Patent Grant
12123033
This application is filed with a Computer Readable Form of a Sequence Listing in accordance with 37 C.F.R. § 1.821(c). The text file submitted by EFS, “013670-9060-US02_sequence_listing_18-DEC-2020_ST25.K” was created on Dec. 18, 2020, contains 235 sequences, has a file size of 86.4 Kbytes, and is hereby incorporated by reference in its entirety.
Described herein are compositions and methods for improving homology directed repair (HDR) efficiency and reducing homology-independent integration following introduction of double strand breaks with engineered nucleases. Additionally, modifications to double stranded DNA donors to improve the donor potency and efficiency of homology directed repair following introduction of double stranded breaks with programmable nucleases.
Genome editing with programmable nucleases allows the site-specific introduction of DNA into target genomes of interest. A number of systems permit targeted genomic editing and these systems include transcription activator-like effector nucleases (TALENs), zinc fingers (ZFNs), or clustered, regularly interspaced, short palindromic repeat (CRISPR).
The CRISPR-Cas9 system has been widely utilized to perform site-specific genome editing in eukaryotic cells. A sequence specific guide RNA is required to recruit Cas9 protein to the target site, and then the Cas9 endonuclease cleaves both strands of the target DNA creating a double stranded break (DSB). This DSB is corrected by the cell's innate DNA damage repair pathways. Two of the main pathways of DSB repair are the error prone non-homologous end joining (NHEJ) pathway, which can lead to random insertions or deletions (indels) in the target DNA, and the homology directed repair (HDR) pathway, which uses a single or double stranded DNA molecule with homology to either side of the DSB as a repair template to generate a desired mutation in the target DNA [1].
Various forms of DNA can be used as the repair template for HDR experiments such as plasmid DNA, double stranded linear DNA (dsDNA), or single stranded DNA (ssDNA). Both dsDNA and ssDNA donors can induce an innate immune response in mammalian tissue culture cells. For short insertions (generally 120 bp) or mutations, a chemically synthesized oligonucleotide such as an IDT® Ultramer™ ssDNA can be used as the single stranded oligo donors (ssODN) for HDR experiments. The use of synthetic ssDNA allows for chemical modifications to be placed in the molecule to potentially improve HDR efficiency. Templates for larger insertions (generally >120 bp) are limited due to the increased complexity of synthesis. Generation of long ssDNA can be a labor intensive and costly process, while linear dsDNA can be generated quickly and in large quantities. Because, the more prevalent NHEJ repair pathway facilitates the ligation of blunt ends, a linear dsDNA donor has a higher risk for homology-independent integration into any DSB present in the cell (including the on-target Cas9 cleavage site, any Cas9 off-target sites, and any endogenous DSB) [2, 3]. When homology-independent integration occurs at the on-target site, the entire donor is incorporated including the homology arms leading to the duplication of one or both homology arm regions.
It has been reported that the addition of a 5′-biotin modification on a linear dsDNA donor can reduce the formation of concatemers and integration via the NHEJ pathway [4]. Similarly, another group reported that biotin or ssDNA overhangs on the 5′-terminus can reduce blunt insertions [5]. Another group suggested that TEG and 2′-OMe ribonucleotide adapters on the 5′-termini of dsDNA donors could potentially increase HDR rates by limiting access of the NHEJ machinery to the free ends of the donor but did not demonstrate any reduction in blunt integration [6].
There is a need for compositions of modified dsDNA templates for HDR and methods thereof that increase the efficiency of HDR and reduce undesired homology-independent integration (both at the targeted site and potential off-target or endogenous DSBs) that is typically associated with linear dsDNA donors.
One embodiment described herein is a double stranded DNA homology directed repair (HDR) donor comprising: a first homology arm region, an insert region, and a second homology arm region; wherein the first homology arm region and the second homology arm region comprise modifications to one or more nucleotides at or near the 5′-termini. In one aspect, the modifications comprise: modifications to the 2′-position of one or more nucleotides at or near the 5′-terminus of the first homology arm region and modifications to the 2′-position of one or more nucleotides at or near the 5′-terminus of the second homology arm region. In another aspect, the modifications comprise modifications to the 2′-position of the 5′-terminal nucleotide, the 5′-penulimate nucleotide, the 5′-antepenultimate (third) nucleotide, or a combination of the nucleotides at or near the 5′-terminus of the first homology arm region and the second homology arm region. In another aspect, the modifications at or near the 5′-termini of the double stranded DNA HDR donor comprise one or more of: 2′-O-methyl (2′-OMe), 2′-fluoro (2′-F), or 2′-O-methoxylethyl (2′-MOE). In another aspect, the modifications at or near the 5′-termini of the double stranded DNA HDR donor comprise 2′-MOE. In another aspect, the modification at or near the 5′-termini are non-template mismatches relative to a target DNA. In another aspect, the first homology arm region and the second homology arm region are 40 to 150 nucleotides in length. In another aspect, the first homology arm region and the second homology arm region are at least 100 nucleotides in length. In another aspect, the double stranded DNA HDR donor further comprises universal primer sequences. In another aspect, the insert region is greater than 100 bp. In aspect, the insert region is greater than 0.25 kb, greater than 0.5 kb, greater than 1 kb, greater than 2 kb, greater than 3 kb, greater 4 kb, greater than 5 kb, greater than 6 kb, greater than 7 kb, greater than 8 kb, greater than 9 kb, or greater than 10 kb. In another aspect, the double stranded HDR donor comprises a hairpin at either the 5′-terminus or the 3′-terminus. In another aspect, the double stranded HDR donor comprises a hairpin at both the 5′-terminus and the 3′-terminus. In another aspect, the double stranded DNA HDR donor improves homology directed repair efficiency and reduces homology-independent integration in a programmable nuclease system.
Another embodiment described herein is a programmable nuclease system comprising: a modified double stranded DNA homology directed repair (HDR) donor, a programmable nuclease enzyme, and a gRNA, wherein the gRNA molecule is capable of targeting the programmable nuclease molecule to a target nucleic acid. In one aspect, the modified double stranded DNA HDR donor comprises a first homology arm region, an insert region, and a second homology arm region; wherein the first homology arm region and the second homology arm region comprises modifications to one or more nucleotides at or near the 5′-termini. In another aspect, the modified double stranded DNA HDR donor comprises modifications to the 2′-position of the 5′-terminal nucleotide, the 5′-penulimate nucleotide, the 5′-antepenultimate (third) nucleotide, or a combination of the nucleotides at or near the 5′-terminus of the first homology arm region and the second homology arm region. In another aspect, the modified double stranded DNA HDR donor comprises at least one 2′-OME, 2′-F, or 2′-MOE modifications one or more nucleotides at or near the 5′-termini. In another aspect, the modified double stranded DNA HDR donor comprises one or more 2′-MOE modifications at or near the 5′-termini. In another aspect, the modified double stranded DNA HDR donor comprises universal primer sequences. In another aspect, the modified double stranded DNA HDR donor improves homology directed repair efficiency and reduces homology-independent integration in a programmable nuclease system. In another aspect, the programmable nuclease system comprises one or more of transcription activator-like effector nucleases (TALENs), zinc fingers (ZFNs), or clustered, regularly interspaced, short palindromic repeat (CRISPR). In another aspect, the programmable nuclease system is CRISPR. In another aspect, the programmable nuclease enzyme is CRISPR associated-9 (Cas9). In another aspect, the programmable nuclease system further comprises one or more HDR enhancers.
Another embodiment described herein is a method for increasing homology directed repair (HDR) rates and reducing homology-independent integration in a programmable nuclease system comprising targeting a candidate editing target site locus with an active programmable nuclease system and a modified double stranded DNA HDR donor. In one aspect, the modified double stranded DNA HDR donor comprises a first homology arm region, an insert region, and a second homology arm region; wherein the first homology arm region and the second homology arm region comprises modifications to one or more nucleotides at or near the 5′-termini. In another aspect, the modified double stranded DNA HDR donor comprises modifications to the 2′-position of the 5′-terminal nucleotide, the 5′-penulimate nucleotide, the 5′-antepenultimate (third) nucleotide, or a combination of the nucleotides at or near the 5′-terminus of the first homology arm region and the second homology arm region. In another aspect, the modified double stranded DNA HDR donor comprises at least one 2′-OME, 2′-F, or 2′-MOE modifications one or more nucleotides at or near the 5′-termini. In another aspect, the modified double stranded DNA HDR donor comprises one or more 2′-MOE modifications at or near the 5′-termini. In another aspect, the modified double stranded DNA HDR donor comprises universal primer sequences. In another aspect, the method further comprises one or more HDR enhancers. In another aspect, the modified double stranded DNA HDR donor improves homology directed repair efficiency and reduces homology-independent integration in a programmable nuclease system.
Another embodiment described herein is the use of modified double stranded DNA HDR donors for increasing homology directed repair (HDR) rates and reducing homology-independent integration in a programmable nuclease system, wherein the modified double stranded DNA HDR donor comprises a first homology arm region, an insert region, a second homology arm region; and optionally, one or more universal priming sequences; wherein the first homology arm region and the second homology arm region comprise modifications to one or more nucleotides at or near the 5′-termini. In one aspect, the modification comprises at least one 2′-OME, 2′-F, or 2′-MOE modifications one or more nucleotides at or near the 5′-termini of the double stranded DNA HDR donor.
Another embodiment described herein is a method for manufacturing a modified double stranded DNA HDR donor, the method comprising synthesizing an oligonucleotide comprising a first homology arm region, an insert region, a second homology arm region; and optionally, one or more universal priming sequences; wherein the first homology arm region and the second homology arm region comprise modifications to one or more nucleotides at or near the 5′-termini. In one aspect, the modification comprises at least one 2′-OME, 2′-F, or 2′-MOE modifications one or more nucleotides at or near the 5′-termini of the double stranded DNA HDR donor.
Another embodiment describe herein is a method for manufacturing a modified double stranded DNA HDR donor, the method comprising amplifying a target nucleic sequence comprising a first homology arm region, an insert region, a second homology arm region with one or more universal primers, wherein the universal priming sequences comprise modification to one or more nucleotides at or near the 5′-termini. In one aspect, the modification comprises at least one 2′-OME, 2′-F, or 2′-MOE modifications at one or more nucleotides at or near the 5′-termini of the universal primer.
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. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the present invention.
As used herein, the terms “amino acid,” “nucleotide,” “polypeptide,” “polynucleotide,” and “vector” have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or R) are used herein.
As used herein, the terms such as “include,” “including,” “contain,” “containing,” “having,” and the like mean “comprising.” The disclosure also contemplates other embodiments “comprising,” “consisting of,” and “consisting essentially of,” the embodiments, aspects, or elements presented herein, whether explicitly set forth or not.
As used herein, the term “a,” “an,” “the” and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, “a,” “an,” or “the” means “one or more” unless otherwise specified.
As used herein, the term “or” can be conjunctive or disjunctive.
As used herein, the term “substantially” means to a great or significant extent, but not completely.
As used herein, the term “about” or “approximately” as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of the measurement system. In one aspect, the term “about” refers to any values, including both integers and fractional components that are within a variation of up to ±10% of the value modified by the term “about.” Alternatively, “about” can mean within 3 or more standard deviations, per the practice in the art. Alternatively, such as with respect to biological systems or processes, the term “about” can mean within an order of magnitude, in some embodiments within 5-fold, and in some embodiments within 2-fold, of a value. As used herein, the symbol “˜” means “about” or “approximately.”
All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1, 0.2, 0.3, 0.4 . . . 2.0. If the end points are modified by the term “about,” the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points.
As used herein, the terms “control,” or “reference” are used herein interchangeably. A “reference” or “control” level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. “Control” also refers to control experiments or control cells.
As used herein, the phrase “an effective amount” of a compound described herein refers to an amount of the compound described herein that will elicit the biological response, for example, reduction or inhibition of an enzyme or a protein activity, or ameliorate symptoms, alleviate conditions, slow or delay disease progression, or prevent a disease, etc.
As used herein, the terms “inhibit,” “inhibition,” or “inhibiting” refer to the reduction or suppression of a given condition, symptom, or disorder, or disease, or a significant decrease in the baseline activity of a biological activity or process.
As used herein, the term “universal primer” refers to a sequence that does not have a known alignment to a target sequence. Universal primers permit the sequence independent amplification of target sequences.
Disclosed herein are methods and compositions of dsDNA donor templates for improving HDR efficiency and reducing blunt integration events. In various embodiments the disclosed methods and compositions allow for a reduction in homology-independent integration following genomic editing with programmable nucleases. In some embodiments bulky modifications are placed at the 5′-terminus of the linear dsDNA donor. In further embodiments bulky modifications are placed at or near the 5′ end of the linear dsDNA donor. Additionally, modifications may be placed at the 2′-position of the DNA (e.g., 2′-MOE, 2′-OME, or 2′-F nucleotides) of a nucleotide at or near the 5′-nucleotide or nucleotides of the dsDNA donor. These modifications demonstrate an improved efficacy at reducing homology-independent integration. Furthermore, this reduction does not seem to be mediated through increased donor stability, as other modifications that have the established ability to block nuclease degradation (PS, etc.) do not also reduce the blunt integration rate to the same extent as other 2′-modifications.
When homology-independent integration occurs at the on-target site, the entire donor is incorporated including the homology arms (
In some embodiments chemical modifications are introduced to the 5′-terminus of linear dsDNA donors. These chemical modifications are used to reduce the risk for NHEJ integration and improve their utility as repair templates in HDR experiments. In some embodiments bulky or large modifications are introduced to the 5′-terminal end of the dsDNA donor. In additional embodiments the modifications may be introduced to the terminal or 5′-DNA nucleotide of the dsDNA oligonucleotide. In some embodiments the modification may be introduced at or near the 5′-terminus of the dsDNA oligonucleotide. In some embodiments the DNA nucleotide or nucleotides at or near the 5′-terminus of the dsDNA oligonucleotide may be modified. In some embodiments, the modifications include biotin, phosphorothioate (PS), triethylene glycol (TEG), Locked Nucleic Acid (LNA, a 2′-oxygen-4′-carbon methylene linkage), hexaethylene glycol (Sp18), 1,3-propanediol (SpC3), 2′-O-methoxyethyl (MOE) ribonucleotides, 2′-O-methyl ribonucleotides (2′-OMe), 2′-fluoro (2′-F) nucleotides, or ribonucleotides. In some embodiments the modification is placed on the 5′-terminal nucleotide, the 5′-penulimate nucleotide, the 5′-antepenultimate (third) nucleotide, or a combination of the nucleotides at or near the 5′-terminus of the dsDNA donor. In additional embodiments the modification is placed at the 2′-position of the 5′-terminal nucleotide, the 5′-penulimate nucleotide, the 5′-antepenultimate (third) nucleotide, or a combination of the nucleotides at or near the 5′-terminus of the dsDNA donor. In yet an additional embodiment the modification is placed at the 2′-position of the 5′-terminal nucleotide, the 5′-penulimate nucleotide, the 5′-antepenultimate (third) nucleotide, or a combination of the nucleotides at or near the 5′-terminus of the dsDNA donor.
The use of 2′-modified ribonucleotides, particularly 2′-O-methoxyethyl (MOE), was found to give the optimal improvement when compared to biotin or other modifications. Additional experiments establishing the use of these modifications with donors mediating large insertions are described herein.
Further improvements to the manufacturing process of the dsDNA donors were evaluated. Universal priming sequences were selected to have no homology to common genomes (human, mouse, rat, zebrafish). Previous work by our group has established the utility of these priming sequences in cloning applications (i.e., highly efficient, reliable amplification). Significant improvements to (1) amplification success with a wide variety of sequences and (2) overall amplification yields can be achieved by incorporating these universal sequences into the donor manufacturing process. Described herein is testing of these universal sequences when placed flanking the complete HDR donor sequence. Due to the lowered risk of homology-independent integration when using the 5′-dsDNA modifications, these sequences do not adversely impact correct HDR rates with modified donors and are only rarely incorporated during blunt integration.
The methods and compositions disclosed herein are of dsDNA donor templates for use in improving HDR efficiency and reducing homology-independent events (blunt integration events or multimerization events). In various embodiments the disclosed methods and compositions allow for a reduction in homology-independent integration or increase in homology-dependent integration following genomic editing with programmable nucleases. In some embodiments bulky nucleotide modifications are placed at the 5′-terminus of the linear dsDNA donor. In additional embodiments modifications placed at the 2′-position of the nucleotide (e.g., 2′-MOE, 2′-OMe) of the 5′-terminal nucleotide or nucleotides near the 5′-terminus of the dsDNA demonstrate an improvement in efficacy at reducing homology-independent integration. Furthermore, this reduction does not seem to be mediated through increased donor stability, as other modifications that have the established ability to inhibit nuclease degradation (PS, etc.) do not also reduce the blunt integration rate to the same extent as other 2′-modifications.
In some embodiments chemical modifications are introduced to the 5′-terminus of linear dsDNA donors. These chemical modifications are used to reduce the risk for NHEJ integration and improve their utility as repair templates in HDR experiments. In some embodiments bulky or large modifications are introduced. In additional embodiments the modifications may be introduced near the 5′-terminus of the dsDNA oligonucleotide donor. In some embodiments the modification may be introduced at or near the 5′-terminus of the dsDNA oligonucleotide donor. In some embodiments the nucleotides at or near the 5′-terminus of the dsDNA oligonucleotide may be modified. In additional embodiments modifications include, but are not limited to: biotin (B); phosphorothioate (PS, *); triethylene glycol (TEG); Locked Nucleic Acid, e.g., a 2′-oxygen-4′-carbon methylene linkage (LNA); hexaethylene glycol (Sp18); 1,3-propanediol (SpC3); 2′-O-methoxyethyl (MOE) ribonucleotides, 2′-O-methyl ribonucleotides (2′-OMe), 2′-fluoro (2′-F) nucleotides, and ribonucleotides.
In further embodiments, the use of hairpin structures on the ends of the dsDNA donor similarly reduces blunt integration.
In one embodiment the end modified dsDNA donor templates would be suitable for use following introduction of double strand breaks by programmable nucleases. In further embodiments the programmable nucleases include transcription activator-like effector nucleases (TALENs), zinc fingers (ZFNs), or clustered, regularly interspaced, short palindromic repeat (CRISPR). In one embodiment, the programmable nuclease system is CRISPR. In one aspect, the programmable nuclease enzyme is CRISPR associated-9 (Cas9).
In one embodiment 5′-terminal modified dsDNA donors are generated by PCR amplification. Primers modified with biotin, phosphorothioate (PS) linkages, TEG, LNA, spacer 18 (SP18), C3 spacers (SpC3), or MOE are used to amplify insert regions and generate end modified dsDNA donors. In some embodiments the insert region is greater than 120 bp. In some embodiments the insert region is at least 1 kb insert regions. In some embodiments the insert region is greater than 1 kb, greater than 2 kb, greater than 3 kb, greater 4 kb, greater than 5 kb, greater than 6 kb, greater than 7 kb, greater than 8 kb, greater than 9 kb, or greater than 10 kb.
In additional embodiments, modifications at or near the 5′-terminus include biotin, phosphorothioate (PS), triethylene glycol (TEG), Locked Nucleic Acid, e.g., a 2′-oxygen-4′-carbon methylene linkage (LNA), hexaethylene glycol (Sp18), 1,3 propanediol (SpC3), 2′-O-methoxyethyl ribonucleotides (MOE), 2′-O-methyl ribonucleotides (2′-OMe), 2′-fluoro (2′-F) nucleotides, and ribonucleotides.
In further embodiments the modification at or near the 5′-terminus includes modifications of the 2′-position of the DNA nucleotide at or near the 5′-terminus of the double stranded DNA donor. In some embodiments the 2′-modification is 2′-MOE, 2′-OMe, or 2′-fluoro and the modification of the nucleotide occurs at or near the 5′-terminus of the double stranded DNA donor. In some embodiments the 5′-terminus modification is on the 5′-terminal nucleotide of the double stranded DNA donor. In additional embodiments the 5′-terminus modification is positioned at the 5′-terminal nucleotide, the 5′-penulimate nucleotide, the 5′-antepenultimate (third) nucleotide, or a combination of the nucleotides at or near the 5′-terminus of the dsDNA donor. In other embodiments the 5′-terminal modification is positioned at 5′-terminus modification is positioned at the 5′-terminal nucleotide, the 5′-penulimate nucleotide, or the 5′-antepenultimate (third) nucleotide. In yet another embodiment the 5′-terminal modification is a 2′-MOE modified ribonucleotide positioned at the terminal 5′-position, the penultimate nucleotide position from the 5′-terminus, the antepenultimate (third) nucleotide position from the 5′-terminus, or a combination thereof. In still a further embodiment the 5′-terminal modification is a 2′-MOE ribonucleotide positioned at the terminal 5′-position, the penultimate nucleotide position from the 5′-terminus, the antepenultimate (third) nucleotide position from the 5′-terminus, or a combination thereof.
In an additional embodiments HDR donors comprise homology arms on either side of an insert. The homology arms are complementary to the sequences flanking the double-stranded break introduced by the programmable nuclease. In some embodiments the homology arms vary in length from at least 20 nucleotides in length to 500 nucleotides in length. In some embodiments the homology arms are at least 40, 50, 60 70, 80, 90, 100, 150, 200, 300, 400, or 500 nucleotides in length. In some embodiments the homology arm length may be greater than 500 nucleotides in length. In additional embodiments the homology arms are preferably at least 40 nucleotides in length and more preferably at least 100 nucleotides in length.
In some embodiments the inserts are placed between homology arms. In some embodiments the inserts are greater than 20 nucleotides in length. In some embodiments the inserts are from at least 1 nucleotide in length to 4 kb in length. In some embodiments the inserts range from 1-2 kb in length. In some embodiments the inserts may be at least 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb in length. In yet an additional embodiment the insert may be 10 kb or longer in length.
In an additional embodiment HDR donor comprise homology arms on either side of an insert where the insert may include SNPs, MNPs, or deletions. In some embodiments the inserts are from at least 1 nucleotide in length to 4 kb in length. In some embodiments the inserts range from 1-2 kb in length. In some embodiments the inserts may be at least 1 bp, 2 bp, 3 bp, 4 bp, 5 bp, 6 bp, 7 bp, 8 bp, 9 bp, 10 bp, 20 bp, 30 bp, 40 bp, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, 100 bp, 200 bp, 300 bp, 400 bp, 500 bp, 600 bp, 700 bp, 800 bp, 900 bp, 1 kb, 2 kb, 3 kb, 4 kb, 5 kb, 6 kb, 7 kb, 8 kb, 9 kb in length. In yet an additional embodiment the insert may be 10 kb or longer in length.
The polynucleotides described herein include variants that have substitutions, deletions, and/or additions that can involve one or more nucleotides. The variants can be altered in coding regions, non-coding regions, or both. Alterations in the coding regions can produce conservative or non-conservative amino acid substitutions, deletions, or additions. Especially preferred among these are silent substitutions, additions, and deletions, which do not alter the properties and activities of the binding.
Further embodiments described herein include nucleic acid molecules comprising polynucleotides having nucleotide sequences about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical, and more preferably at least about 90-99% identical to (a) nucleotide sequences, or degenerate, homologous, or codon-optimized variants thereof; or (b) nucleotide sequences capable of hybridizing to the complement of any of the nucleotide sequences in (a).
By a polynucleotide having a nucleotide sequence at least, for example, 90-99% “identical” to a reference nucleotide sequence is intended that the nucleotide sequence of the polynucleotide be identical to the reference sequence except that the polynucleotide sequence can include up to about 10 to 1 point mutations, additions, or deletions per each 100 nucleotides of the reference nucleotide sequence.
In other words, to obtain a polynucleotide having a nucleotide sequence about at least 90-99% identical to a reference nucleotide sequence, up to 10% of the nucleotides in the reference sequence can be deleted, added, or substituted, with another nucleotide, or a number of nucleotides up to 10% of the total nucleotides in the reference sequence can be inserted into the reference sequence. These mutations of the reference sequence can occur at the 5′- or 3′-terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence. The same is applicable to polypeptide sequences about at least 90-99% identical to a reference polypeptide sequence.
In some embodiments the programmable nucleases (e.g., CRISPR enzyme) or components (e.g. gRNA) can be introduced into the cell using various approaches. Examples include plasmid or viral expression vectors (which lead to endogenous expression of either the enzyme, the gRNA, or both), delivery of the enzyme with separate gRNA/crRNA transfection, or delivery of the enzyme with the gRNA or crRNA as a ribonucleoprotein (RNP) complex.
It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, apparata, assemblies, and applications described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions, apparata, assemblies, and methods provided are exemplary and are not intended to limit the scope of any of the disclosed embodiments. All the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, apparata, assemblies, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences described herein. The compositions, formulations, apparata, assemblies, or methods described herein may omit any component or step, substitute any component or step disclosed herein, or include any component or step disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof.
HDR Rates are Increased, and NHEJ Insertions are Reduced with Modified dsDNA Donors.
Initial tests were performed to compare the homology-independent (i.e., blunt) integration relative to HDR insertion rates of unmodified linear dsDNA, donors containing 5′-biotin modification, or donors with alternative modifications on or near the 5′-termini. dsDNA donors were generated by PCR amplification of a plasmid containing a 1 kb insert with 100 bp of flanking homology arms targeting the human SERPINC1 gene (100-1000-100; SEQ ID NO: 1; see Table 1 for amplification primer sequences; SEQ ID NO: 2-21). Amplification primers were designed with either unmodified sequence or the indicated modifications. Purified dsDNA donors were delivered at 100 nM (1 μg) in a final volume of 28 μL nucleofection buffer with 2 μM Cas9 V3™ RNP (IDT, Coralville, Iowa) targeting SERPINC1 into 3.5×105 HEK-293 cells using Lonza nucleofection (Lonza, Basel, Switzerland). The SC1 (SERPINC1) protospacer sequence used can be found in Table 1 (SEQ ID NO: 22). Cells were lysed after 48 hours using QuickExtract™ DNA extraction solution (Lucigen, Madison, WI). HDR and blunt integration rates were assessed by digital-droplet PCR (ddPCR) (Bio-Rad, Hercules, CA) using PCR assays with primers flanking the junction between the target DNA and insert (Table 1; SEQ ID NO: 23-27). Both HDR and blunt junction assays contained one primer external to the homology arm sequence to avoid amplification from non-integrated donor. The HDR assay probe (SEQ ID NO: 25) covered the junction of the target site and insert sequence. The blunt assay probe (SEQ ID NO: 27) covered the junction between the target site and integrated homology arm sequence.
dsDNA donors containing known nuclease resistant modifications, such as phosphorothioate linkages (2×PS, 3×PS, or 6×PS) or an LNA nucleotide on the 5′-terminus did not improve the HDR:Blunt ratio above unmodified (unmod) donors, as the modifications increased the rates for both HDR and blunt integration (
5′-Modifications Demonstrate Lower Off-Target Integration when Using Shorter Donor Templates.
Homology-independent integration rates depend on the total donor length, with blunt insertion increasing with decreased donor size. To determine whether 5′-terminal modification would reduce blunt insertion rates with a smaller 42 bp insert (compared with the 1 kb insert tested in Example 1), modified dsDNA donors were generated targeting the SERPINC1 locus described in Example 1 (SC1-166S; SEQ ID NO: 22). Donors consisted of a 42 bp insert containing an EcoRI restriction site with 40 bp homology arms (SC1 40-42-40; SEQ ID NO: 28) and were generated by PCR amplification of a plasmid containing the 42 bp insert with 100 bp of flanking homology arms (see Table 2 for amplification primer sequences; SEQ ID NO: 29-44).
Three modifications (Biotin, Sp18, and MOE) from Example 1 were selected for additional testing, while a 6×PS modification was included as a moderately performing control. Donors with three 2′-MOE ribonucleotides (3×MOE) on the 5′-termini were also included to determine if blunt integration could be further reduced by additional modified residues. A 2′-MOE ribonucleotide was also tested at varying distances from the 5′-termini (Int MOE-3 and -5, with a 2′-MOE positioned 3 or 5 nucleotides from the 5′-terminus; SEQ ID NO: 41-44). Modified and unmodified donors were delivered at 500 nM (1.1 μg) with 2 μM Cas9 V3™ (IDT, Coralville, IA) RNP targeting the SERPINC1 locus into 3.5×105 HEK-293 cells in a 28 μL final volume using Lonza nucleofection (Lonza, Basel Switzerland). DNA was extracted after 48 hours using QuickExtract™ DNA extraction solution (Lucigen, Madison, WI). Integration rates were assessed by RFLP using EcoRI digestion, using a Fragment Analyzer™ machine for band quantification (Advanced Analytical, Ames, IA). HDR and blunt integration events could be distinguished by a 40 bp size difference due to the homology arm duplication.
1×MOE and 3×MOE modified donors resulted in the greatest improvement in the HDR:Blunt ratio (4.1- and 4.6-fold improvement over unmod respectively). See
2′-MOE Modifications Lower Integration at Non-Homologous Double Strand Breaks
In addition to blunt integration at the targeted cleavage site, double-strand donors can potentially integrate at any other double-stranded break in the genome, including off-target Cas9 cleavage sites and endogenous breaks in dsDNA. To assess the impact of the 2′-MOE modification on donor integration at potential non-homologous DSBs, dsDNA donors with either unmodified or modified 5′-termini (unmod, 1×MOE, or 6×PS) were generated by PCR amplification (see Table 3 for amplification primer sequences; SEQ ID NO: 48-53) and co-delivered with Cas9 complexed with either the target gRNA (SC1-166S; SEQ ID NO: 22) or a mock “off-target” gRNA with no homology to the donor (AAVS1-670AS; SEQ ID NO: 54; HPRT 38087; SEQ ID NO: 55).
Donors consisted of a 42 bp insert containing an EcoRI restriction site and 50 bp homology arms targeting the SERPINC1 locus (SC1 50-42-50; SEQ ID NO: 47). Donors were delivered at a 100 nM dose (0.3 μg) with 2 μM Cas9 V3™ RNP (IDT, Coralville, IA) and 2 μM Alt-R™ Cas9 Electroporation Enhancer™ into 3×105 K562 cells in a final 28 μL volume by Lonza nucleofection (Lonza, Basel Switzerland). DNA was extracted after 48 hrs using QuickExtract™ DNA extraction solution (Lucigen, Madison, WI). Integration rates were assessed by RFLP using EcoRI digestion, with each band quantified on a Fragment Analyzer™ machine (Advanced Analytical, Ames, IA). HDR and blunt integration events could be distinguished by a 50 bp size difference due to the homology arm duplication (
Blunt integration rates >9% were observed for unmodified dsDNA at the on-target Cas9 site and both “off-target” Cas9 sites. See
Reduction of Off-Target Integration is not the Result of Increased Nuclease Protection, and Specific 2′-Modifications are Required for Efficient Reduction
To determine whether the ability of 2′-MOE modification to reduce blunt integration was specific or a general function of modifications at the 2′-position of the 5′-most nucleotides, additional 2′-modifications were tested (RNA, LNA, 2′-OMe, or 2′-F), as well as a non-template 2′-MOE ribonucleotide (SEQ ID NO: 70-71) on the 5′-termini. (Non-template ribonucleotide defined as non-homologous to the target DNA sequence.) Donors consisted of the sequence previously described in Example 2 (SC1 40-42-40; SEQ ID NO: 28), a 42 bp insert containing an EcoRI restriction site and with 40 bp homology arms targeting the SerpinC1 locus. Donors were generated by PCR amplification as previously described (primer sequences unique to Example 4 listed in Table 4; SEQ ID NO: 60-71).
MGATTCCAATGTGATA
MGAAACCCAGTAGGGG
dsDNA donors were co-delivered with Cas9 complexed with either the target gRNA (SC1-166S; SEQ ID NO: 22) or a gRNA with no homology to the donor (TNPO3; SEQ ID NO: 72). Donors were delivered at a 250 nM dose (0.6 μg) with 2 μM Cas9 V3 RNP into HEK-293 cells in a final 28 μL volume by Lonza nucleofection (Lonza, Basel Switzerland). DNA was extracted after 48 hrs using QuickExtract™ DNA extraction solution (Lucigen, Madison, WI). Integration rates were assessed by RFLP using EcoRI, with each band quantified on a Fragment Analyzer™ machine (Advanced Analytical, Ames, IA).
While HDR was either not impacted or slightly boosted by the various modifications, blunt integration was decreased at both the on-target and off-target DSBs whenever a 2′-MOE modification was present (
Taken together, these data suggest that the ability of 2′-MOE modifications to reduce homology-independent integration is (a) not a function of increased stability by promoting nuclease resistance as other stabilizing modifications do not result in a similar outcome and (b) not a generalized function of 2′-modifications on the 5′-most ribonucleotide as other 2′-modifications do not result in a similar outcome.
Use of Hairpins as Blocking Groups to Reduce Homology-Independent Integration.
In addition to chemical modifications, the use of DNA hairpins at the ends of dsDNA donors can be used to reduce homology-independent integration. Generation of these hairpin-blocked donors is achieved in several methods (
The use of hairpins on chemically synthesized short oligos was functionally tested in cells. A 66-nt sequence was designed to mediate a 6 base GAATTC insertion in the SERPINC1 locus. This sequence and its reverse complement were synthesized as ssODNs that were either fully unmodified (Table 5; SEQ ID NO: 75-76), contained an unmodified hairpin at the 5′-termini (SEQ ID NO: 77-78), contained a MOE-modified hairpin at the 5′ end (SEQ ID NO: 79-80), or contained a non-template MOE-modified base at the 5′-termini (SEQ ID NO: 81-82). Paired ssODNs were diluted to 100 μM and then mixed at a 1:1 ratio to generate a 50 μM final duplex. The oligo mixtures were heated at 95° C. for 1 min and then slow cooled to room temperature to allow the strands to anneal. Duplexed dsDNA donors were co-delivered with Cas9 complexed with the target gRNA (SEQ ID NO: 22). Donors were delivered at a 2 μM concentration e with 2 μM Cas9 V3 RNP and 2 μM Alt-R® Cas9 Electroporation Enhancer into HEK-293 cells in a final 28 μL volume by Lonza nucleofection (Lonza, Basel Switzerland). DNA was extracted after 48 hrs using QuickExtract™ DNA extraction solution (Lucigen, Madison, WI). Integration rates were assessed by RFLP using EcoRI, with each band quantified on a Fragment Analyzer™ machine (Advanced Analytical, Ames, IA) (
CMGMAGATAGGAACTG
CMGMAGGGGCAGGCAA
MAGATAGGAACTGTAA
MAGGGGCAGGCAAGGG
Use of the shorter 66 bp unmodified dsDNA donor resulted in efficient integration through the NHEJ pathway relative to the HDR pathway (55.5% Blunt vs. 27.6% HDR). Introduction of the DNA-only hairpin to the ends of the donor provided an improvement in the repair profile (42.4% Blunt vs. 33.0% HDR). Inclusion of MOE modifications within the hairpin significantly improved this function to similar levels observed with the single 1×MOE on the 5′-termini (58.4% HDR vs. 8.9% Blunt for MOE-modified hairpin; 66.5% HDR vs. 6.9% Blunt for 1×MOE). Additional optimization to the modified hairpins (i.e., ligation, stem-loop length optimization, etc) could be implemented to further improve this function.
2′-MOE Modifications Improve Desired Repair Outcomes at Multiple Sites and in Multiple Cell Lines.
In the following experiments, all guides were tested as Alt-R™ crRNA:tracrRNA complexed to Alt-R™ S. pyogenes Cas9 nuclease. RNP complexes and dsDNA donors were delivered to cells of interest using Lonza nucleofection following recommended protocols.
To further validate the ability of the 2′-MOE modification to drive correct repair through higher HDR rates and reduced blunt integration, unmodified and 1×MOE modified dsDNA donors were tested at 4 additional genomic loci (HPRT, AAVS1 670, AAVS1 T2, EMX1) in 2 cell lines (HEK293, K562). Donors were designed to mediate a 42 bp insert and had 40 bp homology arms (SEQ ID NO: 83-86). Donors were generated by PCR amplification as previously described (primer sequences unique to Example 6 listed in Table 6, SEQ ID NO: 95-142). Donors were delivered at 250 nM in a final volume of 28 μL nucleofection buffer with 2 μM Cas9 V3™ RNP (IDT, Coralville, Iowa) and 2 μM Alt-R™ Cas9 Electroporation Enhancer™ into the indicated cell lines using recommended protocols for Lonza nucleofection (Lonza, Basel, Switzerland). The protospacer sequences used can be found in Table 6 (SEQ ID NO: 135-138). Cells were lysed after 48 hours using QuickExtract™ DNA extraction solution (Lucigen, Madison, WI). Repair events were quantified by NGS amplicon sequencing (rhAmpSeg™) on the Illumina MiSeq platform (locus specific sequencing primers listed in Table 6, SEQ ID NO: 139-146) and data analysis was performed using IDT's in-house data analysis pipeline (CRISPAltRations), described in U.S. patent application Ser. No. 16/919,577, which is incorporated herein by reference for such teachings (
As previously observed, the 5′-modifications all improved HDR rates over unmodified dsDNA donors (A). While the fold-improvement in HDR over an unmodified dsDNA varied across sites and cell lines, the average improvement in HDR rates were relatively similar for all modifications tested (ranging from a 1.2 to 1.3-fold improvement). In contrast, MOE modified donors displayed a greater reduction in blunt integration compared to the 2×PS and Biotin donors (B). On average, the fold reductions in blunt integration relative to unmodified dsDNA were 1.6 (2×PS), 2.3 (Biotin), 2.9 (1×MOE), 3.2 (1×MOE, 2×PS), and 3.3 (3×MOE). When the improvements in HDR and blunt integration were assessed together for each site (C, reported as the ratio of HDR:Blunt repair events), MOE modified dsDNA donors outperformed other modifications. The average fold change over unmodified dsDNA were 2.3 (2×PS), 3.1 (Biotin), 3.6 (1×MOE), 4.1 (1×MOE 2×PS), and 4.3 (3×MOE). Taken together, these results demonstrate that MOE modifications are the most efficient at driving the correct repair event following CRISPR editing.
HDR Rates are Increased, and NHEJ Insertions are Reduced with Modified dsDNA Donors Mediating Large Insertions.
As a follow-up to the work with short insertions, experiments were performed to compare the HDR and blunt integration rates when using dsDNA donors mediating 300 bp, 500 bp, or 1000 bp insertions at two genomic loci (SERPINC1 and EMX1; see Table 7 for donor sequences and amplification primers, SEQ ID NO: 147-154). Donors were generated by PCR amplification of plasmids containing the desired inserts with 100 bp of flanking homology arms. Amplification primers were designed with either unmodified sequence or the indicated modifications. Long ssDNA (Megamers™) were ordered for comparison at the SERPINC1 locus. Donors were delivered at 100 nM in a final volume of 28 μL nucleofection buffer with 2 μM Cas9 V3™ RNP (IDT, Coralville, Iowa) targeting SERPINC1 or EMX1 into 3.5×105 HEK-293 cells using Lonza nucleofection (Lonza, Basel, Switzerland). Cells were treated with the IDT Alt-R™ HDR Enhancer V2 (1 μM) for 24 hrs post-transfection. The protospacer sequences used is shown in Table 1 (SEQ ID NO: 22) and Table 7 (SEQ ID NO: 161).
Cells were lysed after 48 hours using QuickExtract™ DNA extraction solution (Lucigen, Madison, WI). HDR and blunt integration rates were assessed by long-read sequencing using the MinION™ platform (Oxford Nanopore Technologies, Oxford, UK). Locus specific amplification primers used are listed (Table 7, SEQ ID NO: 162-165). Final sequencing libraries were prepared with the PCR Barcoding Expansion and Ligation Sequencing Kit following the manufacturer's recommended protocols. Final data analysis was performed using IDT's in-house data analysis pipeline (CRISPAltRations) (
1×MOE modified donors resulted in higher HDR rates (on average from 28.6% to 30.9% at EMX1 and 44.4% to 53.6% at SERPINC1) and lower blunt integration rates (on average from 3.2% to 1.1% at EMX1 and 8.4% to 2.5% at SERPINC1) when compared to unmodified dsDNA donors mediating large insertions at both genomic loci. Long ssDNA donors mediating the same insertions at the SERPINC1 locus resulted in extremely low blunt integration rates (<1%). The long ssDNA donor mediated the highest rates of HDR for the 300 bp insert (71.3% vs. 54.9% with a modified dsDNA donor). However, the modified dsDNA donor performed as well or better than the long ssDNA for HDR with the larger insertions (55.2% vs. 53.3% for a 500 bp insert, 50.8% vs. 29.4% for a 1000 bp insert). Similar trends were observed for the SERPINC1 samples when an orthologous method of assessment was used (
Utilization of Universal Priming Sequences for Manufacturing Modified dsDNA Donors does not Adversely Affect HDR Repair.
To assess the impact of incorporating universal priming sequences into the donor template, dsDNA donors mediating a 500 bp insert at EMX1 and SERPINC1 (see Table 7, SEQ ID NO: 148 and 151) were prepared with either locus specific primers or with universal primers (Table 7 SEQ ID NO: 153-160; Table 8 SEQ ID NO: 166-181). Placement of the universal priming sequences relative to the donor is shown in
HDR and blunt integration rates were relatively similar for dsDNA donors generated with or without the universal priming sequences. For donors without universal priming sequences, the improvement to HDR and reduction in blunt rates were similar across the various modifications. The major exception to this trend was the 3×MOE modification for the SERPINC1 site, where blunt insertion was still reduced relative to an unmodified dsDNA but HDR was not improved to the same extent as 1×MOE or Biotin5×PS (unmod: 45.1% HDR, 9.0% Blunt; 1×MOE: 55.2% HDR, 2.3% Blunt, 3×MOE: 44.5% HDR, 1.2% Blunt). In contrast, a much larger difference in performance was observed with the modified donors manufactured with the universal priming sequences. Interestingly, the unmodified dsDNA donors for both sites had lower integration rates when universal priming sites were included in the donor sequence (EMX1: 28.8% vs 24.8% HDR, SERPINC1: 45.2% vs 34.4% HDR). For both sites, the 1×MOE modification offered the greatest improvement to the HDR rate when incorporated into donors containing the universal sequences.
Further analysis of the HDR reads from the 1×MOE modified donors was conducted in the Integrative Genomics Viewer [7] (IGV, Broad Institute, Cambridge, MA). When HDR reads were aligned against either a reference amplicon containing the correct HDR sequence (
Use of Modified dsDNA Donors Manufactured with Universal Priming Sequences to Generate GFP Fusions in Human Cell Lines.
To assess the functional performance of modified dsDNA donor templates in applications such as protein tagging, donors were designed to generate GFP tagged GAPDH (C-terminal fusion), CLTA (N-terminal fusion), and RAB11a (N-terminal fusion). Donors were manufactured with universal priming sequences as previously described, using either unmodified or 1×MOE modified primers. Guide and donor sequences used are listed in Table 9. Donors were delivered at 50 nM in a final volume of 28 μL nucleofection buffer with 2 μM Cas9 V3™ RNP (IDT, Coralville, Iowa) targeting GAPDH, CLTA, or RAB11a into 3.5×105 K562 cells using Lonza nucleofection (Lonza, Basel, Switzerland). Following the transfection, cells were plated in duplicate wells. For one set of wells, cells were treated with the IDT Alt-R™ HDR Enhancer V2 (1 μM) for 24 hrs post-transfection. Cells were passaged for 7 days, at which point HDR rates were assessed by flow cytometry. Briefly, cells were washed in PBS and then resuspended at 1-2×106 cells/mL. Hoechst 33258 was added to the cell suspension at a final concentration of 4 μg/ml shortly before analysis for viability staining. Cells were analyzed on a Becton Dickinson LSR II cytometer (BD Bioscience, San Jose, CA) to assess GFP expression levels (
Overall HDR rates varied across the sites tested, with maximum GFP positive rates of 17.2% (GAPDH), 44.9% (CLTA), and 64% (RAB11a) achieved under optimal conditions. No GFP signal was observed in cells that received a dsDNA donor without RNP (data not shown). HDR rates were increased with modified dsDNA donor templates in both untreated conditions (1.6, 1.3, and 1.2-fold improvement over unmodified dsDNA for GAPDH, CLTA, and RAB11a respectively) and in HDR Enhancer treated conditions (1.4, 1.4, and 1.1-fold improvements over unmodified dsDNA respectively). On average, use of 1×MOE modified dsDNA donors increased HDR rates 1.3-fold over unmodified dsDNA donors across all conditions. In comparison, use of the Alt-R HDR Enhancer V2 increased HDR rates on average 2.4-fold across all sites and conditions. The combined use of modified donors and HDR Enhancer boosted HDR rates 3.2-fold on average across all sites. Taken together, this demonstrates the combined utility of using optimal reagents (i.e. modified donors and small molecule enhancers) in HDR experiments.
Use of Universal Priming Sequences Enables Greater Consistency and Improved Yields when Manufacturing dsDNA HDR Templates
In order to assess the impact of universal priming sequences on the manufacturing process of dsDNA HDR templates, 24 sequences were generated using either universal priming sequences (Table 8, SEQ ID NO: 172-181) or gene specific primers (Table 10, SEQ ID NO: 188-235) with varying modifications. As previously described, all donors were produced by amplification from a plasmid (pUCIDT Amp or pUCIDT Kan vectors) containing the sequence of interest. PCR amplifications were conducted using KOD Hot Start DNA Polymerase (EMD) according to the manufacturer's recommendations, with 200 nM primers and 10 ng plasmid DNA in a 50 μL final reaction volume. Thermocycling was conducted using a Bio-Rad S1000 thermal cycler with the following cycling conditions: a 3 min incubation at 95° C., followed by 36 amplification cycles (95° C. for 20 sec; 65° C. for 10 sec; 70° C. for 20-30 sec/kb). Annealing temperatures were adjusted according to the gene specific primer melting temperatures. Following a SPRI bead cleanup, all products were analyzed using Fragment Analyzer (Agilent) and sequence verified by NGS using the Illumina-Nextera DNA Library Preparation Kit. Overall amplification efficiencies from universal primers or gene specific primers were assessed by measuring final yields, reported as ng/μL (
Owing to differences in the yields for short (<500 bp) and long (>500 bp) amplicons, overall yields following amplification with either universal or gene specific primers were assessed separately for 12 short and 12 long HDR templates (
This application claims priority to U.S. Provisional Patent Application No. 62/925,366, filed on Oct. 24, 2019, which is incorporated by reference herein in its entirety.
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