ ID NO 001 with K1865T) as determined according to Example 7 of the present specification.

Another aspect of the invention relates to a nucleic acid sequence encoding the PE polypeptide variant according to the first aspect of this invention. The nucleic acid may be a DNA or RNA. In particular embodiments, the nucleic acid may be a DNA expression vector encoding the PE polypeptide under transcriptional control of an mRNA (Poll 11) polymerase operable in a mammalian cell. In other particular embodiments, the nucleic acid may be an mRNA molecule.

Likewise, the PE molecule may be encoded by a viral vector, such as a attenuated herpesvirus, a lentivirus, or any other viral vector used in medical gene transfer.

Another aspect of the invention relates to combination of polypeptides that contain split intein elements which allow expressing and reconstituting the full prime editor polypeptide from the combination. This allows bypassing size restrictions of PE enzymes in gene delivery technology.

According to this aspect of the invention, the combination consists of a first split PE variant polypeptide comprising an N-terminal part of a split intein component at its C-terminus, and a second split PE variant polypeptide comprising a C-terminal part of a split intein component at its N-terminus. The first split PE variant polypeptide and the second split PE variant polypeptide, when both are present within a target cell, are capable of forming a fusion polypeptide comprising a functional prime editing polypeptide according to the first aspect set forth herein.

In certain embodiments, the combination according to this aspect of the invention comprises a first split PE variant polypeptide that is characterized by SEQ ID NO 004, or a sequence at least 95% identical to SEQ ID NO 004 and having -when joined to the second split PE variant polypeptide- the same biological activity as SEQ ID NO 004 when joined to SEQ ID NO 005; and a second split PE variant polypeptide that comprises SEQ ID NO 005, or a sequence at least 95% identical to SEQ ID NO 005 and having -when joined to the first split PE variant polypeptide- the same biological activity as SEQ ID NO 004 when joined to SEQ ID NO 005. The biological activity of SEQ ID NO 004 joined to SEQ ID NO 005 is determined according to Example 7 of the current specification.

In certain particular embodiments, said N-terminal part of a split intein component and said C- terminal part of a split intein component are, or are derived from, a split intein system found in an organism of the group comprising the cyanobacterium Nostoc punctiforme (Npu), Mxe intein from Mycobacterium xenopi GyrA, DnaE and Rma intein from Rhodothermus marinus.

In more particular embodiments, the split intein is the Nostoc punctiforme split intein.

Another aspect of the invention relates to a combination medicament, in other words to a pharmaceutical composition or kit of parts that comprises a first viral vector and a second viral vector. The first viral vector encodes the first split PE variant polypeptide as specified in any one of the preceding embodiments laid out herein; the second viral vector encoding said second split PE variant polypeptide as specified in any one of the preceding embodiments laid out herein. The combination medicament may further comprise a viral vector encoding a pegRNA capable of functionally interacting with said functional prime editing polypeptide. This viral vector that encodes the pegRNA may be the same vector that encodes one or the other of the split variant peptides, or it may be a separate vector. Practicality suggests that encoding the pegRNA along with either of the polypeptides may be a particularly simple way to achieve expression of the fully active PE enzyme.

In certain embodiments, the pegRNA comprises, or essentially consists of, from 5’ to 3’ end, a guide RNA sequence tract capable of hybridizing to a genomic DNA target adjacent sequence, a structural RNA sequence tract facilitating interaction with the PE variant polypeptide and trans-activation of the PE variant polypeptide, a template sequence tract containing a sequence reverse complementary to an edited target sequence and a hybridizing sequence tract; and

The pegRNA is capable of interacting with the PE variant polypeptide to yield a prime editing protein RNA complex.

In certain embodiments of the combination medicament, the first viral vector and the second viral vector are both an Adeno-associated virus-based vector. In certain particular embodiments thereof, the first viral vector and the second viral vector are both an AAV2 vector.

In certain other embodiments of the combination medicament, the first viral vector and the second viral vector are both a nonintegrating lentiviral vector.

In certain embodiments of the PE variant according to the first aspect of the invention, or of the combination of split PE variants, or the combination medicament, the SpCAS9 nickase is streptococcus pyogenes CAS9 H840A; and the engineered MMLV RT is a Moloney murine leukemia virus reverse transcriptase lacking the RNAseH domain.

Another aspect of the invention relates to a prime editing protein RNA complex comprising a variant according to any one of the aspects and embodiments disclosed herein, wherein the prime editing protein RNA complex comprises a pegRNA comprising the structural RNA sequence tract is SEQ ID NO. 006; and a polypeptide comprising or consisting of SEQ ID NO 002 or SEQ ID NO 003.

Likewise, the invention encompasses a prime editing protein RNA complex comprising a variant characterized by a sequence at least 85% identical, particularly >90% identical, more particularly >95% identical to SEQ ID NO 002 and having at least the same biological activity as SEQ ID NO 003.

The same biological activity as SEQ ID NO 003 is defined as the activity determined in Example 7 by the method described therein.

Another aspect of the invention relates to a combination medicament as described herein, wherein a. the target sequence addressed by the guide RNA sequence tract in the pegRNA is characteristic of a genetic condition in a mammal, particularly a human, characterized by a transition or transversion, or a deletion or insertion mutation of a wild-type sequence, b. and the template sequence tract is characteristic of the reverse complimentary sequence of the wild-type sequence.

Thus, the combination medicament is capable of reverting a disease-associated target sequence to a wild-type, non-disease associated sequence, thereby ameliorating the symptoms of a disease, or treating the disease to curation.

In particular embodiments, the genetic condition is associated to expression of the target sequence in the eye, liver, CNS I brain, myocard, lung, or muscle, particularly in the liver, the brain or the eye.

Another aspect of the invention relates to a nucleic acid encoding a prime editing protein RNA complex, comprising a first nucleic acid sequence encoding a variant of a prime editing polypeptide as specified in the first aspect of the inveiton, and a second nucleic acid sequence encoding a pegRNA, particularly a pegRNA comprising the structural RNA sequence tract SEQ ID NO. 006, both first and second nucleic acid sequences being under control of a promoter operable in a mammalian cell.

An alternative of this aspect of the invention relates to a viral vector comprising the nucleic acid as described in the preceding paragraph. In particular embodiments, the viral vector is an Adenovirus (AdV). In more particular embodiments, the viral vector is a human AdV. In yet even more particular embodiments, the viral vector is a human AdV5.

Yet another aspect of the invention relates to a composition comprising a prime editing (PE) polypeptide variant according to any one of the embodiments laid out as the first aspect of the invention, and a pegRNA. It may be advantageous to provide the PE variant in protein form, and the pegRNA in the form in which it is associated with the PE variant. The pegRNA comprises, or essentially consists of, from 5’ to 3’ end, a guide RNA sequence tract capable of hybridizing to a genomic target adjacent sequence, a structural RNA sequence tract facilitating interaction with the PE variant polypeptide and trans-activation of the PE variant polypeptide, a template sequence tract containing a sequence reverse complementary to an edited target sequence and a hybridizing sequence tract; and the pegRNA being capable of interacting with the PE variant polypeptide to yield a prime editing protein RNA complex.

In certain embodiments, the composition comprises a pegRNA with a guide RNA whose target sequence is characteristic of a genetic condition in a mammal, particularly a human, characterized by a transition or transversion, or a deletion or insertion mutation of a wild-type sequence, and further the pegRNA template sequence tract is characteristic of the reverse complimentary sequence of the wild-type sequence.

In particular embodiments, the genetic condition thus addressed is associated to expression of the target sequence in the eye, liver, CNS I brain, myocard, lung, or muscle, particularly in the liver, the brain or the eye.

Also encompassed is a combination that combines a virus or DNA expression vector, or an mRNA, encoding the PE variant as specified herein, and the pegRNA molecule.

The invention further encompasses the following items:

1 . A variant of a prime editing (PE) polypeptide composed of a SpCas9 nickase fused to an engineered Moloney Murine leukaemia virus reverse transcriptase (RT) wherein said variant is characterized by an enhancing mutation, the mutation being assigned in reference to the numbering of SEQ ID NO 001 , selected from the group consisting of A277D; K1865T; and S237A.

2. The variant according to item 1 , only comprising the enhancing mutations A277D and K1865T. 3. The variant according to item 2, wherein the variant is at least (>) 95% identical to SEQ ID NO 002, particularly wherein the variant is >98%, or even >99% identical to SEQ ID NO 002.

4. The variant according to item 2, wherein the variant is SEQ ID NO 002.

5. The variant according to item 1 , only comprising the enhancing mutations S237A and K1865T.

6. The variant according to item 5, wherein the variant is at least (>) 95% identical to SEQ ID NO 003, particularly wherein the variant is >98%, or even >99% identical to SEQ ID NO 003.

7. The variant according to item 5, wherein the variant is SEQ ID NO 003.

8. The variant according to item 1 , comprising all of the enhancing mutations S237A, A277D and K1865T.

9. The variant according to item 1 , only comprising the enhancing mutation K1865T.

10. A nucleic acid sequence encoding the variant according to any one of items 1 to 9.

11 . The nucleic acid sequence according to item 10, wherein the nucleic acid is a DNA sequence, particularly a DNA sequence under transcriptional control of an POLIII RNA polymerase promoter operable in a mammalian cell.

12. The nucleic acid sequence according to item 10, wherein the nucleic acid is an mRNA sequence.

13. The nucleic acid sequence according to any one of items 10 to 12, wherein the nucleic acid is a viral sequence.

14. A combination of split PE variant polypeptides a. a first split PE variant polypeptide comprising an N-terminal part of a split intein component at its C-terminus; and b. a second split PE variant polypeptide comprising a C-terminal part of a split intein component at its N-terminus; wherein the first split PE variant polypeptide and the second split PE variant polypeptide, when both are present within a target cell, are capable of forming a fusion polypeptide comprising a functional prime editing polypeptide as specified in any one of the preceding items.

15. The combination of split PE variant polypeptides according to item 14, wherein a. said first split PE variant polypeptide is characterized by SEQ ID NO 004, or a sequence at least 95% identical to SEQ ID NO 004 and having -when joined to the second split PE variant polypeptide- the same biological activity as SEQ ID NO 004 when joined to SEQ ID NO 005; and b. said second split PE variant polypeptide comprises SEQ ID NO 005, or a sequence at least 95% identical to SEQ ID NO 005 and having -when joined to the first split PE variant polypeptide- the same biological activity as SEQ ID NO 004 when joined to SEQ ID NO 005.

16. The combination of split PE variant polypeptides according to any one of items 14 or 15, wherein said N-terminal part of a split intein component and said C-terminal part of a split intein component are, or are (i.e. having >85% identity on the amino acid sequence level with), a split intein system found in an organism selected from the group comprising the cyanobacterium Nostoc punctiforme (Npu), Mxe intein from Mycobacterium xenopi GyrA, DnaE and Rma intein from Rhodothermus marinus.

17. The combination of split PE variant polypeptides according to item 16, wherein the split intein is the Nostoc punctiforme split intein.

18. A combination medicament comprising a first viral vector and a second viral vector, a. the first viral vector encoding said first split PE variant polypeptide as specified in any one of items 14 to 17; b. the second viral vector encoding said second split PE variant polypeptide as specified in any one of items 14 to 17; c. the combination medicament further comprising a viral vector, particularly the first or second viral vector, further encoding a pegRNA, the pegRNA comprising, or essentially consisting of, from 5’ to 3’ end, i. a guide RNA sequence tract capable of hybridizing to a genomic target adjacent sequence,

II. a structural RNA sequence tract facilitating interaction with the PE variant polypeptide and trans-activation of the PE variant polypeptide, ill. a template sequence tract containing a sequence reverse complementary to an edited target sequence and iv. a hybridizing sequence tract; and v. the pegRNA being capable of interacting with the PE variant polypeptide to yield a prime editing protein RNA complex.

19. The combination medicament according to item 18, wherein the first viral vector and the second viral vector are a nonintegrating lentiviral vector. 20. The combination medicament according to item 18, wherein the first viral vector and the second viral vector are an Adeno-associated virus-based vector.

21 . The combination medicament according to item 20, wherein the first viral vector and the second viral vector are an AAV2 vector.

22. A prime editing protein RNA complex comprising a variant according to any one of the preceding items 1 to 9, wherein the prime editing protein RNA complex comprises a. a pegRNA comprising the structural RNA sequence tract is SEQ ID NO 006; and b. a polypeptide comprising or consisting of SEQ ID NO 002 or SEQ ID NO 003, or a sequence at least 85% identical, particularly >90% identical, more particularly >95% identical to SEQ ID NO 002 and having at least the same biological activity as SEQ ID NO 003.

23. The combination medicament according to any one of items 18 to 21 , wherein a. the target sequence is characteristic of a genetic condition in a mammal, particularly a human, characterized by a transition or transversion, or a deletion or insertion mutation of a wild-type sequence, b. and the template sequence tract is characteristic of the reverse complimentary sequence of the wild-type sequence.

24. The combination medicament according to item 23, wherein the genetic condition is associated to expression of the target sequence in the eye, liver, CNS I brain, myocard, lung, or muscle, particularly in the liver, the brain or the eye.

25. A nucleic acid encoding a prime editing protein RNA complex, comprising a first nucleic acid sequence encoding a variant of a prime editing polypeptide as specified in any one of items 1 to 8, and a second nucleic acid sequence encoding a pegRNA, particularly a pegRNA comprising the structural RNA sequence tract SEQ ID NO 006, both first and second nucleic acid sequences being under control of a promoter operable in a mammalian cell.

26. A viral vector comprising the nucleic acid according to item 25.

27. The viral vector according to item 26, wherein the viral vector is an Adenovirus (AdV).

28. The viral vector according to item 26, wherein the viral vector is a human AdV, particularly a human AdV5.

29. A composition comprising a prime editing (PE) polypeptide variant according to any one of items 1 to 9, and a pegRNA, the pegRNA comprising, or essentially consisting of, from 5’ to 3’ end, i. a guide RNA sequence tract capable of hybridizing to a genomic target adjacent sequence,

II. a structural RNA sequence tract facilitating interaction with the PE variant polypeptide and trans-activation of the PE variant polypeptide, ill. a template sequence tract containing a sequence reverse complementary to an edited target sequence and iv. a hybridizing sequence tract; and v. the pegRNA being capable of interacting with the PE variant polypeptide to yield a prime editing protein RNA complex.

30. The composition according to item 29, wherein a. the target sequence is characteristic of a genetic condition in a mammal, particularly a human, characterized by a transition or transversion, or a deletion or insertion mutation of a wild-type sequence, b. and the template sequence tract is characteristic of the reverse complimentary sequence of the wild-type sequence.

31 . The composition according to item 30, wherein the genetic condition is associated to expression of the target sequence in the eye, liver, CNS I brain, myocard, lung, or muscle, particularly in the liver, the brain or the eye.

Wherever alternatives for single separable features are laid out herein as “embodiments”, it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

Description of the Figures

Fig. 1 shows evolution of PE variants with enhanced activity in OrthoRep. (a) Parallel evolution of PE by culturing yeast cells in 96 well plates over four subsequent rounds in L-histidine-depleted selection media. In the first round of evolution, outgrowing yeast cells were normalized prior to extraction of p1 via PCR and transformation into fresh host cells for the second round. The same procedure was repeated for the third and fourth round, (b) PE variants containing mutations that increase prime editing rates are enriched in selective conditions: a stop codon in front of the auxotrophic marker gene HIS3 must be repaired by prime editing for successful yeast growth in selective conditions, (c) Effect of evolved PE variants on yeast growth under selective conditions. The assessed variants contained the following mutations: PE_Y17 (nCas9 S219A (S237A in SEQ ID NO 001 ), RT K445T), D2 (nCas9 A259D), A10 (RT Y64W, K373R, R389C, L432M, K445T), E11 (RT Y64W), B4 (RT R44H), H8 (nCas9 S219A, RT K445T), G3 (nCas9 A259D, linker G30S, RT R389C, S606A), H1 (nCas9 A259D, RT Y64W), H3 (nCas9 A259D) E5 (nCas9 S219A, RT K445T), A1 (nCas9 A259D, linker G30S, RT R389C, S606A), A5 (nCas9 S219A, RT Y64C, K445T), A8 (nCas9 S219A, linker G30S, RT K445T), F6 (nCas9 A259D, RT R44H), E3 (nCas9 S320R), B3 (nCas9 R71 C, A259D, linker G30S, RT K445T), A6 (nCas9 S318N, RT R389C, L432M), B8 (nCas9 S320N ,RT K373R), C8 (nCas9 Y132C), G5 (nCas9 A259D.RT K373R), A2 (nCas9 S219A, RT K373R, K445T). (d) Schematic of the in vitro assay to assess PE kinetics (PEKIN). PE variants are subcloned as P2A-GFP fusion proteins and transfected into HEK293T cells. Cells are lysed and PE expression levels are normalized by fluorescence intensity. An in vitro transcribed pegRNA forms the RNP with the PE, which is incubated with a synthetic dsDNA substrate, (e) Schematics illustrating the strategy used to quantify PE activity in PEKIN via qPCR. (f) Quantified prime editing activities of the 21 PE variants isolated after four rounds of evolutions in OrthoRep relative to PEmax. Product formation of the variants is relative to the product formed by PEmax.

Fig. 2 shows in vitro characterization of the activity of PE variants using cell lysates and purified proteins, (a) Quantification of PE activity with increasing concentrations of dsDNA substrate for PEmax (black), five selected PE variants obtained from OrthoRep (solid lines), and recombined versions of these variants (dashed lines), (b) Schematic illustration of constructs that were characterized in PEKIN as purified proteins. PEmax was compared to the evolved variants PE_Y17 and PE_Y18 in a tethered and untethered form, (c) Quantification of prime editing rates of purified proteins in the PEKIN assay in the tethered (solid lines) and untethered form (dashed lines). Virtual products are calculated from respective Ct values, (d) Scheme of the assay used to determine nicking activity via double nicking of a synthetic DNA template. Dark gray illustrates the non-target strand, whereas the target strand is illustrated in light gray, (e) Schematic of the adapted assay to disentangle reverse transcription activity on a ssDNA oligo via a complementary RNA template, (f) Quantification of ACt of double nicked templates relative to the untreated quantified substrate concentrations, (g) Quantification of reverse transcribed product on a ssDNA oligo. Data are displayed as means±s. d. of two independent experiments and were analyzed using a one-way Anova using Tukey’s multiple comparison; (*P<0.05; ns P>0.05).

Fig. 3 shows comparison of editing rates with evolved PE variants in mammalian cell lines,

(a) Correct percentage of substitutions assessed by deep sequencing in cells transfected with plasmids encoding for PEmax, PE_Y17, PE_Y18 together with plasmids encoding for epegRNAs at different time points on site 1 in HEK293T cells, (b) Editing rates of PE_Y17, PE_Y18 and PEmax at other loci in HEK293T cells, analyzed 48h after plasmid transfection, (c) Editing rates of PEmax, PE_Y17 and PE_Y18 on endogenous loci in K562 cells, analyzed 120h after transfection, (d) Editing rates of PEmax, PE_Y17 and PE_Y18 on a self-targeting library in K562 cells. The self-targeting library encoding for epegRNAs and their respective target sites was integrated into cells using lentiviral vectors prior to PE plasmid transfection and analysis of editing rates by deep sequencing after 120h. (e) Editing rates with PE variants encoded on mRNA and nucleofected into HEK293T cell line expressing an epegRNA targeting site 12. (f) Nucleofection of PEmax, PE_Y17 or PE_Y18 protein into HEK293T cells expressing an epegRNA targeting site 12. (g) Nucleofection of PE RNPs with a chemically modified pegRNA targeting site 4 in HEK293T cells. Data are displayed as means±s.d. of three independent experiments and were analyzed using a two-way ANOVA using Tukey’s multiple comparisons (*P<0.05; **P<0.005; ***P<0.0005; ****P<0.0001 ; ns P>0.05).

Fig. 4 shows in-vivo comparison of PE_Y18 and PEmax delivered via AAV or mRNA-LNP. (a) Experimental setup and editing rates at the targeted Adrbl locus in cortices. Intein- split PEmaxARnH and PE_Y18ARnH were packaged into AAV-PHP.eB capsids and injected intracerebroventricular into P1 mice. Each dot represents a single mouse (b) Editing rates at the targeted Dnmtl locus, assessed in different tissues at days 7 and 21 after temporal vein injection of the intein-split PE_Y18ARnH and PEmaxARnH packaged in AAV9. Each dot represents a single mouse, (c) Four weeks before single administration of mRNA-LNPs (2 mg/kg PEmax or PE_Y18 mRNA) via the tail vein, mice were injected with an scAAV9 expressing the epegRNA that targets the Dnmtl locus. Editing rates after LNP administration were assessed in the tail, liver tissue, and isolated hepatocytes. Each dot represents a single mouse. Data are displayed as meansis. d. of the indicated and were analyzed using a two-way ANOVA using Tukey’s multiple comparisons (*P<0.05; **P<0.005; ***P<0.0005; ****P<0.0001 ; ns P>0.05).

Fig. 5 shows characterization of the yeast cell growth in the OrthoRep PE selection approach. The yeast strain GRY333 containing the auxotrophic marker gene HIS3 on a multicopy nuclear plasmid with- and without a stop codon is incubated in L-histidine depleted media. Growth is drastically reduced in yeast cells containing the HIS3 copy with the “unedited” stop codon.

Fig. 6 shows prime editing of the HIS locus by PE1 during OrthoRep selection, (a) Sanger sequencing of the targeted region (T-to-C transversion; SEQ ID NO 013) in the auxotrophic marker gene HIS3. (b) Prime editing rates after a 5-day incubation period where yeast cells are grown in selective conditions. The chromatograms were analyzed with the base editing analysis tool (BEAT)(XuLi, et al., The CRISPR Journal 2, 223-229 (2019

Fig. 7 shows editing rates with PE2 variants in the HEK293T rSTOP-R2 cell line after a single round of evolution. Editing rates of PE2 variants isolated after a single round of evolution tested on rSTOP-R2 in HEK293T cells (Bock, D. et al., Sci Transl Med 14, 9238 (2022)). Data are displayed as means ±s.d. of three independent experiments and were analyzed using a one-way Anova using Tukey’s multiple comparison; (***P<0.0005; ****P<0.0001 ).

Fig. 8 shows the correlation of yeast growth (under selection) and DNA flap generation in PEKIN of different PE variants. OD600 at the final time point (22.6 h) of PEmax variants (Fig 1 c) and respective PEKIN activities (Fig 1f) were plotted against each other. The yeast growth in selective conditions correlated with the relative product formation in PEKIN, P < 0.0001 and R2 = 0.67.

Fig. 9 shows SDS-PAGE analysis of purified Prime Editor proteins. Gel confirming size, purity, and integrity of isolated proteins. Proteins were expressed in E.coli from following plasmids (in order): pLYW320, pLYW320-H, pLYW320-Y18, pLYW321 , pLYW321-H, pLYW321-Y18, pLYW322, pLYW322-H).

Fig. 10 shows Adapted PEKIN assay to assess nicking and reverse transcription activity of purified PE variants PE_Y17 and PE_Y18 compared to PEmax. (a) Scheme of the assay used to determine nicking activity via double nicking of a synthetic DNA template. Dark gray illustrates the non-target strand, whereas the target strand is illustrated in light gray, (b) Schematic of the adapted assay to disentangle reverse transcription activity on a ssDNA oligo via a complementary RNA template, (c) Quantification of ACt of doublenicked templates relative to the untreated quantified substrate concentrations, (d) Quantification of reverse transcribed product on a ssDNA oligo. Data are displayed as means±s.d. of two independent experiments and were analyzed using a one-way Anova using Tukey’s multiple comparison (*P<0.05; ns P>0.05).

Fig. 11 shows indel rates with evolved PE variants in mammalian cell lines, (a) Indels at site 1 in HEK293T cells after PE plasmid delivery, (b) Indels in HEK293T cells after PE plasmid delivery, (c) Indels in K562 cells after PE plasmid delivery, (d) Indels at site 12 in HEK293T cells expressing the respective pegRNA and nucleofected with mRNA encoding PEmax, PE_Y17 or PE_Y18. (e) Indels at site 12 in HEK293T cells expressing the respective pegRNA and nucleofected with PEmax-, PE_Y17- or PE_Y18 protein, (f) Indels at site 4 in HEK293T cells nucleofected with a PEmax-, PE_Y17- or PE_Y18 RNP. Data are displayed as meansis. d. of three independent experiments and were analyzed using a one-way Anova using Tukey’s multiple comparison (ns P>0.05).

Fig. 12 shows off-target activity assessed by amplicon sequencing in HEK293T cells. Data are displayed as means ± s.d. of three independent experiments and were analyzed using a one-way Anova using Tukey’s multiple comparison (*P<0.05; **P<0.005; ***P<0.0005; ns P>0.05).

Fig. 13 shows schematic of the AAV vector designs used in Fig. 4. Shown are the intein- split designs including hSynl (i-ii) and CBh (iii-iv) promoter for PEmaxARnH. The designs for PE_Y18ARnH are identical but include the A259D mutation in nCas9 and the K445T mutation in the M-MLV RT respectively. scAAV2/9 design for RFP expression under the p3 promoter and epegRNA Dnmtl expression under the hU6 promoter. Linkers: SEQ ID NO 014, 015.

Fig. 14 shows indel rates with evolved PE variants in vivo in mice. Indel rates were quantified from in vivo samples targeting the (a) Adrbl and (b) Dnmtl site, respectively. The number of individual replicates is the following for (a) 5-35 days for PEmaxARnH: 4, 4, 4, 4, 3, 4, 3 and for PE_Y18ARnH: 4, 4, 3, 4, 4, 4, 4. The number of individual replicates for (b) is: 7 for PEmaxARnH treated mice at day 7, 10 for PE_Y18ARnH treated mice at day 7, and 5 for PEmax and PE_Y18ARnH at day 21 . The untreated controls for (a) and (b) are three mice that were sacrificed at the age of 5 weeks. For the treated groups data are displayed as means±s.d. of at least three animals per timepoint and were analyzed using a one-way Anova using Tukey’s multiple comparison (*P<0.05; **P<0.005; ****P<0.0001 ; ns P>0.05).

Fig. 15 shows comparison of expression levels of PE variants. Expression levels of n- terminal and c-terminal AAV constructs were normalized to murine and human GAPDH expression respectively, (a) Expression levels of PEmaxARnH and PE_Y18ARnH in the brain, (b) Expression levels of PEmaxARnH and PE_Y18ARnH in the liver, (c) Intended editing at site 12 with PEmax, PE_Y17 and PE_Y18 in HEK293T cells 24, 48 and 72 h after transfections, and the corresponding expression levels of PE variants assessed by RT-qPCR (d). Data are displayed as means±s.d. of at least 3 replicates per timepoint and were analyzed using a one-way Anova using Tukey’s multiple comparison (*P<0.05; ***P<0.0005; ns P>0.05).

Fig. 16 shows chemical modification to omit scaffold insertions, (a) A plot of the percentage of reads obtained through NGS that aligned to the reverse transcribed region. Prior to the scaffold, modifications are integrated to inhibit read-through and compared to native RNA. (b) Chemical modifications: Left: a basic site in vicinity to a phosphoroth ioate AbS I s. Right: C3-linker between an adenine and a cytidine bound to a ribonucleic acid, (c) Scheme of the in vitro control to assess the effect of chemical modifications on the intended stop of the reverse transcription. The RNA template containing the chemical modifications is reverse transcribed via the PE protein on a ssDNA phosphorylated at the 5’ end. The products are circularized via ligation prior to selective amplification for NGS and analysis, (d) Top: Cu(l) catalyzed Click-chemistry was used to generate pegRNAs. Bottom: Chemical group (L) resulting after the reaction of a /3AzideN/ group bound to the 5’ end of Seq ID NO 11 and with a /5Hexynyl/ bound to the 3’ end of the Seq ID NO 12 (RNA), C3-linker) and AbS I s. (e) Left: correct editing rate after electroporation of constructs (mRNA PEmax). For the negative control, PEmax mRNA was administered without pegRNAs. Right: Percentage of scaffold insertions (5 bp) quantified via NGS of the correctly edited cells.

Description of the Tables

Table 1 shows mutations generated during PE evolution in OrthoRep and identified through Sanger sequencing. List of identified mutations from Sanger sequencing of subcloned variants after visible outgrowth of yeast cells in 96 well plates in selective conditions.

Table 2 shows identified mutations in PE variants via long read nanopore sequencing. PE2 variants were isolated from a single well after selection in OrthoRep and subjected to nanopore sequencing to analyze evolutionary trajectories. Unique molecular identifiers (UMIs) were clustered and used to assess respective variant frequencies.

Table 3 shows overview of parallel OrthoRep evolutions with PEmax.

Table 4 shows overview of the number of wells showing visible yeast cell growth with

PEmax using different types of edits.

Examples

Example 1: Development of an OrthoRep selection logic for PE evolution

OrthoRep is a yeast-based directed evolution system that employs an orthogonal error-prone DNA polymerase to trigger hypermutations (1 O'⁵ per base per generation) on a linear plasmid (p1 ) while ensuring that the mutation rate of the host genome remains unaffected. To develop an OrthoRep evolution approach for selecting PE variants with increased activity, we generated GYR333 yeast strains containing linear p1 plasmids expressing PE1 (Anzalone, A.V. et al., Nature 576, 149-157 (2019)), PE2 (Anzalone, A.V. et al., (2019) ibid), or PEmax (Chen, P.J. et al., Cell 184, 5635- 5652. e29 (2021 )), respectively. These strains were co-transformed with a plasmid encoding for the orthogonal, error-prone DNA polymerase TP-DNAP1 (L477V, L640Y, I777K, W814N) (Ravikumar, A., et al., Cell 175, 1946-1957. e13 (2018)), resulting in hypermutation of the PE variants. To link prime editing rates to yeast cell growth in selective conditions, after a five-day mutagenic drift period yeast strains were further transformed with a multicopy nuclear plasmid expressing an inactive version of the essential auxotrophic marker gene HIS3, and a pegRNA that enables the conversion of the inactive HIS3 variant back into an active variant (Fig 1 a-b, Fig 5).

To first validate our selection approach, we started with the evolution of PE1 (Anzalone, A.V. et al., (2019) ibid). PE1 lacks the activity-enhancing mutations that are present in the M-MLV RT domain of PE2 and PEmax, leading to substantially lower editing rates. Yeast cells were transformed with the different plasmids as described above and seeded in a 96 well plate. After a five-day growth period in L-histidine depleted medium we visually observed yeast outgrowth in 5% of the wells (5 of 96). Verifying functionality of the evolution logic, sanger sequencing of the HIS3 locus revealed 71 % editing in yeast cells grown under selection as compared to 10% editing in control wells where yeast cells were grown in non-selective media (Fig 6). In addition, yeast cells that were not transfected with the pegRNA-encoding plasmid did not exhibit growth under selection (0 out of 96 wells). Finally, when we sequenced PE1 variants subcloned from yeast cells grown under selection, we identified two of the mutations that were previously reported in PE2, D200N and T330P (Table 1 ).

Next, we employed the same experimental setup to evolve PE2. Yeast cells were transformed with the different OrthoRep plasmids for the PE selection logic and again cultured under selection for five days. PE2 variants were isolated from outgrowing cells via PCR extraction and subcloned prior to analysis by Sanger sequencing and Oxford Nanopore sequencing (Zurek, P.J., et al., Nat Commun 11 , 6023 (2020)) (Table 1 and 2). To assess whether these variants show higher prime editing activity than PE2, three clones were randomly chosen and subcloned into the pCMV-PE2 mammalian expression vector. Transfection into HEK293T cells indeed revealed an up to a 2.6- fold increase in editing efficiency for isolated variants as compared to PE2 (Fig 7).

Example 2: Evolution and in vitro characterization of novel PEmax variants

After validating the functionality of our prime editing selection logic in OrthoRep, we attempted to evolve PEmax. This PE variant has been established from PE2 and shows higher editing activity due to mammalian-optimized codon usage, improved domain architecture, and two mutations in the nickase domain of Cas9. Yeast cells were transformed with the different plasmids for PEmax evolution following a protocol that again allows for a five-day mutagenic drift period. Subsequently, they were cultured under selective conditions in a 96-well plate for five days (Table 3). Wells that showed visual outgrowth of yeast cells were normalized to an OD of 0.5, pooled and subjected to PCR amplification prior to transformation into fresh host cells for the next round of evolution. Consistent with the hypothesis that more active PEmax variants outcompete other variants, we observed an increase in the number of wells with visual yeast growth already after three days in the second selection round (Table 3). During the third round of evolution, we observed yeast growth in 94 of 96 wells after three days, indicating the need to further intensify the selection pressure. Therefore, we replaced the HIS3 selection cassette containing a stop mutation with a modified version, which requires the incorporation of a 501 bp segment into the HIS3. While the stringent selection pressure of this cassette did not enable yeast growth when the original PEmax variant was transfected, PEmax variants isolated from the third round of evolution led to visible yeast growth in six out of 96 wells after five days (Table 3 and 4).

To verify increased prime editing activity of variants selected over the four rounds of evolution, 21 clones were extracted by PCR and cloned on the linear p1 plasmid. These plasmids were transformed into yeast cells containing the wild-type TP-DNAP1 polymerase (to prevent further mutagenesis) (Ravikumar, A. et al, Nat Chem Biol 10, 175-177 (2014)), the HIS3 selection cassette, and the respective pegRNA to repair the inactivating stop codon. Importantly, 16 out of the 21 evolved variants conveyed higher fitness to yeast growth in selective conditions as compared to PEmax (Fig. 1 c).

To next benchmark the activity of the evolved PEmax variants in mammalian cell-lysates, we developed a Prime Editing KINetics assay (PEKIN), which quantifies the ability of a PE to nick a dsDNA and generate a DNA flap. In brief, the 21 extracted PEmax variants were cloned into a mammalian expression vector, in which they were C-terminally linked to a P2A-GFP fusion protein with self-cleavage capabilities (Walton, R.T., et al., Nature Protoc. 16, 1511-1547 (2021 )). After expression in HEK293T cells followed by cell lysis, PE protein levels were normalized via GFP fluorescence. Subsequently, PE variants were complexed with an in vitro transcribed pegRNA and incubated with a synthetic double-stranded DNA template (Fig. 1d). Nicking of the DNA template followed by the extension from the pegRNA RTT through reverse transcription was subsequently quantified by qPCR (Fig. 1e). Notably, of the 21 tested variants, 15 showed substantially greater flap generation than PEmax (Fig. 1f), with a pronounced correlation to the activity increase observed in the yeast growth assay (Fig. 8).

The five variants that exhibited greatest flap generation were further evaluated using a range of substrate concentrations (Fig. 2a). With the exception of B4, every variant demonstrated higher activity than PEmax across all concentrations. Subsequently, we explored the possibility of an additive effect by cumulating all mutations discovered in the top five variants in a single variant (PE_Combo; S219A, A259D in nCas9 and R44H, Y64W, K373R, R389C, L432M, K445T in the RT). However, the activity of PE_Combo was lower than the activity of PE_Y17, which was the most active variant selected by OrthoRep and contains a S219A amino acid change in nCas9 and a K445T amino acid change in the M-MLV-RT. Notably, PE_D2, the second best performing variant, only contained a mutation in nCas9 (A259D) but not in the RT, prompting us to generate a variant where we combined the A259D mutation in nCas9 with the RT K445T mutation of PE_Y17. Importantly, the resulting variant, termed PE_Y18, showed substantially higher activity than PE_Y17 (Fig. 2a).

To validate results obtained from normalized cell lysates, we next repeated the PEKIN assay with recombinantly expressed and purified PE_Y17, PE_Y18 and PEmax proteins (Fig. 9). Confirming increased activity of PE_Y17 and PE_Y18, both variants led to increased flap formation compared to PEmax with all tested dsDNA substrate concentrations (Fig. 2c). To gain a deeper insight into the functional role of the mutations in PE_Y17 and PE_Y18, we also introduced them into untethered PEmax, in which the nCas9 and M-MLV RT were not fused. Interestingly, in this context we observed no increase in product formation compared to the control (untethered PEmax) (Fig. 2c). Therefore, we adapted the PEKIN assay to determine if either the nicking activity of SpCas9 or the reverse transcription activity of the M-MLV-RT are increased in full-length PE_Y17 and PE_Y18. To assess DNA nicking activity, we supplied the PE RNP with a guide RNA that targets a dsDNA template on both strands (Fig. 2d). To assess reverse transcription activity, we supplied the PE RNP with an ssDNA and an RNA containing a PBS for the ssDNA and an RTT template (Fig. 2d). Quantification of product formation by qPCR, however, revealed no increase in DNA nicking or reverse transcription activity of PE_Y17 or PE_Y18 compared to PEmax (Fig. 2e and 2f). Thus, our data indicates that the identified mutations do not directly influence the activity of the Cas9 nickase or the M-MLV RT, but instead enhance the activity of the PE fusion complex in a more complex manner, for example by enhanced priming of the PBS specifically within the R-loop.

Example 3: Validation of PE Y17 and PE Y18 in mammalian cells

To determine if the enhanced activity of evolved PEmax variants also leads to higher editing in mammalian cells, we first transfected plasmids expressing PE_Y17, PE_Y18, or PEmax together with an epegRNA that induces a C-to-T transition mutation at site 1 in HEK293T cells. After 24, 48 and 72 h cells were harvested and editing rates were assessed by NGS. At all three timepoints we observed substantially higher editing with PE_Y17 and PE_Y18 as compared to PEmax (on average 1.6-fold with PE_Y17 and 2.3-fold with PE_Y18; Fig. 3a), without detecting an increase in indel rates (Fig. 10a). We then targeted additional loci in HEK293T and K562 cells with epegRNAs introducing substitutions, insertions, or deletions (Extended data table 6). Again, improved editing without enhanced indel rates was observed for PE_Y17 and PE_Y18, with an average 1.2-fold increase with PE_Y17 and 1.4-fold increase with PE_Y18 in HEK293T cells (Fig. 3b), and an average 1 ,2-fold increase with PE_Y17 and 1 ,4-fold increase with PE_Y18 in K562 cells (Fig. 3c). To further assess editing rates with PE_Y17 and PE_Y18 at a larger number of target sites, we generated a self-targeting epegRNA library containing 200 different sequences (Mathis, N. et al., Nature Biotechnology 2023 1-9, (2023)). Lentiviral vectors containing epegRNA expression cassettes and the respective target sites were integrated into K562 cells prior to the transfection with PE variants. Deep sequencing of target sites again revealed a trend for increased editing with PE_Y17 and significantly increased editing with PE_Y18 (1 .3-fold increase to PEmax; Fig. 3d). To next assess if the evolved PE variants lead to increased off-target editing, we targeted two loci with pegRNAs that have known off-target activity. However, while on-target editing was increased at both sites, deep-sequencing of the off-target sites did not reveal elevated activity (Fig. 11 ).

To characterise the evolved PEmax variants further, we next delivered in vitro transcribed nucleoside-modified mRNAs encoding for PEmax, PE_Y17 or PE_Y18 into HEK293T cells that constitutively expressed an epegRNA introducing a single base pair deletion in site 12. Confirming our results from plasmid transfections, we observed a trend for higher editing with PE_Y17 and significantly higher editing (1.3-fold) for PE_Y18 compared to PEmax (Fig. 3e). Similarly, nucleofection of the purified PE proteins instead of mRNA into the same cell line led to a trend for higher editing at site 12 with PE_Y17 and a significantly higher editing (2-fold) with PE_Y18 (Fig. 3f). Finally, electroporation of RNPs consisting of the PE complexed with a synthetic pegRNA targeting site 4 demonstrated significantly higher editing rates for both evolved variants (1.9-fold for PE_Y17 and 3.5-fold for PE_Y18; Fig. 3g).

Example 4: PE Y18 enhances prime editing rates in vivo

Recently, prime editing approaches for introducing or correcting mutations in vivo have been developed. Therefore, we assessed if our most active variant, PE_Y18, also increases prime editing rates in mouse tissues. Using adeno-associated viral (AAV) vectors, we first delivered PEmax and PE_Y18 into the murine brain. Both PE variants were expressed from the neuronspecific human synapsin 1 (hSynl ) promoter, together with an epegRNA that installs a C-to-T transition mutation in the Adrbl locus. Due to the limited packaging capacity of AAV, both PE variants were also shortened by removing the RnaseH domain and split into two parts using the intein-split systems as described previously (Bock, D. et al. (2022) ibid) (Fig. 12). After packaging both vectors into AAV-PHP.eB21 capsids, they were injected into the ventricles in 1-day-old pups (P1 ). Importantly, deep sequencing of isolated brain tissues revealed significantly higher editing rates with PE_Y18ARn/-/ compared to PEmax at all analysed time points (5-35 days post injection; on average 4.7-fold increase; Fig. 4a), with a non-significant increase in indels and comparable expression (Fig. 13a and Fig. 14a). Next, we exchanged the epegRNA on the AAV vector with an epegRNA that installs a T-to-G transversion in the Dnmtl locus, and the hSyn promoter with the ubiquitous CBh promoter. Vectors were packaged into AAV9 capsids, which enable transduction of various tissues including the liver and heart, and systemically delivered via the temporal vein into P1 mice. After 7- and 21-days genomic DNAwas isolated from the liver and heart and analysed for editing rates. In line with our previous results, we observed significantly higher editing with PE_Y18ARn/-/ in the liver (1.4-fold increase at day 7 and a 2.3-fold increase at day 21 ), and a statistically non-significant trend for higher editing in the heart (1 .5-fold increase at day 7 and a 1 .1 - fold increase at day 21 ), without observing an increase in indel rates or expression levels (Fig 4b; Fig. 13b and 14b).

AAV delivery leads to prolonged PE expression, which is a limitation for clinical application due to potential immune responses to bacterial and viral components of the PE. Therefore, we also benchmarked PEmax with PE_Y18 in a transient in vivo prime editing approach where nucleoside- modified mRNAs encoding for both PE variants were encapsulated into lipid nanoparticles (LNPs). Systemic delivery of 2mg/kg mRNA-LNP into mice pre-treated with self-complementary AAV9 (scAAV9) encoding for the Dnmtl epegRNA resulted in a 1.3-fold increase in editing rates with PE_Y18, which, nevertheless, was not statistically significant (P=0.1306) (Fig. 4c). Example 5: Discussion

In this study we demonstrate proof-of-concept for using OrthoRep to evolve PE variants with increased activity. After four rounds of evolutions, we isolated a number of PE variants exhibiting increased editing activity in yeast and higher DNA-flap generation in our in vitro PEKIN assay. The two most efficient PE variants, PE_Y17 and recombinant PE_Y18, also show higher activity at various target sites when delivered into cells. PE_Y18, moreover, led to higher prime editing rates in vivo in the brain and liver of mice. Interestingly, the activity-enhancing mutations did not show an effect when introduced into an untethered PE, where the nCas9 and M-MLV RT are not fused. This highlights the advantage of directly evolving full-length PEs for higher activity instead of evolving the individual components (nicking activity of nCas9 or reverse transcription activity of the RT). Since the linear plasmid p1 allows for encoding proteins with sizes up to 22 kb, in principle even larger multi-component genome editing tools such as CRISPR-associated transposons could be evolved for higher activity in OrthoRep. Moreover, our selection approach to evolve PE variants is highly versatile, and stringency could be easily adapted. For example, instead of linking yeast growth to the installation of a single edit it could be linked to the requirement of installing several consecutive edits, either on the same or on different auxotrophic marker genes.

One drawback of our selection approach, however, is the necessity to extract the PE variants after each selection round in order to retransform them into fresh host cells with unedited selection plasmids. Such manual intervention is not required during PACE, where a phage carrying the genome editor infects fresh host cells with selection plasmids every 20 minutes. Nevertheless, while PACE has been successfully employed to optimize various genome editing tools, selections occur in the bacterial cytoplasm. OrthoRep, in contrast, functions in eukaryotic cells, which has certain advantages. For example, it could be employed to screen for genome editors with more efficient nuclear import, or to evolve enzymes that require eukaryotic posttranslational modifications. Thus, it would be highly valuable to develop continuous selection approaches for genome editors in OrthoRep. This could be achieved by pairing the system with another editor that continuously reverts the essential mutation, or by incorporating abortive mating to continuously pass PE variants to fresh host cells.

Continuous PE evolution in OrthoRep could potentially lead to variants with further improved activity than PE_Y18. This would be beneficial for the clinical translation of prime editing, since in vivo editing rates with PE_Y18 did not reach levels that are typically achieved with Cas9 nucleases or base editors, and AAV doses above what would be deemed safe for human application had to be used. Another limitation for efficient prime editing is the stability of the pegRNA, particularly the 3’end that is not protected by Cas9 and readily degraded by exo- and endonucleases. Therefore, future research should also be directed on refining pegRNA chemistry or design. Hopefully, the combination of improvements on the pegRNA and the PE protein will facilitate successful clinical implementation of prime editing. Example 6: Materials and Methods:

CloningPlasmids containing the P1 landing pad pTBL1201_pUC_FDP, pTBL963_pcDNA3_1 and pAR-Ec633 (Addgene #130873) were gifts from Chang Liu. PE1 (pLYW118), PE2 (pLYE094) and PEmax (pLYW200) were integrated into pTBL1201_pUC_FDP using PCR amplification from psZ157 CRISPEY RT/Cas9 (Addgene #114454), pCMV-PE1 (Addgene # 132774) and pCMV-PE2 (Addgene # 132775) pCMV-PEmax (Addgene #174820) using HiFi DNA Assembly Master Mix [New England Biolabs (NEB)]. All PCRs were performed using Q5 High-Fidelity DNA Polymerase (NEB). Multi-copy nuclear plasmid pLYW105 for target sites of yeast evolutions is based on pCEV- G1-Ph (Addgene # 46814) and pTBL963_pcDNA3_1 and was assembled by PCR and HiFi DNA Assembly Master Mix containing different epegRNAs and target sites.

Plasmids containing epegRNAs were created by ligation of the annealed spacer, scaffold, and 3' extension oligos into the Bsal-digested pU6-pegRNA-GG-acceptor (Addgene #13277), pU6- tevopreQI -GG-acceptor (Addgene #174038) with Golden Gate assembly as previously describedl ,5. To generate intein-split PE plasmids, inserts were ordered as gBIocks from Integrated DNA Technologies (IDT) or amplified from pCMV-PEmax plasmids using PCR. Inserts were cloned into the Notl- and EcoRI-digested pCMV-PEmax backbone using HiFi DNA Assembly Master Mix (NEB). For the cloning of the PiggyBac reporter plasmids for the Adrbl , Dnmtl , PKU and PCSK9 locus, inserts with homology overhangs for cloning were ordered from IDT and cloned into the Xbal- and EcoRI-digested pPB-Zeocin backbone using HiFi DNA Assembly Master Mix (NEB).

To prepare plasmids for AAV production, inserts with homology overhangs were either ordered as gBIocks (IDT) or generated by PCR. Inserts were cloned into Xbal- and Notl-digested AAV backbones using HiFi DNA Assembly Master Mix (NEB).

All plasmids were sequenced by Sanger Sequencing. The amino acid sequences of intein-split PEmax p.713/p.714 constructs are listed as SEQ ID NO 004 and 005.

Yeast cell culture OD measurements, transformations, and selection

Saccharomyces cerevisiae strain GRY333 was a gift from Chang C. Liu with the genotype F102-2, leuAO, ura3A0, HIS4+, his3Al , flolAO wt-pGKL1/2. All strains were grown at 30 °C in selective complete media or yeast extract peptone (YPD). For selection with Zeocin [100 ug/mL], the pH of the medium was adjusted to 7.5. The plasmids pAR-Ec633 and pLYW105 were transformed into GRY333 as previously described and selected on uracil-depleted plates containing Zeocin (Gietz, R.D. et al., Nature Protocols, 31-34, (2007)). Integration of PE1 , PE2 and PEmax on P1 was achieved as previously described with PCR product instead of restriction digested plasmid (Ravikumar, A. et al., (2014) ibid). After the appearance of colonies, cells were picked and passaged in uracil and leucine-depleted medium containing Zeocin for seven days. The presence and identity of P1 were confirmed by gel electrophoresis of extracted P1 and PCR on the yeast culture. To initiate selections, cultures were diluted 1 :1000 in L-histidine-depleted selective complete media. Evolutions were performed in parallel by incubating 200 μL diluted cultures in 96 well plates at 30°C without shaking. The successful growth of emerging cells was optically identified in the respective wells. Cultures from individual wells were normalized to an OD600 of 0.5 and pooled for PCR amplification of the P1 with the Phire Plant direct PCR master mix (Thermo Fisher). The resulting PCR products were gel purified and transformed into new host cells for subsequent rounds of directed evolution, Oxford Nanopore sequencing, or directly subcloned into P2A-GFP fusion plasmid for further characterizations.

Characterizations of selection plasmid in yeast cell culture

Selection cassettes pLYW105 with and without essential edit were characterized in L-histidine depleted media containing Zeocin by continuous culturing and measurement of absorbance at 600 nm in flat 96 well plates (Greiner) in a Tecan Infinite 200Pro at 30°C (Fig. 5).

Expression and purification of tethered and untethered prime editor constructs

The tethered and untethered Prime Editor constructs were expressed in Escherichia coli Rosetta 2 (DE3) for 18 h at 18 °C as fusion proteins with an N-terminal His6-MBP-TEV tag. Bacterial pellets were resuspended and lysed in 20 mM HEPES-KOH pH 7.8, 500 mM NaCI, 10 mM imidazole, and 5% (v/v) glycerol supplemented with protease inhibitors. Cell lysates were clarified by ultracentrifugation, loaded on a 10 mL Ni-NTA Superflow column (QIAGEN) and washed with 5-7 column volumes of 20 mM HEPES-KOH pH 7.5, 500 mM NaCI, 15 mM imidazole. The tagged proteins were eluted with 7-10 column volumes of 20 mM HEPES-KOH pH 7.5, 250 mM NaCI, 250 mM imidazole. The proteins were then loaded on an equilibrated HiTrap Heparin HP column (GE Healthcare). The column was washed with 5 column volumes of 20 mM HEPES-KOH pH 7.5, 250 mM NaCI, 1 mM DTT, and the proteins were eluted with 30 column volumes of 20 mM HEPES- KOH pH 7.5, 1 M NaCI, 1 mM DTT, in a 0-100% gradient. The NaCI concentration was adjusted to 400 - 500 mM NaCI by dilution and His6-MBP tag was removed by TEV protease cleavage at 4 °C. The proteins were then concentrated and further purified by gel filtration, eluting in 20 mM HEPES pH 7.5, 500 mM NaCI, 1mM DTT. pLYW320 (PEmax), pLYW320_Y18 (PE_Y18), pLYW320H (PE_Y17), and pLYW321 (PEmax nCas9) were purified using a Superdex 200 16/600 column (GE Healthcare). pLYW321 H (PE_Y17 nCas9) and pLYW321_Y18 (PE_Y18 nCas9) were purified using a Superdex 20026/600 column (GE Healthcare). pLYW322 (PEmax M-MLV RT) and pLYW322H (PE_Y17 I PE_Y18 M-MLV RT) were loaded on a Superdex 75 16/600 gel filtration column (GE Healthcare). Pure fractions were concentrated to 1 .4 - 23.3 mg/mL, analyzed by SDS- PAGE (Fig. 9), and flash frozen in liquid nitrogen for storage at -80 °C.

Mammalian cell culture, transfection, nucleofections and genomic DNA preparation

HEK293T [American Type Culture Collection (ATCC) CRL-3216] cells were maintained in DMEM plus GlutaMAX (Thermo Fisher Scientific), supplemented with 10% (v/v) fetal bovine serum (FBS) (DMEM++) (Sigma Aldrich) and 1 % penicillin/strepromycin (Thermo Fisher Scientific) at 37°C and 5% CO2. K562 cells (ATCC CCL-243) were maintained in RPMI++ (RPMI 1640 Medium with GlutaMAX Supplement (Thermo Fisher Scientific), supplemented with 10% (v/v) FBS and 1 % penicillin/streptomycin. Cells were passaged every 3 to 4 days at a confluency below 90%.

Cells were seeded in 96-well and 48-well cell culture plates at 70% confluency (Greiner) six hours prior to lipofection. Cells were transfected as previously reported and harvested by lysis at respective time points post-transfection with direct lysis buffer: 10 uL of 4x lysis buffer (10 mM Tris- HCI pH 8, 2% Triton™ X-100, 1 mM EDTA, 1 % Proteinase K [20 mg/mL]) (Anzalone, A.V. et al., (2019) ibid). When intein-split PEs were transfected, 300 ng of each PE half was used.

Nucleofections of HEK293T cells were performed using the Neon™ transfection system using 10 μL tips. Cells were harvested and washed 3 x with phosphate-buffered saline (PBS) prior to counting. Cells were repeatedly spun down and resuspended in R buffer to a concentration of ~ 2*10⁵ cells per 5 μL. Reactions were prepared in PBS by the respective addition of mRNA, proteins, or RNPs consisting of synthetic pegRNA and proteins. For nucleofections 0.125 pmol mRNA and 5 pmol of RNP 1 :1 protein :pegRNA molar ratio was used. RNPs were assembled for 10 minutes in PBS at 37 °C. Synthetic pegRNA was ordered at Axolabs. For mRNA one pulse of 1400 mV and 20 mS pulse width was used and for proteins and RNP one pulse of 1700 mV, 20 mS was applied. After nucleofection, cells were cultured in 200 uL of DMEM++ for 48 hours prior to harvesting.

Genomic DNA from cortices was isolated by phenol/chloroform extraction. First, tissue samples were incubated overnight in lysis buffer (50 mM Tris-HCI pH 8.0, 100 mM EDTA, 100 mM sodium chloride, and 1 % SDS; Thermo Fisher Scientific) at 55°C and 300 rpm. Subsequently, phenol/chloroform/isoamyl alcohol (25:24:1 , Thermo Fisher Scientific) was added and samples were centrifuged (5 min, maximum speed). The upper phase was transferred to a clean tube and DNA was precipitated using 100% ethanol (Sigma-Aldrich). Samples were centrifuged (5 min, maximum speed) and pellets were washed using cold 70% ethanol (-20°C).

Generation of reporter cell lines

To generate site 1 (Adrbl ), site 6 (PKU) and self-targeting site 5 (PCSK9) reporter cell lines with the PiggyBac transposon, HEK293T cells were seeded into a 48-well cell culture plate (Greiner) and transfected at 70% confluency with 225 ng of the PiggyBac-transposon and 25 ng of the transposase using Lipofectamine 2000 (Thermo Fisher Scientific) according to the manufacturer's instructions. Three days after transfection, cells were enriched for 10 days using Zeocin selection [750 pg/ml], rSTOP-R2 was generated as preciously described (Bock, D. et al. ,(2022) ibid).

Self-targeting libraries

The custom oligonucleotide pool containing pegRNAs and corresponding target sequence was ordered from Twist Bioscience and cloned into the Lenti_gRNA-Puro plasmid (Addgene #84752) as previously described (Mathis, N. et al., (2023), ibid)), (Kim, N. et al., Nat Biotechnology, 38, 1328-1336 (2020)). Cell pools were harvested 120 hours after plasmid transfection without antibiotic selection prior to analysis by deep sequencing (Chen, P.J. et al., (2021 ) ibid). AAV production

AAV9 serotype PHP.eB were produced by the Viral Vector Facility of the Neuroscience Center Zurich. Briefly, AAV vectors were ultracentrifuged and diafiltered.

To generate Pseudotyped AAV9 vectors, packaging, capsid, and helper plasmids (Addgene No. 112865 and 112867) were co-transfected and incubated for five days until harvest. The vectors were then precipitated using PEG and NaCI and subjected to gradient centrifugation with OptiPrep (Sigma-Aldrich) for further purification, following the previously described method. Subsequently, the concentrated vectors were obtained using Vivaspin® 20 centrifugal concentrators (VWR). Physical titers (vector genomes per milliliter, vg/mL) were determined using a Qubit 3.0 fluorometer (Thermo Fisher Scientific) as previously done (During, D.N. et al., Cell Rep 33, (2020)). The identity of the packaged genomes of each AAV vector was confirmed by PCR. AAV9 viruses were stored at -80°C until they were used. If required, they were diluted using phosphate-buffered saline (PBS) from Thermo Fisher Scientific. mRNA synthesis and LNP production

An mRNA production plasmid was used to subclone the coding sequences of PEmax, PE_Y17 and PE_Y18, employing HiFi DNA Assembly Master Mix from NEB. Modified nucleoside-containing mRNA was generated using N1 mM^J-5'-triphosphate (TriLink) instead of UTP. Co-transcriptional addition of the trinucleotide cap1 analog, CleanCap (TriLink), was used to cap the in vitro transcribed mRNAs. The mRNA was purified by cellulose (Sigma-Aldrich) and analyzed using agarose gel electrophoresis prior to storage at -20 °C. mRNA-LNPs were synthesized by means of nanoprecipitation as reported previously. Briefly, lipids dissolved in ethanol are rapidly mixed with m1 Psi modified mRNA dissolved in an aqueous buffer of low pH using a special microfluidic device. mRNA-LNPs were similar in composition to those of the BioNTech vaccine and contained the ionizable lipid ALC-0315 (proprietary to Acuitas Therapeutics), DSPC, Cholesterol and DMG- PEG2000 at 46.3:9.4:42.7:1.6 mokmol ratio, encapsulated at an RNA to total lipid ratio of -0.04 (wt/wt). The LNP formulations were dialyzed overnight at 4 °C against 1 x PBS, 0.2-pm sterile filtered and stored at 4 °C. Encapsulation efficiencies of mRNA were measured by the Quant-iT Ribogreen Assay (Life Technologies) and LNP sizes were determined with a Malvern Zetasizer (Malvern Panalytical). Polydispersity indexes (PDIs) were determined to be around 0.14 with a Z- average of around 130 nm.

Animal Studies

Animal experiments were performed in accordance with protocols approved by the Kantonales Veterinaramt Zurich and in compliance with all relevant ethical regulations. C57BL/6J mice were housed in a pathogen-free animal facility at the Institute of Pharmacology and Toxicology of the University of Zurich. Mice were kept in a temperature- and humidity-controlled room on a 12-hour light-dark cycle. Mice were fed a standard laboratory chow (Kliba Nafag no. 3437 with 18.5% crude protein). To target Adrbl in the brain, newborn mice (P1 ) received 5.0 x 10¹⁰ vg per animal and construct via intracerebroventricular injection. For Dnmtl , newborn mice (P1 ) received 1.7 x 10¹⁰ vg per animal and construct (Fig. 13 i -iv) via temporal vein injections. Adult mice were injected

10 with 5 x 10 vg per animal of scAAV9 (Fig. 13 v). After four weeks animals were dosed with 2mg/kg (LNP) via the tail vein.

Brain Isolation

Mice were euthanized with CO2, followed by decapitation. The skull was removed with scissors and tweezers without inflicting damage to the underlying tissue. The brain was removed using a spatula. The cortex was identified based on the mouse brain atlas and separated from the remaining brain regions for genomic DNA isolation (Paxinos, G. & Franklin, K. B. J. The Mouse Brain in Stereotaxic Coordinates, 2nd edition. Academic Press Preprint at (2001 )).

Isolation of hepatocytes

The process of isolating primary hepatocytes involved a two-step perfusion method. Initially, the liver was subjected to pre-perfusion with Hanks' buffer, supplemented with EDTA and Hepes, by inserting the cannula through the superior vena cava and cutting the portal vein. This was followed by a low-flow perfusion with digestion buffer containing freshly added Liberase, which lasted for approximately 10 minutes. The digestion was halted using isolation buffer, and the cells were gently scraped away from the matrix using a cell scraper. Subsequently, the cell suspension was filtered through a 100-pm filter from Corning, and the hepatocytes were purified using two low-speed centrifugation steps that lasted for 2 minutes at 50g.

Isolation genomic DNA from mouse samples and short read deep sequencing

Genomic DNA from mouse samples was isolated by direct lysis (cells, tissues and isolated hepatocytes) or phenol/chloroform extraction (brain tissue). Specific primers were used to generate targeted amplicons for deep sequencing. Input genomic DNA was first amplified in 10μL reactions for 30 cycles using NEBNext High-Fidelity 2xPCR Master Mix (NEB). Amplicons were then purified using AMPure XP beads (Beckman Coulter) and subsequently amplified for eight cycles using primers with sequencing adapters. Approximately equal amounts of PCR products were pooled, gel purified, and quantified using a Qubit 3.0 fluorometer and the dsDNA HS Assay Kit (Thermo Fisher Scientific). Paired-end sequencing of purified libraries was performed on an Illumina Miseq.

HTS data analysis

Sequencing reads were demultiplexed with the Miseq Reporter (Illumina). Next, amplicons were aligned to the respective reference sequence using CRISPResso2 (Clement 2019). Prime editing efficiencies were calculated as percentage of (number of reads containing only the desired edit)/(number of total aligned reads). Indel rates were calculated as percentage of (number of indel- containing reads)/(total aligned reads). Reference amplicons are listed in extended data table 6. Analysis for self-targeting libraries was performed with a custom Python script which will be deposited on GitHub before publication. Oxford Nanopore sequencing

Oxford Nanopore Sequencing was adapted from an established protocol (Zurek, P.J. et al., Nat Common 11 , 6023 (2020)). PE2 variants were extracted by direct PCR amplification from cultured yeast cells from a single 96 well with primers including the respective unique molecular identifiers (UMIs). 25 cycles were performed prior to gel extraction and HiFi DNA Assembly into the pUC19 (New England Biolabs) which was previously digested (Kpnl-HF, SpHI-HF). For each PCR amplification, approximately 1000 colonies were inoculated prior to plasmid purification. 100 ng of the plasmid pool was PCR amplified with 15 cycles NEBNext High Fidelity 2x PCR Master Mix and corresponding primers with binding regions outside of the UMIs and experiment specific barcodes. Thereafter, Oxford Nanopore sequencing was performed on the as previously described. Consensus reads were created by a previously described Python script (Zurek, P.J., et al. (2020) ibid) and mutations were counted and identified with a custom Python script that will be deposited on GitHub prior to publication.

Statistical analysis

All statistical analyses were performed using GraphPad Prism 9.5.0 for macOS. If not stated differently data represents biological replicates and are depicted as means±s.d. Statistical analyses are indicated in the respective figure legends. Same applies for the sample sizes and the statistical tests performed respectively. For all analyses, p<0.05 was considered statistically significant.

Example 7: Screening and validation of enzymatic activities

PE variants were subcloned from normalized yeast cultures from the last round of directed evolutions into the vector pLYW315. 2000 ng of the constructs were transfected into HEK293T in triplicates in 48 well plates. Cells were lysed as previously described with lysis buffer (20 mM Hepes pH7.5, 100 mM KCI, 5mM MgCh, 5%(v/v) glycerol, 1 mM DTT, 0.1 % (v/v) Triton X-100 and protease inhibitor as previously reported (Walton et al., Nature Protocols 2021 16:3 16, 1511-1547 (2021 )). In vitro transcription was performed on PCR amplified and gel purified pegYW060 (SEQ ID NO 011 ) with the HiScribe T7 High Yield RNA synthesis kit (NEB). RNA integrity was identified by gel electrophoresis. For lysates, GFP expression levels were normalized with lysis buffer to an equivalent of 150 nM fluorescein as previously described. RNP complexes were formed at room temperature with 1 μL of 2.5 pM pegRNA and 1.25 μL of the normalized lysates respectively. For the purified proteins, 2.5 pM of each construct in 10 mM HEPES, 150 mM NaCI, pH 7.5 was incubated with 2.5 pM pegRNA. The dsDNA substrate was annealed in nuclease-free water by heating to 98°C and cooling down by > 5 °C per minute. Per reaction 0.5 μL of the annealed dsDNA substrate at varying concentrations was mixed with 0.5 μL of cleavage buffer (100 mM HEPES, pH 7.5, 1 .5 M NaCI and 50mM MgCI2) and 1 .75 μL of nuclease-free water. The reactions were carried out for 1 h at 37°c by combining the 2.25 μL of the RNP solution and the 2.75 μL of the substrate solution. The reactions were stopped by the addition of 5 μL 1 % Proteinase K (20 mg/mL) followed by incubation at 60°C for 1 h and 10 minutes at 95 °C. Quantification by qPCR

Reverse transcribed cDNA was quantified by qPCR after the enzymatic reactions were stopped. qPCR was performed using the FIREPol qPCR Master Mix (Solis BioDyne) and analyzed using a Lightcycler 480 system (Roche). Quantifications of virtual products were performed with the formula: 10E^A((Ct-14.87/-3.48) *1 '000'000.

Cited references:

Anzalone, A. V et al. Search-and-replace genome editing without double-strand breaks or donor DNA. Nature 576, 149-157 (2019).

Chen, P. J. et al. Enhanced prime editing systems by manipulating cellular determinants of editing outcomes. Cell 184, 5635-5652. e29 (2021 ).

Mathis, N. et al. Predicting prime editing efficiency and product purity by deep learning. Nature Biotechnology 2023 1-9 (2023) doi:10.1038/s41587-022-01613-7.

Ravikumar, A., Arrieta, A. & Liu, C. C. An orthogonal DNA replication system in yeast. Nat Chem Biol 10, 175-177 (2014).

Ravikumar, A., Arzumanyan, G. A., Obadi, M. K. A., Javanpour, A. A. & Liu, C. C. Scalable, Continuous Evolution of Genes at Mutation Rates above Genomic Error Thresholds. Cell 175, 1946-1957. e13 (2018).

Zurek, P. J., Knyphausen, P., Neufeld, K., Pushpanath, A. & Hollfelder, F. UMI-linked consensus sequencing enables phylogenetic analysis of directed evolution. Nat Commun 11 , 6023 (2020).

Walton, R. T., Hsu, J. Y., Joung, J. K. & Kleinstiver, B. P. Scalable characterization of the PAM requirements of CRISPR-Cas enzymes using HT-PAMDA. Nature Protocols 2021 16:3 16, 1511-1547 (2021 ).

XuLi, LiuYakun & HanRenzhi. BEAT: A Python Program to Quantify Base Editing from Sanger Sequencing, https://home.liebertpub.com/crispr 2, 223-229 (2019).

Bock, D. et al. In vivo prime editing of a metabolic liver disease in mice. Sci Tran si Med 14, 9238 (2022).

Gietz, R. D. & Schiestl, R. H. High-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nature Protocols 2007 2:1 2, 31-34 (2007).

Kim, N. et al. Prediction of the sequence-specific cleavage activity of Cas9 variants. Nature Biotechnology 2020 38:11 38, 1328-1336 (2020).

Paxinos, G. & Franklin, K. B. J. The Mouse Brain in Stereotaxic Coordinates, 2nd edition. Academic Press Preprint at (2001 ). During, D. N. et al. Fast Retrograde Access to Projection Neuron Circuits Underlying Vocal Learning in Songbirds. Cell Rep 33, (2020).

All scientific publications and patent documents cited in the present specification are incorporated by reference herein.

SEQUENCES:

In the event of discrepancies between the sequences shown in the present specification and those of the enclosed sequence protocol according to WIPO standard ST.26, the sequences shown herein shall prevail.

SEQ ID NO 001: reference (unmutated) PE polypeptide

MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLF DSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQL FEENPINASGVDAKAILSARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQL SKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKA LVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLKREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFE EWDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVL TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEM ARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS DYDVDAIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNLTKAER GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR EINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFK TEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK LIARKKDWDPKKYGGFDSPTVAYSVLWAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE

VKKDLI IKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE QHKHYLDEI IEQI SEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTI DRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGS TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLI IPLKATSTPVSIKQYPMSQEAR LGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPS HQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQ HPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEAR KETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAP ALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKL TMGQPLVILAPHAVEALVKQPPDRWLSNARMTHYQALLLDTDRVQFGPWALNPATLLPLPEEGLQHNCLD ILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQ ALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLS I IHCPGHQ KGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFESPKKKRKVGSGPAAKRVKL D

SEQ ID NO 002: Y_18 A277D (A259D in Cas9) and K1865T (K445T in M-MLV RT)

MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLF DSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQL FEENPINASGVDAKAILSARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLDEDAKLQL SKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKA LVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLKREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFE EWDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVL TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEM ARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS DYDVDAIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNLTKAER GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR EINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFK TEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK LIARKKDWDPKKYGGFDSPTVAYSVLWAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLI IKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE QHKHYLDEI IEQI SEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTI DRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGS TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLI IPLKATSTPVSIKQYPMSQEAR LGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPS HQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQ HPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEAR KETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAP ALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKL TMGQPLVILAPHAVEALVTQPPDRWLSNARMTHYQALLLDTDRVQFGPWALNPATLLPLPEEGLQHNCLD ILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQ ALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLS I IHCPGHQ KGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFESPKKKRKVGSGPAAKRVKL D

SEQ ID NO 003: Y_17 S237A (S219A relative to the nCas9 sequence) and K1865T

MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLF DSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQL FEENPINASGVDAKAILSARLSKARKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQL

SKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKA

LVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLKREDLLRKQRTFDNG

SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFE

EWDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD

LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVL

TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN

FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEM

ARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS

DYDVDAIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNLTKAER

GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR

EINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFK

TEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK

LIARKKDWDPKKYGGFDSPTVAYSVLWAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE

VKKDLI IKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE

QHKHYLDEI IEQI SEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTI

DRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGS

TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLI IPLKATSTPVSIKQYPMSQEAR

LGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPS

HQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQ

HPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEAR

KETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAP

ALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKL

TMGQPLVILAPHAVEALVTQPPDRWLSNARMTHYQALLLDTDRVQFGPWALNPATLLPLPEEGLQHNCLD

ILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQ

ALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLS I IHCPGHQ

KGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFESPKKKRKVGSGPAAKRVKL D

SEQ ID NO 004: Y_18 split, N-terminal part

MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLF

DSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD

EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQL

FEENPINASGVDAKAILSARLSKSRKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLDEDAKLQL

SKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKA

LVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLKREDLLRKQRTFDNG

SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFE

EWDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD

LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVL

TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN FMQLIHDDSLTFKEDIQKAQVGGGGSGGGGSGGGGSCLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDN NGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNLPNSGGS KRTADGSEFEPKKKRKV

SEQ ID NO 005: Y_18 split, C-terminal part

MKRTADGSEFESPKKKRKVIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASNGGGGSGGGGSGGGGSSG QGDSLHEHIANLAGSPAIKKGILQTVKWDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEE GIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDAIVPQSFLKDDSIDNKVL TRSDKNRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQIT KHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAWGTALIK KYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETG EIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAY SVLWAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLI IKLPKYSLFELENGRKR

MLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEI IEQISEFSKRVILA DANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTT IDRKRYTSTKEVLDATLIHQSITG LYETRIDLSQLGGDSGGSSGGSKRTADGSEFESPKKKRKVSGGSSGGSTLNIEDEYRLHETSKEPDVSLGS TWLSDFPQAWAETGGMGLAVRQAPLI IPLKATSTPVS IKQYPMSQEARLGIKPHIQRLLDQGILVPCQSPW NTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPSHQWYTVLDLKDAFFCLRLHPTSQ PLFAFEWRDPEMGISGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQHPDLILLQYVDDLLLAATSELDC QQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEARKETVMGQPTPKTPRQLREFLGKA GFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAPALGLPDLTKPFELFVDEKQGYAK GVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKLTMGQPLVILAPHAVEALVKQPPD RWLSNARMTHYQALLLDTDRVQFGPWALNPATLLPSGGSKRTADGSEFESPKKKRKVGSGPAAKRVKLD

SEQ ID NO 006: pegRNA structural RNA sequence tract: gscscsrUrArArUrGrUrArCrUrGrUrGrUrGrCrArG rGrUrUrUrCrArGrAgcuaugcuggaaaca gcauagcrArArGrUrUrGrArArArUrArArGrGrCrUrArGrUrCrCrGrUrUrArUrCrAacuugaaa aaguggcaccgagucggugcr UrUrCrCrGrArGrUrCrUrUrUrCrArCrUrGrCrArCrArCrArGrUr ArCrAususas rN (capital font): RNA residues n (lower case font): 2'-O-methyl residues s: phosphoroth ioate backbone modification underlined: protospacer: italic: scaffold', underlined italic, reverse transcription template & primer binding site.

SEQ ID NO 007: _nCas9 (position S219A, A259D bold font)

MKRTADGSEFESPKKKRKVDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLF

DSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVD EVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQL FEENPINASGVDAKAILSARLSKARKLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLDEDAKLQL SKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKA LVRQQLPEKYKEI FFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLKREDLLRKQRTFDNG SIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFE EWDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVD LLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKI IKDKDFLDNEENEDILEDIVL TLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRN FMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEM ARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLS DYDVDAIVPQSFLKDDS IDNKVLTRSDKNRGKSDNVPSEEWKKMKNYWRQLLNAKLITQRKFDNLTKAER GGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVR EINNYHHAHDAYLNAWGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFK TEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK LIARKKDWDPKKYGGFDSPTVAYSVLWAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKE VKKDLI IKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVE QHKHYLDEI IEQI SEFSKRVILADANLDKVLSAYNKHRDKPIREQAENI IHLFTLTNLGAPAAFKYFDTTI DRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD

SEQ ID NO 008: _M-MLV RT (position K445T bold font)

TLNIEDEYRLHETSKEPDVSLGSTWLSDFPQAWAETGGMGLAVRQAPLI IPLKATSTPVSIKQYPMSQEAR LGIKPHIQRLLDQGILVPCQSPWNTPLLPVKKPGTNDYRPVQDLREVNKRVEDIHPTVPNPYNLLSGLPPS HQWYTVLDLKDAFFCLRLHPTSQPLFAFEWRDPEMGI SGQLTWTRLPQGFKNSPTLFNEALHRDLADFRIQ HPDLILLQYVDDLLLAATSELDCQQGTRALLQTLGNLGYRASAKKAQICQKQVKYLGYLLKEGQRWLTEAR KETVMGQPTPKTPRQLREFLGKAGFCRLFIPGFAEMAAPLYPLTKPGTLFNWGPDQQKAYQEIKQALLTAP ALGLPDLTKPFELFVDEKQGYAKGVLTQKLGPWRRPVAYLSKKLDPVAAGWPPCLRMVAAIAVLTKDAGKL TMGQPLVILAPHAVEALVTQPPDRWLSNARMTHYQALLLDTDRVQFGPWALNPATLLPLPEEGLQHNCLD ILAEAHGTRPDLTDQPLPDADHTWYTDGSSLLQEGQRKAGAAVTTETEVIWAKALPAGTSAQRAELIALTQ ALKMAEGKKLNVYTDSRYAFATAHIHGEIYRRRGWLTSEGKEIKNKDEILALLKALFLPKRLS I IHCPGHQ KGHSAEARGNRMADQAARKAAITETPDTSTLLIENSSPSGGSKRTADGSEFES

SEQ ID NO 009: oYW_741(fwd synthetic target)

AACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGC GTTGCGCTC

SEQ ID NO 010: oYW_742(rev synthetic target)

GAGCGCAACGCAATTAATGTGAGTTAGCTCACTCATTAGGCACCCCAGGCTTTACACTTTATGCTTCCGGC TCGTATGTT

SEQ ID NO 011: pegYWOBO rGrCrUrCrArCrUrCrArUrUrArGrGrCrArCrCrCrCrGrUrUrUrCrArGrArGrCrUrArUrGrCr UrGrGrArArArCrArGrCrArUrArGrCrArArGrUrUrGrArArArUrArArGrGrCrUrArGrUrCrC rGrUrUrArUrCrArArCrUrUrGrArArArArArGrUrGrGrCrArCrCrGrArGrUrCrGrGrUrGrC SEQ ID NO 012: Linker joining SpCasO and M-MLV RT in SEQ ID NO 001

SGGSSGGSKR TADGSEFESP KKKRKVSGGS SGGS

Table 1

Table 2 Table3 Table 4

Claims

Claims

1 . A variant of a prime editing (PE) polypeptide composed of a SpCas9 nickase fused to an engineered Moloney Murine leukaemia virus reverse transcriptase (RT) wherein said variant is characterized by an enhancing mutation selected from the group consisting of A277D; K1865T; and S237A, wherein the numbering of mutated positions is in reference to SEQ ID NO 001 .

2. The variant according to claim 1 , only comprising the enhancing mutations A277D and K1865T.

3. The variant according to claim 2, wherein the variant is at least (>) 95% identical to SEQ ID NO 002, particularly wherein the variant is >98%, or even >99% identical to SEQ ID NO 002.

4. The variant according to claim 2, wherein the variant is SEQ ID NO 002.

5. The variant according to claim 1 , only comprising the enhancing mutations S237A and K1865T.

6. The variant according to claim 1 , comprising all of the enhancing mutations S237A, A277D and K1865T.

7. The variant according to claim 1 , only comprising the enhancing mutation K1865T.

8. A nucleic acid sequence encoding the variant according to any one of claims 1 to 7.

9. A combination of split PE variant polypeptides a. a first split PE variant polypeptide comprising an N-terminal part of a split intein component at its C-terminus having a sequence at least 95% identical to SEQ ID NO 004; and b. a second split PE variant polypeptide comprising a C-terminal part of a split intein component at its N-terminus; having a sequence at least 95% identical to SEQ ID NO 005; wherein the first split PE variant polypeptide and the second split PE variant polypeptide, when both are present within a target cell, are capable of forming a fusion polypeptide comprising a functional prime editing polypeptide as specified in any one of the preceding claims.

10. The combination of split PE variant polypeptides according to claim 9, wherein a. said first split PE variant polypeptide is characterized by SEQ ID NO 004, or a sequence at least 97% identical to SEQ ID NO 004 and having -when joined to the second split PE variant polypeptide- the same biological activity as SEQ ID NO 004 when joined to SEQ ID NO 005; and b. said second split PE variant polypeptide comprises SEQ ID NO 005, or a sequence at least 97% identical to SEQ ID NO 005 and having -when joined to the first split PE variant polypeptide- the same biological activity as SEQ ID NO 004 when joined to SEQ ID NO 005.

11 . The combination of split PE variant polypeptides according to any one of claims 9 or 10, wherein said N-terminal part of a split intein component and said C-terminal part of a split intein component are, or share >85% identity on the amino acid sequence level with, a split intein system found in an organism selected from the group comprising the cyanobacterium Nostoc punctiforme (Npu), Mxe intein from Mycobacterium xenopi GyrA, DnaE and Rma intein from Rhodothermus marinus.

12. A combination medicament comprising a first viral vector and a second viral vector, a. the first viral vector encoding said first split PE variant polypeptide as specified in any one of claims 9 to 11 ; b. the second viral vector encoding said second split PE variant polypeptide as specified in any one of claims 9 to 11 ; c. the combination medicament further comprising a viral vector, particularly the first or second viral vector, further encoding a pegRNA; the pegRNA comprising, from 5’ to 3’ end, i. a guide RNA sequence tract capable of hybridizing to a genomic target adjacent sequence,

II. a structural RNA sequence tract facilitating interaction with the PE variant polypeptide and trans-activation of the PE variant polypeptide, ill. a template sequence tract containing a sequence reverse complementary to an edited target sequence and iv. a hybridizing sequence tract; and v. the pegRNA being capable of interacting with the PE variant polypeptide to yield a prime editing protein RNA complex.

13. A prime editing protein RNA complex comprising a variant according to any one of the preceding claims 1 to 7, wherein the prime editing protein RNA complex comprises a. a pegRNA comprising the structural RNA sequence tract is SEQ ID NO 006; and b. a polypeptide comprising or consisting of SEQ ID NO 002 or SEQ ID NO 003, or a sequence at least 85% identical, particularly >90% identical, more particularly >95% identical to SEQ ID NO 002 and having at least the same biological activity as SEQ ID NO 003.

14. The combination medicament according to claim 12, wherein a. the target sequence is characteristic of a genetic condition in a mammal, particularly a human, characterized by a transition or transversion, or a deletion or insertion mutation of a wild-type sequence, b. and the template sequence tract is characteristic of the reverse complimentary sequence of the wild-type sequence.

15. The combination medicament according to claim 14, wherein the genetic condition is associated to expression of the target sequence in the eye, liver, CNS I brain, myocard, lung, or muscle, particularly in the liver, the brain or the eye.