Patent Application
20220411777
Described herein are fusion proteins containing cytidine deaminases (e.g. human or rat APOBECs, pmCDA1 or AID) or adenosine deaminases (e.g. E. coli TadAs) or a combination thereof, catalytically impaired CRISPR-Cas proteins (e.g. Cas9, CasX or Cas12 nucleases), linkers, nuclear localization signals (NLSs) and a human or E. coli uracil-n-glycosylase (UNG) and/or REV1 protein that enable the CRISPR-guided programmable introduction of C-to-G and G-to-C transversions in DNA. The UNG may be fused to the deaminase-Cas fusion or not, in which case endogenous UNG may be recruited using molecular machinery that is integrated into the deaminase-Cas fusion architecture, e.g. using peptide or RNA aptamers or scFVs, sdABs or Fabs.
DNA base editors represent a new class of genome editing tools that enable the programmable installation of single or multiple base substitutions. Current generations of cytosine base editors (CBE) and adenine base editors (ABE) allow for the targeted deamination of cytosines and adenines that get exposed on ssDNA by RNA-guided CRISPR-Cas proteins1-4. The majority of disease-associated genetic perturbations known to date are point mutations, also known as single nucleotide variants (SNVs). Current iterations of CBEs and ABEs can target disease-relevant transition mutations and revert them to the original genotype, e.g. correcting G-to-A (C-to-T) mutations using ABE. However, a relevant fraction of disease-associated SNVs represent C-to-G and G-to-C substitutions that cannot be targeted using current BEs.
Described herein are CRISPR-guided C-to-G transversion base editors (CGBE) that enable the installation of cytosine-to-guanine and guanine-to-cytosine base edits in the ssDNA bubble generated by RNA-guided fusion proteins that contain adenine (e.g. E. coli TadA) and/or cytosine (e.g. rat APOBEC1) deaminases as well as CRISPR-Cas proteins (e.g. S. pyogenes Cas9) and/or REV1 or UNG proteins that are directly fused and/or recruited to the deaminase-Cas fusion protein. CGBE comprises a programmable DNA-binding domain (e.g. catalytically impaired dead or nicking Cas9) fused to a cytosine and/or adenosine deaminase. The adenosine deaminase can be a wild type (WT) or mutant E. coli TadA or previously described engineered TadA variants in the form of monomers, homodimers or heterodimers thereof, to decrease RNA editing activity while still preserving DNA editing activity (SECURE or RRE variants, Grünewald et al, NBT 2019—in press). The cytidine deaminase can be, e.g. rat APOBEC1, A3A, AID or pmCDA1, or previously described engineered variants of these deaminases (e.g. rAPOBEC1 with mutations from SECURE-BE3) with reduced RNA editing activity and preserved DNA editing capabilities5-9. In some embodiments, CGBE comprises one or more uracil-N-glycosylases (UNGs) fused to the N and/or C-terminus of the CBE or ABE fusion protein without uracil-N-glycosylase inhibitors (UGIs) and potentially with fused REV1 proteins. In some embodiments, CGBE comprise a linker between the adenosine or cytidine deaminase and the programmable DNA binding domain as well as between the deaminase domain and the UNG or the DNA binding domain and the UNG. In some embodiments the TadA domain can be monomeric, homodimeric or heterodimeric and contain all combinations of wild type (WT) E. coli TadA, or mutant variants of TadA).
Thus, provided herein are C-to-G transversion base editors (CGBEs) comprising a cytidine deaminase, a programmable DNA binding domain, and further comprising one or more nuclear localization sequences (NLS), and optionally one or more human or E. coli or other uracil-n-glycosylases (UNGs) or SMUG1, preferably wherein the CGBE does not comprise a uracil-N-glycosylase inhibitors (UGI).
In some embodiments, the cytidine deaminase comprises an active cytidine deaminase domain, preferably a monomeric domain, from a wild type and/or engineered rat APOBEC1 (rAPOBEC1), human APOBEC3A, human APOBEC3G, human AID, pmCDA1 (e.g., shown in Tables A and B) or variations thereof bearing mutations that reduce RNA or DNA off-target editing while retaining efficient DNA base editing.
In some embodiments, the cytidine deaminase comprises one or more mutations corresponding to mutations in rAPOBEC1, human APOBEC3A, human APOBEC3G, human AID or pmCDA1 or in any homologue or orthologue thereof (optionally those in Tables A and B).
In some embodiments, the cytidine deaminase is a rAPOBEC1 or any one of its ortho- or paralogues listed in Tables A or B, comprises one or more mutations that decrease RNA editing activity while preserving DNA editing activity, wherein the mutations are at amino acid positions that correspond to residues R33, P29, K34, E181, and/or L182 of rAPOBEC1 (SEQ ID NO:67) or to W90Y, R126E, R132E, W90Y+R126E (double mutant), R126E+R132E (double mutant), W90Y+R132E (double mutant), W90Y+R126E+R132E (triple mutant) (see, e.g., Ref. 16).
In some embodiments, the one or more mutations comprises a mutation at amino acid position that correspond to: (1) residue R33 of WT rAPOBEC1 or evoAPOBEC1; or (2) residue R13 in evoFERNY-APOBEC1; or (3) residue R12 in FERNY-APOBEC1.
In some embodiments, the mutation at amino acid position that correspond to residue R33 is a R33A substitution mutation.
In some embodiments, the CGBE comprises N- or C-terminal fusions of one or more human or E. coli UNG or SMUG1 or other orthologues of UNG or SMUG1 (e.g. as shown in Table J).
In some embodiments, the one or more UNGs are E. coli UNGs.
In some embodiments, the UNG(s) is absent, e.g., to minimize indel formation and reduce the size/length of the editor (e.g. miniCGBE1).
In some embodiments, the cytidine deaminase is a wildtype or engineered rAPOBEC1 (or any one of its ortho- or paralogues listed in Tables A or B) and the cytidine deaminase bears one or more mutations at positions: P29F, P29T, R33A, K34A, R33A+K34A (double mutant), E181Q and/or L182A of rAPOBEC1 (SEQ ID NO:67).
In some embodiments, the CGBE further includes one or more mutations at its cytidine deaminase rAPOBEC1 (or any one of its ortho- or paralogues listed in Tables A or B) residues corresponding to E24, V25; R118, Y120, H121, R126; W224-K229; P168-1186; L173+L180; R15, R16, R17, to K15-17 &A15-17; Deletion E181-L210; P190+P191; Deletion L210-K229 (C-terminal); and/or Deletion S2-L14 (N-terminal) of SEQ ID NO:67.
In some embodiments, the CGBE does not comprise one or more UNGs and/or the CGBE further comprises translesion polymerase REV1 (SEQ ID NO: 200) on either the N- or C-terminus or on both. In some embodiments, the CGBE comprises one or more UNGs and the tvBE further comprises a translesion polymerase REV1 (SEQ ID NO: 200). In some embodiments, the translesion polymerase REV1 (SEQ ID NO: 200) is fused to either the N- or C-terminus or both.
In some embodiments, the CGBE includes a linker between the cytosine deaminase monomer and/or between the cytosine deaminase monomer or single-chain dimers and the programmable DNA binding domain.
Exemplary Constructs Include:
1. CGBE1:
bpNLS-E.coliUNG-LINKER-rAPOBEC1(R33A)-LINKER-SpCas9(D10A)-bpNLS
2. miniCGBE1:
bpNLS-rAPOBEC1(R33A)-LINKER-SpCas9(D10A)-bpNLS
In some embodiments, the programmable DNA binding domain is selected from the group consisting of an engineered C2H2 zinc-finger, a transcription activator effector-like effector (TALE), and a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas RNA-guided nuclease (RGNs) and variants thereof.
The CGBE of any one of claims 1-15, wherein the CRISPR RGN is a ssDNA nickase or a catalytically inactive CRISPR Cas RNA-guided nuclease (e.g., a Cas9 or Cas12a that has ssDNA nickase activity or is catalytically inactive); in some embodiments, the Cas RGN is from SpCas9-NG or VRQR-Cas9.
Also provided herein are base editing systems comprising:
(i) a CGBE as described herein, wherein the programmable DNA binding domain is a CRISPR Cas RGN or a variant thereof; and
(ii) at least one guide RNA compatible with the base editor comprising a spacer sequence that directs the base editor to a target sequence, preferably wherein the target sequence comprises a cytosine at position 4-8, 5-7, or position 6 (with 1 being the most PAM-distal position).
Also provided herein are isolated nucleic acids encoding a CGBE as described herein, vectors comprising the isolated nucleic acids, and isolated host cells, preferably mammalian host cells (but also plant, bacterial, etc), comprising the nucleic acids or the vectors described herein. In some embodiments, the isolated host cell expresses the CGBE of any one of claims 1-17.
Additionally provided herein are methods for generating a cytosine-to-guanine and guanine-to-cytosine alteration in a nucleic acid, the method comprising contacting the nucleic acid with the CGBE of any one of claims 1-17, or the base editing system of claim 18.
In some embodiments, the CGBE achieves at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, least 50%, at least 55%, at least 60%, or at least 63% C-to-G conversions in a target sequence.
In some embodiments, the target sequence is a sequence within or adjacent to one of the genes in Table E1 or Table E2.
Also provided herein are methods for generating a cytosine-to-guanine and guanine-to-cytosine alteration in a selected nucleotide of a target region of a nucleic acid. The methods include contacting the nucleic acid with:
(i) a C-to-G transversion base editor (CGBE) comprising an adenosine deaminase, e.g., a wild type and/or engineered (e.g. ABEs 0.1, 0.2, 1.1, 1.2, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 2.10, 2.11, 2.12, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 4.1, 4.2, 4.3, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 5.10, 5.11, 5.12, 5.13, 5.14, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 7.10, ABEmax) E. coli TadA monomer, or variations of homo- or heterodimers thereof, bearing one or more mutations in either or both monomers that decrease RNA editing activity while preserving DNA editing activity, wherein the mutations are at amino acid positions that correspond to residues of E. coli TadA as listed in Table H, a programmable DNA binding domain comprising a ssDNA nickase or a catalytically inactive CRISPR Cas RNA-guided nuclease; and
(ii) at least one guide RNA compatible with the base editor and comprising a spacer that directs the base editor to the target sequence, preferably wherein the target sequence comprises a cytosine at position 4-8, 5-7, or position 6 (with 1 being the most PAM-distal position).
In some embodiments, the cytosine-to-guanine or guanine-to-cytosine alteration is listed in Table D.
Also provided herein are compositions comprising a CGBE or base editing systems as described herein, optionally including one or more ribonucleoprotein (RNP) complexes.
Additionally provided herein are the CGBE or base editing systems described herein, for use in generating a cytosine-to-guanine and guanine-to-cytosine alteration in a cell, wherein the alteration corrects a specific disease-related mutation provided in Tables E1 and E2.
In some embodiments, the CGBE does not comprise a UNG, and the CGBE recruits endogenous UNG with the help of a peptide aptamer fused to the CGBE.
In some embodiments, the CGBE does not comprise a UNG, and CGBE recruits endogenous UNG with the help of RNA aptamers fused to the gRNA.
In some embodiments, the CGBE does not comprise a UNG, and the CGBE recruits endogenous UNG with the help of a Fab, scFV or sdAb elements fused to the CGBE.
In some embodiments, the CGBE does not comprise a UNG, and wherein the CGBE recruits endogenous REV1 translesion polymerase.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
ABEs install A-to-G substitutions in DNA while CBEs allow for the introduction of C-to-T mutations. However, both these types of mutations represent transitions and the extensive subset of disease-associated transversion mutations—e.g. C-to-G mutations-cannot be directly targeted with neither CBEs nor ABEs.
We sought to engineer a C-to-G transversion base editor (CGBE) that enables the programmable installation of C-to-G and G-to-C mutations. Based on our finding that ABE proteins that do not comprise UGIs can reproducibly induce C-to-G editing at position 6 of the spacer (with 1 being the most PAM-distal position) at mutliple genomic sites (
Thus, described herein are variants of base editor fusion proteins that enable the programmable introduction of transversion base edits, specifically C-to-G and G-to-C. A table of potentially actionable codon and amino acid changes are shown in Table D and a list of potential disease targets (using Cas proteins compatible with NGG, NG, and NGA-PAMs) is shown in Tables E1-E3.
In some embodiments, the cytidine deaminase is pmCDA1 (sea lamprey) or APOBEC1 from rat, or from a different species (Table A), e.g., a different mammalian species such as H. sapiens. The APOBEC, AICDA (AID) and CDA1 family members have high sequence homology and represent potential candidates for CGBE architectures (Table B)2,15-18.
Specifically, reduced RNA editing variants of rAPOBEC1, enhanced human A3A, and human AID are candidates for inclusion into CGBE architectures.
In some embodiments, CGBE described herein can be a wild-type BE4max or SECURE-BE4max-R33A as well as eA3A variants with truncated UGIs and additional N- or C-terminal fusion of a human or E. coli UNG.
In some embodiments, the cytidine deaminases in Anc-BE4max, evoAPOBEC1-BE4max (SEQ ID 205), FERNY-BE4max, evoFERNY-BE4max (SEQ ID 204), CDA1-BE4max, and evoCDA1-BE4max may be used in a BE4max architecture with truncated UGIs and optionally also have UNGs (human or E. coli, N- or C-terminal) added. In other embodiments, the SECURE-CBE R33 and/or K34 residue changes may be introduced in evoAPOBEC1.
In some embodiments, R13 and/or K14 residue changes are introduced in FERNY and evoFERNY-APOBEC1 (these residue changes are embedded in the same amino acid sequence motif as R33 and K34 in WT rat APOBEC1 that was used in BE3, BE4, and BE4max). These modifications (single or double residue change) can greatly reduce RNA off-target editing and enhance on-target C-to-G editing. All of the APOBEC1-based CBEs described herein can used with or without the proposed mutations in the context of a C-to-G transversion base editor.
The cytidine deaminase domain need not include an entire full protein, but can be a variant as described herein that has changes or truncations that do not abolish the cytidine deaminase activity.
In some embodiments, the adenosine deaminase is TadA from E. coli, or an orthologue from a different prokaryote, e.g. S. aureus, or a homologue from the eukaryotic domain, such as yeast TAD1/2 or a mammalian species such as human (e.g. ADAT2; Table C). The tRNA-specific adenosine deaminase family members have high sequence homology and many of these orthologues may be compatible with one or more of the amino acid substitutions in E. coli TadA expected to cause an RRE phenotype and would be desirable in a CGBE architecture.
The wild type sequence of wild type E. coli TadA, available in uniprot at P68398, is as follows:
The engineered E. coli TadA sequence present in ABE7.10 and ABEmax is as follows:
In the most commonly used ABEs (ABE7.10 and ABEmax), these two proteins were fused using a 32 amino acid linker (bolded in sequence below), forming a heterodimer, the sequence of which is as follows:
GGSSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNR
Other exemplary sequences are shown in Table C. These tRNA-specific adenosine deaminase orthologues and homologues also represent candidates for inclusion of the mutations previously described at analogous positions in these proteins.
In some embodiments, the base editors included catalytically dead adenine deaminase variants, e.g. E59A. (Gaudelli et al, 2017, PMID: 29160308) as part of a heterodimer.
The adenine deaminase domain need not include an entire full protein, but can be a variant as described herein that has changes or truncations that do not abolish the adenine deaminase activity.
Cellular molecular pathways are in complete homeostasis within healthy cells. Especially, DNA repair pathways are balanced in ways that potentially mutagenic lesions are repaired at the optimal level. In mammalian cells, there is continuous generation of deamination mutations and repair of deamination reactions occurring in the background. Impairments in this process can lead to disruption of this homeostasis. On the deamination side, aberrant overexpression of deaminases that can induce spontaneous deamination at DNA and RNA levels has been shown to be responsible for inducing different cancers.10,11 On the other hand, expression levels of DNA glycosylases—a family of enzymes responsible for repairing the deaminated bases via the base excision repair (BER) pathway—are also crucial. DNA glycosylases carry out their activity by removing the lesions and creating abasic sites. Overexpression of uracil DNA glycosylase (UNG) has been shown to confer chemotherapy resistance in certain cancers.12 Moreover, overexpression of uracil glycosylase inhibitor (UGI), a component of CBEs, is potentially responsible for the observed levels of toxicity and genome-wide Cas9-independent DNA off-target effects that can be induced by CBEs. In the light of these aforementioned independent observations, it is clear that one needs to control and optimize the expression levels of the exogeneous base editor constructs in order to minimize the potential unwanted side-effects to the target cells and preserve the homeostasis.
In some embodiments of the C-to-G transversion base editors (CGBEs) described herein, Uracil-DNA glycosylase (UNG) is a critical component that carries out the generation of abasic sites after cytosines are deaminated to uracil.
Exemplary UNG/SMUG Sequences for Inclusion in CGBE
In some embodiments, the CGBE fusion proteins described herein include a functional UNG or Single-Strand-Selective Monofunctional Uracil-DNA Glycosylase 1 (SMUG1) domain. Table J provides a list of UNG and SMUG1 orthologues.
Recruiting Endogenous UNG to Target and Edit Genetic Loci
While overexpression of engineered constructs is the first and main strategy to edit genomic loci, it has been well established that overexpression of exogeneous proteins can have unwanted and fatal consequences. In the context of base editors specifically, it has been demonstrated that overexpression of base editors can induce hundreds to thousands of off-target single nucleotide variations (SNVs) on DNA and RNA.6,7,13,14 All in all, there is great need to temporally and spatially control the expression levels of base editors in target cells. To this end, recruiting the endogenous cellular machinery to carry out the enzymatic reactions of interest, instead of exogenously providing a protein in excess, is a prominent bypass to minimize exogeneous components that need to be overexpressed.
It is possible that exogeneous overexpression of human or bacterial UNG may alter the repair pathway balance towards more efficient abasic site generation genome-wide. While more research is warranted to elucidate the impact of such UNG overexpression in mammalian cells, bypassing the need for overexpression of an immunogenic (in the case of E. coli UNG) protein and preserving the natural endogenous expression levels of UNG would be advantageous. To this end, we are proposing to utilize three alternative methods/constructs with the aim of recruiting the endogenous UNG to the target site of deamination.
Peptide aptamers are small amino acid sequences that can be designed and selected against virtually any given protein of interest. Peptide aptamers can have dissociation constants similar to naturally found antibodies. Owing to their small size, ease of production, high specificity, higher stability and solubility, peptide aptamers represent a significant alternative to the antibodies. Starting from an initial randomized library of peptides, peptide aptamers can be selected and further optimized via various methods in vitro and in vivo.
Fusing an engineered peptide aptamer against human UNG into our CGBE constructs would allow us to recruit endogenous UNG bypassing the need to overexpress the protein exogenously. (
Also, various peptide aptamers can be engineered from scratch against human UNG by methods including but not limited to yeast-two-hybrid systems in vivo, and phage-display in vitro systems. Candidate peptide aptamers displaying strong affinity against human UNG will be sequenced and the identified DNA and amino acid sequences will be employed as fusion partners in our next generation CGBE constructs. Optimal conformation of the peptide aptamer fusion will be determined empirically by cloning it into different sites in our constructs with different linkers.
RNA aptamers are short stretches (80-120 nucleotides) of RNA molecules with strong and selective affinity against the target proteins of interest. Candidate RNA aptamers can be chemically synthesized as randomized libraries and several rounds of in vitro and in vivo selections can be applied. Employing the method called Systematic Evolution of Ligands by EXponential enrichment (SELEX), a number of candidate RNA aptamer molecules can be identified against one's target protein of interest.
As an example, the fusion of MS2 aptamers to CRISPR gRNAs is a widely used and well-known example of such a strategy. In this strategy, MS2 RNA aptamers are fused to the ends of gRNA constructs, thereby enabling specific recruitment of MS2 bacteriophage coat protein fused target proteins. Therefore, we propose that fusing an already engineered RNA aptamer against human UNG, if any exists, into the gRNA component of our CGBE constructs would allow us to recruit endogenous UNG bypassing the need to overexpress exogenously. (
Also, various RNA aptamers against human UNG can be engineered by strategies including but not limited to the available in vitro and in vivo SELEX strategies in the literature. Candidate RNA aptamers displaying strong affinity against human UNG will be sequenced and identified RNA sequences will be employed as gRNA fusion partners in our next generation CGBE constructs. Optimal conformation of the RNA aptamer fusion will be determined empirically by cloning it into different sites in our gRNA constructs with different linkers.
Section 3: Fab, scFV, or sdAb Mediated Recruiting of UNG to the Target Site
Antibodies are naturally expressed immunological proteins comprised of two light and two heavy chain proteins expressed from different genes. They are selected against specific parts (epitopes) of specific target proteins (antigens) in immune cells. Therefore, they can selectively bind to target antigens with high affinities. Antibodies are large molecules (˜150 kDa) consisting of a constant region (Fc) and antigen binding regions (Fab) with number of disulfide bonds in between chains. Therefore, it is not practical to generate a single peptide fusion protein fused with a large intact multimeric antibody and one's protein of interest.
However, getting rid of the Fc portion and using a single Fab portion of an antibody is a smaller (˜50 kDa) and more viable option to have than having a UNG fusion partner. Important to note is that the Fab portion still has constant regions of heavy and light chains that can be further resected while retaining the antigen specific binding affinity. This approach produces a shorter fragment (˜25 kdA) called single-chain variable fragment (scFv) that is linked with each other via short peptide linker. scFv consists of variable domains of heavy and light chains. Taking one step further and separating variable domains of heavy and light chains and producing a single chain (thus single variable domain) antibody fragment is called single-domain antibodies (sdAb) or nanobodies. This is the smallest of all antibody fragments (˜12-15 kDa) around 110 amino acids in length.
Given these premises, fusing an Fab, scFv or sdAb raised against human UNG target protein to our CGBE constructs in different conformations would be a viable option to recruit the endogenous human UNG to the target loci.
Also, various new Fabs, scFvs and sdAbs against human UNG can be generated by methods including but not limited to generating a mouse hybridoma clone, then converting full IgG (or IgM) into a scFv, Fab or sdAb; generating an immunized phage display scFv, Fab or sdAb mouse library, then using human UNG to screen the library; screening a premade scFv, Fab or sdAb antibody phage display library; generating synthetic libraries by altering the variable domains of antibodies via introducing random oligonucleotides, then screening against human UNG.
Candidate Fabs, scFvs or sdAbs displaying strong affinity against human UNG will be sequenced and the identified DNA and amino acid sequences will be employed as fusion partners in our next generation CGBE constructs. Optimal conformation of the fusion partners will be determined empirically by cloning it into different sites in our constructs with different linkers.
In some embodiments, the base editors include programmable DNA binding domains such as engineered C2H2 zinc-fingers, transcription activator effector-like effectors (TALEs), and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cas RNA-guided nucleases (RGNs) and their variants, including ssDNA nickases (nCas9) or their analogs and catalytically inactive dead Cas9 (dCas9) and its analogs (e.g., as shown in Table F), and any engineered protospacer-adjacent motif (PAM) or high-fidelity variants (e.g., as shown in Table G). A programmable DNA binding domain is one that can be engineered to bind to a selected target sequence.
Although herein we refer to Cas9, in general any Cas9-like nickase could be used (including the related Cpf1/Cas12a enzyme classes), unless specifically indicated. These orthologs, and mutants and variants thereof as known in the art, can be used in any of the fusion proteins described herein. See, e.g., WO 2017/040348 (which describes variants of SaCas9 and SpCas 9 with increased specificity) and WO 2016/141224 (which describes variants of SaCas9 and SpCas 9 with altered PAM specificity).
The Cas9 nuclease from S. pyogenes (hereafter simply Cas9) can be guided via simple base pair complementarity between 17-20 nucleotides of an engineered guide RNA (gRNA), e.g., a single guide RNA or crRNA/tracrRNA pair, and the complementary strand of a target genomic DNA sequence of interest that lies next to a protospacer adjacent motif (PAM), e.g., a PAM matching the sequence NGG or NAG (Shen et al., Cell Res (2013); Dicarlo et al., Nucleic Acids Res (2013); Jiang et al., Nat Biotechnol 31, 233-239 (2013); Jinek et al., Elife 2, e00471 (2013); Hwang et al., Nat Biotechnol 31, 227-229 (2013); Cong et al., Science 339, 819-823 (2013); Mali et al., Science 339, 823-826 (2013c); Cho et al., Nat Biotechnol 31, 230-232 (2013); Jinek et al., Science 337, 816-821 (2012)). The engineered CRISPR from Prevotella and Francisella 1 (Cpf1, also known as Cas12a) nuclease can also be used, e.g., as described in Zetsche et al., Cell 163, 759-771 (2015); Schunder et al., Int J Med Microbiol 303, 51-60 (2013); Makarova et al., Nat Rev Microbiol 13, 722-736 (2015); Fagerlund et al., Genome Biol 16, 251 (2015). Unlike SpCas9, Cpf1/Cas12a requires only a single 42-nt crRNA, which has 23 nt at its 3′ end that are complementary to the protospacer of the target DNA sequence (Zetsche et al., 2015). Furthermore, whereas SpCas9 recognizes an NGG PAM sequence that is 3′ of the protospacer, AsCpf1 and LbCp1 recognize TTTN PAMs that are found 5′ of the protospacer (Id.).
In some embodiments, the present system utilizes a wild type or variant Cas9 protein from S. pyogenes or Staphylococcus aureus, or a wild type or variant Cpf1 protein from Acidaminococcus sp. BV3L6 or Lachnospiraceae bacterium ND2006 either as encoded in bacteria or codon-optimized for expression in mammalian cells and/or modified in its PAM recognition specificity and/or its genome-wide specificity. A number of variants have been described; see, e.g., WO 2016/141224, PCT/US2016/049147, Kleinstiver et al., Nat Biotechnol. 2016 August; 34(8):869-74; Tsai and Joung, Nat Rev Genet. 2016 May; 17(5):300-12; Kleinstiver et al., Nature. 2016 Jan. 28; 529(7587):490-5; Shmakov et al., Mol Cell. 2015 Nov. 5; 60(3):385-97; Kleinstiver et al., Nat Biotechnol. 2015 December; 33(12):1293-1298; Dahlman et al., Nat Biotechnol. 2015 November; 33(11):1159-61; Kleinstiver et al., Nature. 2015 Jul. 23; 523(7561):481-5; Wyvekens et al., Hum Gene Ther. 2015 July; 26(7):425-31; Hwang et al., Methods Mol Biol. 2015; 1311:317-34; Osborn et al., Hum Gene Ther. 2015 February; 26(2):114-26; Konermann et al., Nature. 2015 Jan. 29; 517(7536):583-8; Fu et al., Methods Enzymol. 2014; 546:21-45; and Tsai et al., Nat Biotechnol. 2014 June; 32(6):569-76, inter alia. Concerning rAPOBEC1 itself, a number of variants have been described, e.g. Chen et al, RNA. 2010 May; 16(5):1040-52; Chester et al, EMBO J. 2003 Aug. 1; 22(15):3971-82.: Teng et al, J Lipid Res. 1999 April; 40(4):623-35.; Navaratnam et al, Cell. 1995 Apr. 21; 81(2):187-95.; MacGinnitie et al, J Biol Chem. 1995 Jun. 16; 270(24):14768-75.; Yamanaka et al, J Biol Chem. 1994 Aug. 26; 269(34):21725-34. The guide RNA is expressed or present in the cell together with the Cas9 or Cpf1. Either the guide RNA or the nuclease, or both, can be expressed transiently or stably in the cell or introduced as a purified protein or nucleic acid.
In some embodiments, the Cas9 also includes one of the following mutations, which reduce nuclease activity of the Cas9; e.g., for SpCas9, mutations at D10A or H840A (which creates a single-strand nickase).
In some embodiments, the SpCas9 variants also include mutations at one of each of the two sets of the following amino acid positions, which together destroy the nuclease activity of the Cas9: D10, E762, D839, H983, or D986 and H840 or N863, e.g., D10A/D10N and H840A/H840N/H840Y, to render the nuclease portion of the protein catalytically inactive; substitutions at these positions could be alanine (as they are in Nishimasu al., Cell 156, 935-949 (2014)), or other residues, e.g., glutamine, asparagine, tyrosine, serine, or aspartate, e.g., E762Q, H983N, H983Y, D986N, N863D, N863S, or N863H (see WO 2014/152432).
In some embodiments, the Cas9 is fused to one or more SV40 or bipartite (bp) nuclear localization sequences (NLSs) protein sequences; an exemplary (bp)NLS sequence is as follows: (KRTADGSEFES)PKKKRKV (SEQ ID NO: 204). Typically, the NLSs are at the N- and C-termini of an ABEmax fusion protein, but can also be positioned at the N- or C-terminus in other ABEs, or between the DNA binding domain and the deaminase domain. Linkers as known in the art can be used to separate domains.
Transcription activator like effectors (TALEs) of plant pathogenic bacteria in the genus Xanthomonas play important roles in disease, or trigger defense, by binding host DNA and activating effector-specific host genes. Specificity depends on an effector-variable number of imperfect, typically ˜33-35 amino acid repeats. Polymorphisms are present primarily at repeat positions 12 and 13, which are referred to herein as the repeat variable-diresidue (RVD). The RVDs of TAL effectors correspond to the nucleotides in their target sites in a direct, linear fashion, one RVD to one nucleotide, with some degeneracy and no apparent context dependence. In some embodiments, the polymorphic region that grants nucleotide specificity may be expressed as a triresidue or triplet.
Each DNA binding repeat can include a RVD that determines recognition of a base pair in the target DNA sequence, wherein each DNA binding repeat is responsible for recognizing one base pair in the target DNA sequence. In some embodiments, the RVD can comprise one or more of: HA for recognizing C; ND for recognizing C; HI for recognizing C; HN for recognizing G; NA for recognizing G; SN for recognizing G or A; YG for recognizing T; and NK for recognizing G, and one or more of: HD for recognizing C; NG for recognizing T; NI for recognizing A; NN for recognizing G or A; NS for recognizing A or C or G or T; N* for recognizing C or T, wherein * represents a gap in the second position of the RVD; HG for recognizing T; H* for recognizing T, wherein * represents a gap in the second position of the RVD; and IG for recognizing T.
TALE proteins may be useful in research and biotechnology as targeted chimeric nucleases that can facilitate homologous recombination in genome engineering (e.g., to add or enhance traits useful for biofuels or biorenewables in plants). These proteins also may be useful as, for example, transcription factors, and especially for therapeutic applications requiring a very high level of specificity such as therapeutics against pathogens (e.g., viruses) as non-limiting examples.
Methods for generating engineered TALE arrays are known in the art, see, e.g., the fast ligation-based automatable solid-phase high-throughput (FLASH) system described in U.S. Ser. No. 61/610,212, and Reyon et al., Nature Biotechnology 30,460-465 (2012); as well as the methods described in Bogdanove & Voytas, Science 333, 1843-1846 (2011); Bogdanove et al., Curr Opin Plant Biol 13, 394-401 (2010); Scholze & Boch, J. Curr Opin Microbiol (2011); Boch et al., Science 326, 1509-1512 (2009); Moscou & Bogdanove, Science 326, 1501 (2009); Miller et al., Nat Biotechnol 29, 143-148 (2011); Morbitzer et al., T. Proc Natl Acad Sci USA 107, 21617-21622 (2010); Morbitzer et al., Nucleic Acids Res 39, 5790-5799 (2011); Zhang et al., Nat Biotechnol 29, 149-153 (2011); Geissler et al., PLoS ONE 6, e19509 (2011); Weber et al., PLoS ONE 6, e19722 (2011); Christian et al., Genetics 186, 757-761 (2010); Li et al., Nucleic Acids Res 39, 359-372 (2011); Mahfouz et al., Proc Natl Acad Sci USA 108, 2623-2628 (2011); Mussolino et al., Nucleic Acids Res (2011); Li et al., Nucleic Acids Res 39, 6315-6325 (2011); Cermak et al., Nucleic Acids Res 39, e82 (2011); Wood et al., Science 333, 307 (2011); Hockemeye et al. Nat Biotechnol 29, 731-734 (2011); Tesson et al., Nat Biotechnol 29, 695-696 (2011); Sander et al., Nat Biotechnol 29, 697-698 (2011); Huang et al., Nat Biotechnol 29, 699-700 (2011); and Zhang et al., Nat Biotechnol 29, 149-153 (2011); all of which are incorporated herein by reference in their entirety.
Zinc finger (ZF) proteins are DNA-binding proteins that contain one or more zinc fingers, independently folded zinc-containing mini-domains, the structure of which is well known in the art and defined in, for example, Miller et al., 1985, EMBO J., 4:1609; Berg, 1988, Proc. Natl. Acad. Sci. USA, 85:99; Lee et al., 1989, Science. 245:635; and Klug, 1993, Gene, 135:83. Crystal structures of the zinc finger protein Zif268 and its variants bound to DNA show a semi-conserved pattern of interactions, in which typically three amino acids from the alpha-helix of the zinc finger contact three adjacent base pairs or a “subsite” in the DNA (Pavletich et al., 1991, Science, 252:809; Elrod-Erickson et al., 1998, Structure, 6:451). Thus, the crystal structure of Zif268 suggested that zinc finger DNA-binding domains might function in a modular manner with a one-to-one interaction between a zinc finger and a three-base-pair “subsite” in the DNA sequence. In naturally occurring zinc finger transcription factors, multiple zinc fingers are typically linked together in a tandem array to achieve sequence-specific recognition of a contiguous DNA sequence (Klug, 1993, Gene 135:83).
Multiple studies have shown that it is possible to artificially engineer the DNA binding characteristics of individual zinc fingers by randomizing the amino acids at the alpha-helical positions involved in DNA binding and using selection methodologies such as phage display to identify desired variants capable of binding to DNA target sites of interest (Rebar et al., 1994, Science, 263:671; Choo et al., 1994 Proc. Natl. Acad. Sci. USA, 91:11163; Jamieson et al., 1994, Biochemistry 33:5689; Wu et al., 1995 Proc. Natl. Acad. Sci. USA, 92: 344). Such recombinant zinc finger proteins can be fused to functional domains, such as transcriptional activators, transcriptional repressors, methylation domains, and nucleases to regulate gene expression, alter DNA methylation, and introduce targeted alterations into genomes of model organisms, plants, and human cells (Carroll, 2008, Gene Ther., 15:1463-68; Cathomen, 2008, Mol. Ther., 16:1200-07; Wu et al., 2007, Cell. Mol. Life Sci., 64:2933-44).
One existing method for engineering zinc finger arrays, known as “modular assembly,” advocates the simple joining together of pre-selected zinc finger modules into arrays (Segal et al., 2003, Biochemistry, 42:2137-48; Beerli et al., 2002, Nat. Biotechnol., 20:135-141; Mandell et al., 2006, Nucleic Acids Res., 34:W516-523; Carroll et al., 2006, Nat. Protoc. 1:1329-41; Liu et al., 2002, J. Biol. Chem., 277:3850-56; Bae et al., 2003, Nat. Biotechnol., 21:275-280; Wright et al., 2006, Nat. Protoc., 1:1637-52). Although straightforward enough to be practiced by any researcher, recent reports have demonstrated a high failure rate for this method, particularly in the context of zinc finger nucleases (Ramirez et al., 2008, Nat. Methods, 5:374-375; Kim et al., 2009, Genome Res. 19:1279-88), a limitation that typically necessitates the construction and cell-based testing of very large numbers of zinc finger proteins for any given target gene (Kim et al., 2009, Genome Res. 19:1279-88).
Combinatorial selection-based methods that identify zinc finger arrays from randomized libraries have been shown to have higher success rates than modular assembly (Maeder et al., 2008, Mol. Cell, 31:294-301; Joung et al., 2010, Nat. Methods, 7:91-92; Isalan et al., 2001, Nat. Biotechnol., 19:656-660). In preferred embodiments, the zinc finger arrays are described in, or are generated as described in, WO 2011/017293 and WO 2004/099366. Additional suitable zinc finger DBDs are described in U.S. Pat. Nos. 6,511,808, 6,013,453, 6,007,988, and 6,503,717 and U.S. patent application 2002/0160940.
In some embodiments, the components of the fusion proteins are at least 80%, e.g., at least 85%, 90%, 95%, 97%, or 99% identical to the amino acid sequence of a exemplary sequence (e.g., as provided herein), e.g., have differences at up to 1%, 2%, 5%, 10%, 15%, or 20% of the residues of the exemplary sequence replaced, e.g., with conservative mutations, e.g., including or in addition to the mutations described herein. Optionally the differences can include truncations or deletions. In preferred embodiments, the variant retains a desired activity of the parent, e.g., deaminase activity, and/or the ability to interact with a guide RNA and/or target DNA, optionally with improved specificity or altered substrate specificity.
To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%. The nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein nucleic acid “identity” is equivalent to nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. Percent identity between two polypeptides or nucleic acid sequences is determined in various ways that are within the skill in the art, for instance, using publicly available computer software such as Smith Waterman Alignment (Smith, T. F. and M. S. Waterman (1981) J Mol Biol 147:195-7); “BestFit” (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)) as incorporated into GeneMatcher Plus™, Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, M. O., Ed, pp 353-358; BLAST program (Basic Local Alignment Search Tool; (Altschul, S. F., W. Gish, et al. (1990) J Mol Biol 215: 403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software. In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the length of the sequences being compared. In general, for proteins or nucleic acids, the length of comparison can be any length, up to and including full length (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). For purposes of the present compositions and methods, at least 80% of the full length of the sequence is aligned.
For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blosum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
Also provided herein are isolated nucleic acids encoding the base editor fusion proteins, vectors comprising the isolated nucleic acids, optionally operably linked to one or more regulatory domains for expressing the variant proteins, and host cells, e.g., mammalian host cells, comprising the nucleic acids, and optionally expressing the variant proteins. In some embodiments, the host cells are stem cells, e.g., hematopoietic stem cells.
In some embodiments, the fusion proteins include a linker between the DNA binding domain (e.g., ZFN, TALE, or nCas9) and the BE domains. Linkers that can be used in these fusion proteins (or between fusion proteins in a concatenated structure) can include any sequence that does not interfere with the function of the fusion proteins. In preferred embodiments, the linkers are short, e.g., 2-20 amino acids, and are typically flexible (i.e., comprising amino acids with a high degree of freedom such as glycine, alanine, and serine). In some embodiments, the linker comprises one or more units consisting of GGGS (SEQ ID NO:135) or GGGGS (SEQ ID NO:136), e.g., two, three, four, or more repeats of the GGGS (SEQ ID NO:137) or GGGGS (SEQ ID NO:138) unit. Other linker sequences can also be used.
In some embodiments, the CGBE fusion protein includes a cell-penetrating peptide sequence that facilitates delivery to the intracellular space, e.g., HIV-derived TAT peptide, penetratins, transportans, or hCT derived cell-penetrating peptides, see, e.g., Caron et al., (2001) Mol Ther. 3(3):310-8; Langel, Cell-Penetrating Peptides: Processes and Applications (CRC Press, Boca Raton Fla. 2002); E1-Andaloussi et al., (2005) Curr Pharm Des. 11(28):3597-611; and Deshayes et al., (2005) Cell Mol Life Sci. 62(16):1839-49.
Cell penetrating peptides (CPPs) are short peptides that facilitate the movement of a wide range of biomolecules across the cell membrane into the cytoplasm or other organelles, e.g. the mitochondria and the nucleus. Examples of molecules that can be delivered by CPPs include therapeutic drugs, plasmid DNA, oligonucleotides, siRNA, peptide-nucleic acid (PNA), proteins, peptides, nanoparticles, and liposomes. CPPs are generally 30 amino acids or less, are derived from naturally or non-naturally occurring protein or chimeric sequences, and contain either a high relative abundance of positively charged amino acids, e.g. lysine or arginine, or an alternating pattern of polar and non-polar amino acids. CPPs that are commonly used in the art include Tat (Frankel et al., (1988) Cell. 55:1189-1193, Vives et al., (1997) J. Biol. Chem. 272:16010-16017), penetratin (Derossi et al., (1994) J. Biol. Chem. 269:10444-10450), polyarginine peptide sequences (Wender et al., (2000) Proc. Natl. Acad. Sci. USA 97:13003-13008, Futaki et al., (2001) J. Biol. Chem. 276:5836-5840), and transportan (Pooga et al., (1998) Nat. Biotechnol. 16:857-861).
CPPs can be linked with their cargo through covalent or non-covalent strategies. Methods for covalently joining a CPP and its cargo are known in the art, e.g. chemical cross-linking (Stetsenko et al., (2000) J. Org. Chem. 65:4900-4909, Gait et al. (2003) Cell. Mol. Life. Sci. 60:844-853) or cloning a fusion protein (Nagahara et al., (1998) Nat. Med. 4:1449-1453). Non-covalent coupling between the cargo and short amphipathic CPPs comprising polar and non-polar domains is established through electrostatic and hydrophobic interactions.
CPPs have been utilized in the art to deliver potentially therapeutic biomolecules into cells. Examples include cyclosporine linked to polyarginine for immunosuppression (Rothbard et al., (2000) Nature Medicine 6(11):1253-1257), siRNA against cyclin B1 linked to a CPP called MPG for inhibiting tumorigenesis (Crombez et al., (2007) Biochem Soc. Trans. 35:44-46), tumor suppressor p53 peptides linked to CPPs to reduce cancer cell growth (Takenobu et al., (2002) Mol. Cancer Ther. 1(12):1043-1049, Snyder et al., (2004) PLoS Biol. 2:E36), and dominant negative forms of Ras or phosphoinositol 3 kinase (PI3K) fused to Tat to treat asthma (Myou et al., (2003) J. Immunol. 171:4399-4405).
CPPs have been utilized in the art to transport contrast agents into cells for imaging and biosensing applications. For example, green fluorescent protein (GFP) attached to Tat has been used to label cancer cells (Shokolenko et al., (2005) DNA Repair 4(4):511-518). Tat conjugated to quantum dots have been used to successfully cross the blood-brain barrier for visualization of the rat brain (Santra et al., (2005) Chem. Commun. 3144-3146). CPPs have also been combined with magnetic resonance imaging techniques for cell imaging (Liu et al., (2006) Biochem. and Biophys. Res. Comm. 347(1):133-140). See also Ramsey and Flynn, Pharmacol Ther. 2015 Jul. 22. pii: S0163-7258(15)00141-2.
Alternatively or in addition, the CGBE fusion proteins can include a nuclear localization sequence, e.g., SV40 large T antigen NLS (PKKKRRV (SEQ ID NO:348)) and nucleoplasmin NLS (KRPAATKKAGQAKKKK (SEQ ID NO:349)). Other NLSs are known in the art; see, e.g., Cokol et al., EMBO Rep. 2000 Nov. 15; 1(5): 411-415; Freitas and Cunha, Curr Genomics. 2009 December; 10(8): 550-557.
In some embodiments, the CGBE fusion proteins include a moiety that has a high affinity for a ligand, for example GST, FLAG or hexahistidine sequences. Such affinity tags can facilitate the purification of recombinant CGBE fusion proteins.
The CGBE fusion proteins described herein can be used for altering the genome of a cell. The methods generally include expressing or contacting the CGBE fusion proteins in the cells; in versions using one or two Cas9s, the methods include using a guide RNA having a region complementary to a selected portion of the genome of the cell. Methods for selectively altering the genome of a cell are known in the art, see, e.g., U.S. Pat. No. 8,993,233; US 20140186958; U.S. Pat. No. 9,023,649; WO/2014/099744; WO 2014/089290; WO2014/144592; WO144288; WO2014/204578; WO2014/152432; WO2115/099850; U.S. Pat. No. 8,697,359; US20160024529; US20160024524; US20160024523; US20160024510; US20160017366; US20160017301; US20150376652; US20150356239; US20150315576; US20150291965; US20150252358; US20150247150; US20150232883; US20150232882; US20150203872; US20150191744; US20150184139; US20150176064; US20150167000; US20150166969; US20150159175; US20150159174; US20150093473; US20150079681; US20150067922; US20150056629; US20150044772; US20150024500; US20150024499; US20150020223; US20140356867; US20140295557; US20140273235; US20140273226; US20140273037; US20140189896; US20140113376; US20140093941; US20130330778; US20130288251; US20120088676; US20110300538; US20110236530; US20110217739; US20110002889; US20100076057; US20110189776; US20110223638; US20130130248; US20150050699; US20150071899; US20150050699; US20150045546; US20150031134; US20150024500; US20140377868; US20140357530; US20140349400; US20140335620; US20140335063; US20140315985; US20140310830; US20140310828; US20140309487; US20140304853; US20140298547; US20140295556; US20140294773; US20140287938; US20140273234; US20140273232; US20140273231; US20140273230; US20140271987; US20140256046; US20140248702; US20140242702; US20140242700; US20140242699; US20140242664; US20140234972; US20140227787; US20140212869; US20140201857; US20140199767; US20140189896; US20140186958; US20140186919; US20140186843; US20140179770; US20140179006; US20140170753; WO/2008/108989; WO/2010/054108; WO/2012/164565; WO/2013/098244; WO/2013/176772; US 20150071899; Makarova et al., “Evolution and classification of the CRISPR-Cas systems” 9(6) Nature Reviews Microbiology 467-477 (1-23) (June 2011); Wiedenheft et al., “RNA-guided genetic silencing systems in bacteria and archaea” 482 Nature 331-338 (Feb. 16, 2012); Gasiunas et al., “Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria” 109(39) Proceedings of the National Academy of Sciences USA E2579-E2586 (Sep. 4, 2012); Jinek et al., “A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity” 337 Science 816-821 (Aug. 17, 2012); Carroll, “A CRISPR Approach to Gene Targeting” 20(9) Molecular Therapy 1658-1660 (September 2012); U.S. Appl. No. 61/652,086, filed May 25, 2012; Al-Attar et al., Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs): The Hallmark of an Ingenious Antiviral Defense Mechanism in Prokaryotes, Biol Chem. (2011) vol. 392, Issue 4, pp. 277-289; Hale et al., Essential Features and Rational Design of CRISPR RNAs That Function With the Cas RAMP Module Complex to Cleave RNAs, Molecular Cell, (2012) vol. 45, Issue 3, 292-302.
For methods in which the CGBE fusion proteins are delivered to cells, the proteins can be produced using any method known in the art, e.g., by in vitro translation, or expression in a suitable host cell from nucleic acid encoding the CGBE fusion protein; a number of methods are known in the art for producing proteins. For example, the proteins can be produced in and purified from yeast, E. coli, insect cell lines, plants, transgenic animals, or cultured mammalian cells; see, e.g., Palomares et al., “Production of Recombinant Proteins: Challenges and Solutions,” Methods Mol Biol. 2004; 267:15-52. In addition, the CGBE fusion proteins can be linked to a moiety that facilitates transfer into a cell, e.g., a lipid nanoparticle, optionally with a linker that is cleaved once the protein is inside the cell. See, e.g., LaFountaine et al., Int J Pharm. 2015 Aug. 13; 494(1):180-194.
To use the CGBE fusion proteins described herein, it may be desirable to express them from a nucleic acid that encodes them. This can be performed in a variety of ways. For example, the nucleic acid encoding the CGBE fusion can be cloned into an intermediate vector for transformation into prokaryotic or eukaryotic cells for replication and/or expression. Intermediate vectors are typically prokaryote vectors, e.g., plasmids, or shuttle vectors, or insect vectors, for storage or manipulation of the nucleic acid encoding the CGBE fusion for production of the CGBE fusion protein. The nucleic acid encoding the CGBE fusion protein can also be cloned into an expression vector, for administration to a plant cell, animal cell, preferably a mammalian cell or a human cell, fungal cell, bacterial cell, or protozoan cell.
To obtain expression, a sequence encoding a CGBE fusion protein is typically subcloned into an expression vector that contains a promoter to direct transcription. Suitable bacterial and eukaryotic promoters are well known in the art and described, e.g., in Sambrook et al., Molecular Cloning, A Laboratory Manual (3d ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990); and Current Protocols in Molecular Biology (Ausubel et al., eds., 2010). Bacterial expression systems for expressing the engineered protein are available in, e.g., E. coli, Bacillus sp., and Salmonella (Palva et al., 1983, Gene 22:229-235). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.
The promoter used to direct expression of a nucleic acid depends on the particular application. For example, a strong constitutive promoter is typically used for expression and purification of fusion proteins. In contrast, when the CGBE fusion protein is to be administered in vivo for gene regulation, either a constitutive or an inducible promoter can be used, depending on the particular use of the CGBE fusion protein. In addition, a preferred promoter for administration of the CGBE fusion protein can be a weak promoter, such as HSV TK or a promoter having similar activity. The promoter can also include elements that are responsive to transactivation, e.g., hypoxia response elements, Gal4 response elements, lac repressor response element, and small molecule control systems such as tetracycline-regulated systems and the RU-486 system (see, e.g., Gossen & Bujard, 1992, Proc. Natl. Acad. Sci. USA, 89:5547; Oligino et al., 1998, Gene Ther., 5:491-496; Wang et al., 1997, Gene Ther., 4:432-441; Neering et al., 1996, Blood, 88:1147-55; and Rendahl et al., 1998, Nat. Biotechnol., 16:757-761).
In addition to the promoter, the expression vector typically contains a transcription unit or expression cassette that contains all the additional elements required for the expression of the nucleic acid in host cells, either prokaryotic or eukaryotic. A typical expression cassette thus contains a promoter operably linked, e.g., to the nucleic acid sequence encoding the CGBE fusion protein, and any signals required, e.g., for efficient polyadenylation of the transcript, transcriptional termination, ribosome binding sites, or translation termination. Additional elements of the cassette may include, e.g., enhancers, and heterologous spliced intronic signals.
The particular expression vector used to transport the genetic information into the cell is selected with regard to the intended use of the CGBE fusion protein, e.g., expression in plants, animals, bacteria, fungus, protozoa, etc. Standard bacterial expression vectors include plasmids such as pBR322 based plasmids, pSKF, pET23D, and commercially available tag-fusion expression systems such as GST and LacZ.
Expression vectors containing regulatory elements from eukaryotic viruses are often used in eukaryotic expression vectors, e.g., SV40 vectors, papilloma virus vectors, and vectors derived from Epstein-Barr virus. Other exemplary eukaryotic vectors include pMSG, pAV009/A+, pMTO10/A+, pMAMneo-5, baculovirus pDSVE, and any other vector allowing expression of proteins under the direction of the SV40 early promoter, SV40 late promoter, metallothionein promoter, murine mammary tumor virus promoter, Rous sarcoma virus promoter, polyhedrin promoter, or other promoters shown effective for expression in eukaryotic cells.
The vectors for expressing the CGBE fusion protein can include RNA Pol III promoters to drive expression of the guide RNAs, e.g., the H1, U6 or 7SK promoters. These human promoters allow for expression of CGBE fusion protein in mammalian cells following plasmid transfection.
Some expression systems have markers for selection of stably transfected cell lines such as thymidine kinase, hygromycin B phosphotransferase, and dihydrofolate reductase. High yield expression systems are also suitable, such as using a baculovirus vector in insect cells, with the gRNA encoding sequence under the direction of the polyhedrin promoter or other strong baculovirus promoters.
The elements that are typically included in expression vectors also include a replicon that functions in E. coli, a gene encoding antibiotic resistance to permit selection of bacteria that harbor recombinant plasmids, and unique restriction sites in nonessential regions of the plasmid to allow insertion of recombinant sequences.
Standard transfection methods are used to produce bacterial, mammalian, yeast or insect cell lines that express large quantities of protein, which are then purified using standard techniques (see, e.g., Colley et al., 1989, J. Biol. Chem., 264:17619-22; Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells are performed according to standard techniques (see, e.g., Morrison, 1977, J. Bacteriol. 132:349-351; Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983).
Any of the known procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, plasmid vectors, viral vectors, both episomal and integrative, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Sambrook et al., supra). It is only necessary that the particular genetic engineering procedure used be capable of successfully introducing at least one gene into the host cell capable of expressing the CGBE fusion protein.
In methods wherein the fusion proteins include a Cas9 domain, the methods also include delivering at least one gRNA that interacts with the Cas9, or a nucleic acid that encodes a gRNA.
Alternatively, the methods can include delivering the CGBE fusion protein and guide RNA together, e.g., as a complex. For example, the CGBE fusion protein and gRNA can be can be overexpressed in a host cell and purified, then complexed with the guide RNA (e.g., in a test tube) to form a ribonucleoprotein (RNP), and delivered to cells. In some embodiments, the CGBE fusion protein can be expressed in and purified from bacteria through the use of bacterial expression plasmids. For example, His-tagged CGBE fusion protein can be expressed in bacterial cells and then purified using nickel affinity chromatography. The use of RNPs circumvents the necessity of delivering plasmid DNAs encoding the nuclease or the guide, or encoding the nuclease as an mRNA. RNP delivery may also improve specificity, presumably because the half-life of the RNP is shorter and there's no persistent expression of the nuclease and guide (as you′d get from a plasmid). The RNPs can be delivered to the cells in vivo or in vitro, e.g., using lipid-mediated transfection or electroporation. See, e.g., Liang et al. “Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection.” Journal of biotechnology 208 (2015): 44-53; Zuris, John A., et al. “Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.” Nature biotechnology 33.1 (2015): 73-80; Kim et al. “Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.” Genome research 24.6 (2014): 1012-1019.
The present invention also includes the vectors and cells comprising the vectors, as well as kits comprising the proteins and nucleic acids described herein, e.g., for use in a method described herein.
The base editors described herein can be used to generate transversion mutations—i.e., C-to-G mutations—in a nucleic acid sequence, e.g., in a cell, e.g., a cell in an animal (e.g., a mammal such as a human or veterinary subject), or a synthetic nucleic acid substrate. The methods include contacting the nucleic acid with a base editor as described herein. Where the base editor includes a CRISPR Cas9 or Cas12a protein, the methods further include the use of one or more guide RNAs that direct binding of the base editor to a sequence to be deaminated.
For example, the base editors described herein can be used for in vitro, in vivo or in situ directed evolution, e.g., to engineer polypeptides or proteins based on a synthetic selection framework, e.g. antibiotic resistance in E. coli or resistance to anti-cancer therapeutics being assayed in mammalian cells (e.g. CRISPR-X Hess et al, PMID: 27798611 or BE-plus systems Jiang et al, PMID: 29875396).
Loxodonta
africana
Protopterus
annectens
Alligator
mississippiensis
Anolis
carolinensis
Corvus
brachyrhynchos
Calypte anna
Tursiops
truncatus
Tyto alba
Pteropus alecto
Rhinopithecus
bieti
Delphlnapterus
leucas
Lonchura striata
domestica
Amazona
aestiva
Saimiri
boliviensis
boliviensis
Pan paniscus
Pongo
pygmaeus
Bos taurus
Myotis brandtii
Felis catus
Cebus capucinus
Cebus capucinus
imitator
imitator
Pan troglodytes
Alligator
sinensis
Cricetulus
griseus
Antrostomus
carolinensis
Propithecus
coquereli
Macaca
fascicularis
Nipponia
nippon
Pelecanus
crispus
Fukomys
damarensis
Myotis davidii
Canis lupus
familiaris
Dryobates
pubescens
Mandrillus
leucophaeus
Balearica
regulorum
gibbericeps
Aptenodytes
forsteri
Enhydra lutris
Enhydra lutris
kenyoni
kenyoni
Mustela
putorius furo
Trichechus
manatus
latirostris
Ailuropoda
melanoleuca
Manacus
vitellinus
Mesocricetus
auratus
Rhinopithecus
roxellana
Chlorocebus
sabaeus
Cavia porcellus
Neomonachus
schauinslandi
Opisthocomus
hoazin
Equus ferus
caballus
Homo sapiens
Nestor notabilis
Egretta garzetta
Aotus
nancymaae
Mus musculus
Heterocephalus
glaber
Merops nubicus
Fulmarus
glacialis
Nomascus
leucogenys
Papio anubis
Monodelphis
domestica
Dipodomys ordii
Odobenus
rosmarus
divergens
Patagioenas
Patagioenas
fasciata monilis
fasciata monilis
Colobus
angolensis
palliatus
Tarsius syrichta
Sus scrofa
Macaca
nemestrina
Oryctolagus
cuniculus
Rattus
norvegicus
Cariama
cristata
Gavia stellata
Macaca mulatta
Acanthisitta
chloris
chloris)
Columba livia
Ovis aries
Otolemur
gamettii
Stylophora
pistillata
Cercocebus
atys
Physeter
macrocephalus
Pongo abelii
Eurypyga helias
Sarcophilus
harrisii
Leptonychotes
weddellii
Erinaceus
europaeus
Haliaeetus
albicilla
Callithrix
jacchus
Bos mutus
Pterocles
gutturalis
Petromyzon
marinus cytosine
Petromyzon
marinus cytosine
E. coli TadA
S. aureus TadA
S. pyogenes TadA
S. typhi TadA
A. aeolicus TadA
S. pombe TAD2
S. cerevisiae TAD1
S. cerevisiae TAD2
A. thaliana TAD2
X. laevis ADAT2
X. tropicalis ADAT2
D. rerio ADAT2
B. taurus ADAT2
M. musculus ADAT2
H. sapiens ADAT2
S. pyogenes Cas9
S. aureus Cas9
S. thermophilus Cas9
S. pasteurianus Cas9
C. jejuni Cas9
F. novicida Cas9
P. lavamentivorans
C. lari Cas9 (ClCas9)
Pasteurella multocida
F. novicida Cpf1
M. bovoculi Cpf1
L. bacterium N2006
S. pyogenes Cas9
S. pyogenes Cas9
S. pyogenes Cas9
S. pyogenes Cas9
S. pyogenes Cas9
S. pyogenes Cas9
S. pyogenes Cas9
S. pyogenes Cas9
S. pyogenes Cas9
S. aureus Cas9
S. aureus Cas9 with PAM interaction
Streptococcus
macacae (Smac) Cas9
N. meningitidis
Caenorhabditis
elegans
Mycobacterium
riyadhense
Enterobacter cloacae
Clostridium oryzae
Lactobacillus apis
Flavobacterium sp.
Delftia lacustris
Lactococcus garvieae
Lactobacillus rodentium
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
All base editor (BE) and prime editor (PE) constructs were cloned into a mammalian expression plasmid backbone under the control of a pCMV promoter (AgeI and NotI restriction digest of parental plasmid Addgene #112101). The wild-type SpCas9 construct (SQT 817; Addgene #53373) is expressed under the control of a CAG promoter. All BE and PE constructs were encoded as P2A-eGFP fusions for co-translational expression of the base/prime editors and eGFP. Gibson fragments with matching overlaps were PCR-amplified using Phusion High-fidelity polymerase (NEB). Fragments were gel-purified and assembled for 1 hour at 50° C. and transformed into chemically competent E. coli (XL1-Blue, Agilent). The UNGs used in our experiments originated either from E. coli (eUNG; UniProtKB-P12295) or Homo sapiens (hUNG; UniProtKB-P13051), were codon-optimized for expression in human cells and synthesized as gblocks (IDT). All guide RNA (gRNA) constructs were cloned into a BsmBI-digested pUC19-based entry vector (BPK1520, Addgene #65777) with a U6 promoter driving gRNA expression. We designed the pegRNAs to implement the same C-to-G changes that the CGBE constructs would install and followed previously described default design rules for designing pegRNAs and ngRNAs15. PegRNAs were cloned into the BsaI-digested pU6-pegRNA-GG-acceptor entry vector (Addgene #132777) and ngRNAs were cloned into the abovementioned BsmBI-digested entry vector BPK1520. Oligos containing the spacer, the 5′phosphorylated pegRNA scaffold, and the 3′ extension sequences were annealed to form dsDNA fragments with compatible overhangs and ligated using T4 ligase (NEB). All plasmids used for transfection experiments were prepared using Qiagen Midi or Maxi Plus kits.
GAATACTAAGCATAGACTCC
GTAAACAAAGCATAGACTGA
GAAGACCAAGGATAGACTGC
ACACACACACTTAGAATCTG
CACACACACTTAGAATCTGT
TTAAGCTGTAGTATTATGAA
CCTGGCCTGGGTCAATCCTT
GAGTCCGAGCAGAAGAAGAA
GTATTCACCTGAAAGTGTGC
GGAATCCCTTCTGCAGCACC
GAACACAAAGCATAGACTGC
GGCCCAGACTGAGCACGTGA
GGCACTGCGGCTGGAGGTGG
CTGGCCTGGGTCAATCCTTG
CAAAGCAGGATGACAGGCAG
ACTTCCACATGAGCGTGGTC
GGCACTCGGGGGCGAGAGGA
GAGCTCACTGAACGCTGGCA
GACCCTCAGCCGTGCTGCTC
GCTGACTCAGAGACCCTGAG
GGGGCTCAACATCGGAAGAG
GCTGGCTCAGGTTCAGGAGA
CTGCTCGGGGTGGGACTCTG
GTCATCTTAGTCATTACCTG
GAGGACGTGTGTGTCTGTGT
TAAGCATAGACTCCAGGATA
TACTCTGAGTGTACAAAAGA
AGTAAACAAAGCATAGACTG
TTTGTGCAAACACAGATTGC
CGGGCATCAGAATTCCCTGG
AAAGTACAAACGGCAGAAGC
GTACAAACGGCAGAAGCTGG
GCTGCAGAAGGGATTCCATG
CGCCGTCTCCAAGGTGAAAG
AGCGATCCAGGTGCTGCAGA
GGAACACAAAGCATAGACTG
TGTGTTCCAGTTTCCTTTAC
TTGTTTGCAGCTATTCAGGC
AAGTCGAGGGAGGGATGGTA
GACACGTGGATTGTGCTGTC
GTCATACACTGGGCTGGCCA
CAAAGTCCAGGACCGGCTGG
GCATGGCTCTAGTGCTTTCC
GGTCATACACTGGGCTGGCC
AAGGAGACAAAGTCCAGGAC
GATTGTGCTGTCAGGAGCTC
ATGACTAAGATGACTGCCAA
TGAGTTACAACGAACACCTC
ACCATCTTTTGTACACTCAG
CACTTCTCTTCCTGCCCTCT
AGCTTCTGCCGTTTGTACTT
CGTCTCATATGCCCCTTGGC
ATAGACTCCAGGATAAGGTA
CTCAACATCGGAAGAGGGGA
TCAATCCTTGGGGCCCAGAC
ATGTTCCAATCAGTACGCAG
GATGACTGCCAAGGGGCATA
AAGTACAAGCACTCAATGTG
ACACACACTTAGAATCTGTG
GCGGACAGTGGACGCGGCGG
GAACACAATGCATAGATTGC
AAACATAAAGCATAGACTGC
CACCCAGACTGAGCACGTGC
GACACAGACTGGGCACGTGA
AGCTCAGACTGAGCAAGTGA
AGACCAGACTGAGCAAGAGA
GAGCCAGAATGAGCACGTGA
TGCACTGCGGCCGGAGGAGG
GGCTCTGCGGCTGGAGGGGG
GGCACGACGGCTGGAGGTGG
GGCATCACGGCTGGAGGTGG
GGCGCTGCGGCGGGAGGTGG
GAGTCTAAGCAGAAGAAGAA
GAGGCCGAGCAGAAGAAAGA
GAGTCCTAGCAGGAGAAGAA
GAGTCCGGGAAGGAGAAGAA
GAGCCGGAGCAGAAGAAGGA
GGAACCCCGTCTGCAGCACC
GGAGTCCCTCCTACAGCACC
AGAGGCCCCTCTGCAGCACC
ACCATCCCTCCTGCAGCACC
GGATTGCCATCCGCAGCACC
TGAATCCCATCTCCAGCACC
CTATATTACTT
ACCTTATCC
ATGAGGAAAG
GGACTAGAGT
GCTGGCCCTG
TAAAGGAAAC
TGAGTTACAA
CGAACACCTC
GGCCCAGACT
GAGCACGTGA
GGAATCCCTT
CTGCAGCACC
STR-authenticated HEK293T (CRL-3216), K562 (CCL-243), HeLa (CCL-2), and U2OS cells (similar match to HTB-96; gain of #8 allele at the D5S818 locus) were used in this study. HEK293T and HeLa cells were grown in Dulbecco's Modified Eagle Medium (DMEM, Gibco) with 10% heat-inactivated fetal bovine serum (FBS, Gibco) supplemented with 1% penicillin-streptomycin (Gibco) antibiotic mix. K562 cells were grown in Roswell Park Memorial Institute (RPMI) 1640 Medium (Gibco) with 10% FBS supplemented with 1% Pen-Strep and 1% GlutaMAX (Gibco). U2OS cells were grown in DMEM with 10% FBS supplemented with 1% Pen-Strep and 1% GlutaMAX. Cells were grown at 37° C. in 5% CO2 incubators and periodically passaged upon reaching around 80% confluency. Cell culture media supernatant was tested for mycoplasma contamination using the MycoAlert mycoplasma detection kit (Lonza) and all tests were negative throughout the experiments.
HEK293T cells were seeded at 1.25×104 cells per well into 96-well flat bottom cell culture plates (Corning) for DNA on-target experiments or at 6.25×104 cells per well into 24-well cell culture plates (Corning) for DNA off-target experiments. 24 hours post-seeding, cells were transfected with 30 ng of control or base/prime editor plasmid and 10 ng of gRNA plasmid (and 3.3 ng nicking gRNA plasmid for PE3) using 0.3 μL of TransIT-X2 (Mirus) lipofection reagent for experiments in 96-well plates, or 150 ng control or base editor plasmid and 50 ng gRNA, and 1.5 μL TransIT-X2 for experiments in 24-well plates. K562 cells were electroporated using the SF Cell Line Nucleofector X Kit (Lonza), according to the manufacturer's protocol with 2×105 cells per nucleofection and 800 ng control or base/prime editor plasmid, 200 ng gRNA or pegRNA plasmid, and 83 ng nicking gRNA plasmid (for PE3). U2OS cells were electroporated using the SE Cell Line Nucleofector X Kit (Lonza) with 2×105 cells and 800 ng control or base/prime editor plasmid, 200 ng gRNA or pegRNA, and 83 ng nicking gRNA (for PE3). HeLa cells were electroporated using the SE Cell Line 4D-Nucleofector X Kit (Lonza) with 5×105 cells and 800 ng control or base/prime editor, 200 ng gRNA or pegRNA, and 83 ng nicking gRNA (for PE3). 72 hours post-transfection, cells were lysed for extraction of genomic DNA (gDNA).
HEK293T cells were washed with 1×PBS (Corning) and lysed overnight by shaking at 55° C. with 43.5 μl of gDNA lysis buffer (100 mM Tris-HCl at pH 8, 200 mM NaCl, 5 mM EDTA, 0.05% SDS) supplemented with 5.25 μl of 20 mg/ml Proteinase K (NEB) and 1.25 μl of 1M DTT (Sigma) per well for experiments in 96-well plates, or with 174 μl DNA lysis buffer, 21 μl Proteinase K, and 5 μL 1M DTT per well for experiments in 24-well plates. K562 cells were centrifuged for 5 min, media removed, and lysed overnight by shaking at 55° C. with 174 μl DNA lysis buffer, 21 μl Proteinase K, and 5 μL 1M DTT per well in 24-well plates. U2OS cells and HeLa cells were washed with 1×PBS and lysed overnight shaking at 55° C. with 174 μl DNA lysis buffer, 21 μl Proteinase K, and 5 μL 1M DTT per well in 24-well plates. Subsequently, gDNA was extracted from lysates using 1-2× paramagnetic beads as previously described7 and eluted in 45 μl of 0.1×EB buffer. DNA extraction was performed using a Biomek FXP Laboratory Automation Workstation (Beckman Coulter).
DNA targeted amplicon sequencing was performed as previously described.7 Briefly, extracted gDNA was quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher). Amplicons were constructed in 2 PCR steps. In the first PCR, regions of interest (170-250 bp) were amplified from 5-20 ng of gDNA with primers containing Illumina forward and reverse adapters on both ends (Supplementary Table 9). PCR products were quantified on a Synergy HT microplate reader (BioTek) at 485/528 nm using a Quantifluor dsDNA quantification system (Promega), pooled and cleaned with 0.7× paramagnetic beads, as previously described.7 In a second PCR step (barcoding), unique pairs of Illumina-compatible indexes (equivalent to TruSeq CD indexes, formerly known as TruSeq HT) were added to the amplicons. The amplified products were cleaned up with 0.7× paramagnetic beads, quantified with the Quantifluor or Qubit systems, and pooled before sequencing. The final library was sequenced on an Illumina MiSeq machine using the Miseq Reagent Kit v2 (300 cycles, 2×150 bp, paired-end). Demultiplexed FASTQ files were downloaded from BaseSpace (Illumina).
Human HEK293T cells were transfected with plasmids encoding nCas9, ABEmax, miniABEmax-K20/R21A, and miniABEmax-V82G (
Given the observation outlined in Example 1 on ABE-mediated C-to-G alterations, we wondered whether we could induce these edits more efficiently by modifying the BE4max CBE8,15, which harbors an enzyme actually intended to deaminate cytosines (the rat APOBEC1 cytidine deaminase)(
We also investigated whether introducing mutations into the APOBEC1 part of BE4maxΔUGI-hUNG might further increase the frequency of C-to-G editing. Although we do not have a mechanistic understanding of how C-to-G edits are induced, we reasoned that altering the deamination dynamics of APOBEC1 might also influence the editing outcome. We focused on the APOBEC1 R33A mutation, a substitution we previously showed can decrease off-target RNA editing while substantially preserving the efficiency and increasing the precision of on-target DNA editing by CBEs5. We found that introduction of R33A into BE4maxΔUGI-hUNG increased C-to-G editing frequencies with three of the seven gRNAs tested in HEK293T cells while leaving editing frequencies essentially unaltered with the other four (
We additionally explored whether replacing the hUNG present in the BE4max(R33A)ΔUGI editor with an orthologous UNG from Escherichia coli (eUNG) might further increase the efficiency of C-to-G edits. We created two additional editors: BE4max(R33A)ΔUGI-eUNG or eUNG-BE4max(R33A)ΔUGI with an eUNG added to the carboxy- or amino-terminal ends, respectively. Testing of these fusions in HEK293T cells revealed that both induced C-to-G edits with higher frequencies than BE4max(R33A)ΔUGI-hUNG for six out of seven gRNAs tested (mean editing frequencies ranging from 3.3-57.0% and 8.5-62.6% for BE4max(R33A)ΔUGI-eUNG and eUNG-BE4max(R33A)ΔUGI, respectively) (
To more comprehensively characterize CGBE1, we tested its activity with 18 additional gRNAs in human HEK293T cells. 12 of the sites targeted by these 18 gRNAs have a C at position 6 (“C6-sites”) (
We explored the impact of deleting the eUNG domain from the CGBE1 editor on its activity. This particular editor architecture, which we named miniCGBE1 (
To more fully characterize the positional preferences within the editing windows of CGBE1 and miniCBGE1, we tested these two editors side-by-side with BE4max and BE4max(R33A) using 23 additional gRNAs that target sites with cytosines at protospacer positions 4, 5, 7, and 8 (
Cas9-dependent DNA off-target profiles of CGBEs was assessed by transfecting HEK 293T cells with nCas9 control, BE4max, BE4max(R33A), CGBE1, and miniCGBE1 using HEK site 2, HEK site 3, HEK site 4, EMX1 site 1, and FANCF site 1 gRNAs. 23 genomic sites that have previously been described as known off-target sites for said gRNAs (Tsai et al, NBT 2014) were sequenced with NGS to detect potential off-target base editing of CGBE constructs. BE4max induced C-to-D (D=A, G, or T) edits at 15 of the 23 off-target sites with BE4max-R33A inducing edits less efficiently at all 15 sites, consistent with previously published observations that introduction of R33A reduces Cas9-dependent DNA off-target edits by the BE3 CBE (
We tested whether we could improve the somewhat more restricted targeting range of CGBEs by using previously described SpCas9-NG and SpCas9-VRQR variants that recognize shorter NG19 and alternative NGA20 PAMs, respectively. We targeted six sites with NGT PAMs using modified CGBE1-NG and miniCGBE1-NG variants and six sites with NGAG PAMs using CGBE1-VRQR and miniCGBE1-VRQR variants. Each of these 12 new sites have a cytosine at position 6 embedded within an AT-rich sequence context to provide an optimal target for C-to-G editing (
We compared our CGBEs with Prime Editing (PE) methods that can introduce a diverse range of different edits and that were published15 while we were completing this project. The PE2 system uses two components: (1) a Prime Editor fusion protein and (2) a prime editing gRNA (pegRNA) (
CGBE architectures described in
Unbiased detection of RNA off-target editing with the help of RNA-seq will be assessed. Cells will be transfected with two different gRNAs and CGBE constructs that are co-translationally expressed with P2A-EGFP in 15 cm dishes and trypsinized 36 hours post-transfection. Subsequently, GFP+ cells will be sorted on a BD FACSAria II and lysed to harvest both DNA and RNA. After efficient on-target editing is confirmed via targeted amplicon sequencing, RNA-seq will be performed using a TruSeq stranded total RNA library prep and sequencing on a NextSeq 500 machine at the MGH or a NovaSeq at the Broad Institute.
Next generation CGBE constructs fused with the candidate peptide aptamers will be assessed by transfection experiments, for example, those using lipofection and nucleofection techniques into human cells such as HEK 293T, U2OS and K562 cell lines. The transfections will be carried out with gRNA constructs with spacer sequences targeting human genomic loci having cytosines in the editing windows that is generated by our CGBE constructs. 72 hours post-transfection, genomic DNA (gDNA) will be harvested, and target loci will be PCR amplified. PCR amplicons will be subjected to targeted next generation sequencing (NGS) to quantify on-target editing efficiencies. The DNA off-target activities of the next generation CGBE constructs will be assessed by analyzing the top in-silico predicted candidate off-target sites using targeted amplicon sequencing (NGS) using the treated gDNAs. In order to assess the potential RNA off-target activities of our next generation CGBE constructs, we will be harvesting total RNA in parallel in the treated cells in order to conduct stranded libraries for transcriptome-wide analysis via RNA sequencing (RNA-seq).
The next generation CGBE constructs will be analyzed using RNA aptamers fused to the gRNA in a series of transfection experiments (using, for example, lipofection and nucleofection techniques) in human cells such as HEK 293T, U2OS and K562 cell lines. The transfections will be carried out with fusion gRNA constructs with spacer sequences targeting human genomic loci having cytosines in the editing windows generated by our CGBE constructs. 72 hours post-transfection, genomic DNA (gDNA) will be harvested, and target loci will be PCR amplified. PCR amplicons will be subjected to targeted next generation sequencing (NGS) to quantify on-target editing efficiencies. In order to test the potential DNA off-target activities of our next generation CGBE constructs, the top in-silico predicted candidate off-target sites will be analyzed with targeted amplicon sequencing (NGS) using the treated gDNAs. In order to assess the potential RNA off-target activities of our next generation CGBE constructs, we will be harvesting total RNAs in parallel in the treated cells in order to conduct transcriptome-wide analysis via RNA sequencing (RNA-seq).
Next generation CGBE constructs fused with the candidate Fab, scFv, or sdAb, will be assessed in a series of transfection experiments (e.g., using lipofection or nucleofection techniques) in human cells such as HEK 293T, U2OS and K562 cell lines. The transfections will be carried out with gRNA constructs with spacer sequences targeting human genomic loci having cytosines in the editing windows generated by CGBE constructs. 72 hours post-transfection, genomic DNA (gDNA) will be harvested, and target loci will be PCR amplified. PCR amplicons will be subjected to targeted next generation sequencing (NGS) to quantify on-target editing efficiencies. DNA off-target activities of the next generation CGBE constructs will be assessed by analyzing the top in silico predicted candidate off target sites using targeted amplicon sequencing (NGS). In order to assess the potential RNA off-target activities of our next generation CGBE constructs, we will be harvesting total RNA in parallel in the treated cells in order to conduct transcriptome-wide analysis via RNA sequencing (RNA-seq).
Human Lung Cancer. Ann. Surg. Oncol. 18, 2084-2092 (2011).
mississippiensis OX = 8496 GN = APOBEC1A PE = 4 SV = 1
striata domestica OX = 299123 GN = APOBEC1 PE = 4 SV = 1
aestiva OX = 12930 GN = AAES_27783 PE = 4 SV = 1
sinensis OX = 38654 GN = LOC102373005 PE = 4 SV = 1
fascicularis OX = 9541 GN = EGM_20518 PE = 4 SV = 1
damarensis OX = 885580 GN = H920_16562 PE = 4 SV = 1
kenyoni OX = 391180 GN = LOC111142361 PE = 4SV = 1
furo OX = 9669 GN = APOBEC1 PE = 2 SV = 1
manatus latirostris OX = 127582 GN = LOC101361717 PE = 4 SV = 1
domestica OX = 13616 GN = APOBEC1 PE = 1 SV = 1
ordii OX = 10020 GN = Apobec1 PE = 4 SV = 1
fasciata monilis OX = 372326 GN = APOBEC1 PE = 4 SV = 1
scrota OX = 9823 GN = APOBEC1 PE = 4 SV = 2
mulatta OX = 9544 GN = EGK_03318 PE = 4 SV = 1
pistillata OX = 50429 GN = APOBEC1 PE = 4 SV = 1
sapiens OX = 9606 GN = APOBEC3B PE = 1 SV = 1
sapiens OX = 9606 GN = APOBEC3C PE = 1 SV = 2
sapiens OX = 9606 GN = APOBEC3D PE = 1 SV = 1
sapiens OX = 9606 GN = APOBEC3G PE = 1 SV = 1
sapiens OX = 9606 GN = APOBEC3H PE = 1 SV = 4
Petromyzon marinus cytosine deaminase (pmCDA1), Genbank ABO15149.1
Petromyzon marinus cytosine deaminase (pmCDA1) R187W, as used in Target-AID,
E. coli TadA, SEQ ID NO: 98
S. aureus TadA, SEQ ID NO: 99
S. pyogenes TadA, SEQ ID NO: 100
S. typhi TadA, SEQ ID NO: 101
A. aeolicus TadA, SEQ ID NO: 102
S. pombe TAD2, SEQ ID NO: 103
S. cerevisiae TAD1, SEQ ID NO: 104
S. cerevisiae TAD2, SEQ ID NO: 105
A. thaliana TAD2, SEQ ID NO: 106
X. laevis ADAT2, SEQ ID NO: 107
X. tropicalis ADAT2, SEQ ID NO: 108
D. rerio ADAT2, SEQ ID NO: 109
B. Taurus ADAT2, SEQ ID NO: 110
M. musculus ADAT2, SEQ ID NO: 111
thaliana OX = 3702 GN = UNG PE = 1 SV = 1
maritimus OX = 29073 GN = UNG PE = 3 SV = 1
bieti OX = 61621 GN = UNG PE = 3 SV = 1
ursinus OX = 29139 GN = UNG PE = 3 SV = 1
pombe (strain 972/ATCC 24843) OX = 284812 GN = ung1 PE = 3 SV = 1
rubripes OX = 31033 GN = ung PE = 3 SV = 1
electricus OX = 8005 GN = ung PE = 3 SV = 1
tropicalis OX = 8364 GN = aoc3 PE = 3 SV = 1
cloacae subsp. cloacae OX = 336306 GN = ung PE = 3 SV = 1
oryzae OX = 1450648 GN = ung PE = 3 SV = 1
apis OX = 303541 GN = ung PE = 3 SV = 1
garvieae OX = 1363 GN = ung PE = 3 SV = 1
rodentium OX = 947835 GN = ung PE = 3 SV = 1
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Patent Application Ser. No. 62/894,628 filed on Aug. 30, 2019; 62/910,912 filed on Oct. 4, 2019; 62/916,654 filed on Oct. 17, 2019; and 63/023,208, filed on May 11, 2020. The entire contents of the foregoing are hereby incorporated by reference.
This invention was made with Government support under Grant No. HG009490 awarded by the National Institutes of Health and contract HR0011-17-2-0042 awarded by the Defense Advanced Research Projects Agency of the Department of Defense. The Government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2020/048777 | 8/31/2020 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63023208 | May 2020 | US | |
| 62916654 | Oct 2019 | US | |
| 62910912 | Oct 2019 | US | |
| 62894628 | Aug 2019 | US |
