WO2023025767A1 - Method for cas9 nickase-mediated gene editing - Google Patents

Method for cas9 nickase-mediated gene editing Download PDF

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WO2023025767A1
WO2023025767A1 PCT/EP2022/073414 EP2022073414W WO2023025767A1 WO 2023025767 A1 WO2023025767 A1 WO 2023025767A1 EP 2022073414 W EP2022073414 W EP 2022073414W WO 2023025767 A1 WO2023025767 A1 WO 2023025767A1
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sequence
dna
gene
dsdna
target
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PCT/EP2022/073414
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French (fr)
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Peter Klaus Georg RAJEWSKY
Van Trung CHU
Ngoc Tung TRAN
Xun Li
Ralf KÜHN
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Max-Delbrück-Centrum Für Molekulare Medizin In Der Helmholtz-Gemeinschaft
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Publication of WO2023025767A1 publication Critical patent/WO2023025767A1/en

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]

Abstract

The present invention is in the field of gene editing. It relates an in vitro method for modifying a double stranded DNA (dsDNA) molecule through single-strand break (SSB)-mediated homology-directed repair (HDR) in a eukaryotic cell, such as a haematopoietic stem and progenitor cell (HSPC) or T cell, the method comprising: 1) introducing into the cell DNA nickase, at least two guide RNAs, exogenous DNA donor; 2) generating at least two single strand breaks (SSB) in the dsDNA molecule to be modified, wherein a first guide RNA/DNA nickase complex introduces a nick cleavage of one strand of the dsDNA within the first target sequence, and a second guide RNA/DNA nickase complex, introduces a nick cleavage of the opposite strand of the dsDNA within the second target sequence, thereby producing a 5' or 3' overhang, wherein the distance (spacer) between two single strand breaks (SSB) on the dsDNA molecule to be modified is 202 base pair (bp) or greater, and replacing a DNA sequence of the dsDNA, positioned in proximity to the two single strand breaks (SSB), with the template sequence.

Description

METHOD FOR CAS9 NICKASE-MEDIATED GENE EDITING
DESCRIPTION
The present invention is in the field of gene editing.
In one aspect, the invention relates to an in vitro method for modifying a double stranded DNA (dsDNA) molecule through single-strand break (SSB)-mediated homology-directed repair (HDR) in a eukaryotic cell, such as a haematopoietic stem and progenitor cell (HSPC) or T cell, the method comprising: introducing into the cell a guide RNA-guided DNA nickase or a nucleic acid molecule encoding a guide RNA- guided DNA nickase, at least two guide RNAs, comprising a first guide RNA capable of hybridizing to a first target sequence in the dsDNA, and a second guide RNA capable of hybridizing to a second target sequence in the dsDNA, and an exogenous DNA donor template comprising a DNA template sequence, generating at least two single strand breaks (SSB) in the opposing strands of the dsDNA molecule to be modified, wherein a first guide RNA/DNA nickase complex, comprising the first guide RNA, introduces a nick cleavage of one strand of the dsDNA within the first target sequence, and a second guide RNA/DNA nickase complex, comprising the second guide RNA, introduces a nick cleavage of the opposite strand of the dsDNA within the second target sequence, thereby producing a 5’ or 3’ overhang, wherein the distance (spacer) between two single strand breaks (SSB) on the dsDNA molecule to be modified is 202 base pair (bp) or greater, and replacing a DNA sequence of the dsDNA, positioned in proximity to the two single strand breaks (SSB), with the template sequence.
The invention also relates to an isolated eukaryotic cell, such as a haematopoietic stem and progenitor cell or T cell, modified by a method of the invention, preferably for use in the treatment of a medical condition, wherein the medical condition is associated with the reduced function of a gene product induced by pathological gene mutations and said mutations have been replaced with a DNA template sequence without said mutations.
Furthermore, the invention relates to a kit comprising reagents required for preforming the method of the invention and the use of the kit for performing the method.
BACKGROUND OF THE INVENTION
CRISPR/Cas9-created locus-specific double-strand breaks (DSBs) are predominantly repaired by the non-homologous end joining (NHEJ) pathway causing micro-insertions/deletions (Indels), and, to a lesser extent, by the homology-directed repair (HDR) pathway, if the homologous template is provided, allowing precise genetic correction 8 W-12 K has been shown that preassembled ribonucleoproteins (RNP) complexes of Cas9 nuclease and synthetic sgRNA in combination with adeno-associated virus (AAV) serotype 6 for donor template delivery have led to high HDR efficiencies in human CD34+ HSPCs and T cells 13'16. The CRISPR/Cas9/AAV6 approach has been used to repair mutations causing several monogenic blood disorders in patient-derived HSPCs 17-19.
Although, CRISPR/Cas9 exhibits great potential for therapeutic applications, its off-target effects are a major concern that is important to minimize. Genome-wide off-targets are significantly reduced by increasing specificity of CRISPR/Cas9-mediated site cleavage. The CRISPR/Cas9 specificity has been improved by using truncated sgRNAs (tru-sgRNAs, 17-18 mers) 9i2°, or extended sgRNA with two additional G nucleotides at the 5’ end 5, or using sgRNAs with high specificity 21. On the other hand, off-target effects of Cas9 nuclease were shown to be lower when high-fidelity SpCas9 mutants (HIFI-SpCas9, SpCas9-HF1 and eSpCas91 .1 ) were used instead of wild-type Cas9 22-24. Furthermore, Tuladhar Rubina et al teaches that CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA mis-regulation (Nature Communications, vol. 10, no. 1 , 01.12.2019).
US 2016/281111 A1 discloses a method for modifying a target gene in a cell. Paired enzymatically active Cas9 molecules are used for generating a double strand break (DSB) with overhangs. However, according to this technology, the DSB of the target gene is repaired by gene conversion.
Quadras et al (Genome Biology, vol. 18, no. 1 , 01 .12.2017) discloses an “Easi-CRISPR” method for generation of conditional transgenic mice by inserting alleles using long ssDNA donors and CRISPR ribonucleoproteins. According to this technology, long ssDNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein complexes into mouse zygotes leading to an in vivo gene knock-in. It appears that a Cas9 endonuclease was used and therefore a blunt double strand break was generated during gene editing. Furthermore, a ssDNA donor was used for homology-directed repair (HDR) to insert the gene of interest. Unwanted NHEJ- mediated on-target mutations are presented at high frequencies and unintended genetic alternations were not assessed in this study.
Another strategy to minimize off-target activities is to use the Cas9 nickase, a mutated version of Cas9 in which the RuvC or HNH nuclease domain has been inactivated by introducing the D10A or H840A mutation, respectively. The Cas9 nickase leads to fewer indels than the Cas9 nuclease, most likely because the single-stranded breaks (SSBs), induced by the Cas9 nickase, are efficiently repaired by the base excision repair pathway 25. As a result, single-nicks lead to low/or no NHEJ events and rare HDR events when a DNA donor template is provided 2627.
A “Genome editing application note” published by Integrated DNA Technologies Inc. ("CRISPR- Cas9 donor DNA template optimization and nickase mutants promote homology-directed repair (HDR) for efficient, high fidelity genome editing”, Genome editing application note, 01.01.2019), discloses (1 ) optimizing the homology arm length of single-stranded (ss) ODN donor templates (in a range from 27 to 122 bp) for inserting restriction enzyme sites or point mutation, and (2) generating staggered DSBs using Cas9 nickases (D10A or H840A) with a pair of PAM-out or PAM-in sgRNAs at a spacer distance of 40 to 130 or 18 to 97 bp, respectively. The authors conclude that Cas9 nickase D10A is more potent in mediating HDR than Cas9 nickase H840A, and single-stranded oligodeoxynucleotides are preferred donor templates for nickase-mediated HDR. However, the staggered DSBs are predominantly repaired by the NHEJ pathway, leading to unwanted on-target mutations. As a consequence, the ratio of HDR:NHEJ events is low.
Ran et al. 27 disclose that in combination with a pair of PAM-out sgRNAs at a distance of 38-68 bps (termed double-nick sgRNAs), the Cas9D10A nickase (Cas9n) creates offset double nicks that induce site-speficific DSBs, termed double-nick approach hereafter 27. As consequence, this double-nick approach facilitates efficient HDR and NHEJ events in human cell lines. The doublenick approach reduced genome-wide off-target effects of 50- to 1000-fold in comparison with Cas9 combining with one or two sgRNAs in human cell lines 27. The limitation of this strategy is that this approach still causes high frequencies of indels leading to nonsense-mutations in the target mRNA 2728.
US10711285B2 includes the data of Ran et al and discloses a method for modifying a genomic locus of interest. In this method, paired Cas9 nickases (D10A) and guide RNAs are used to generate a 5’ overhang in a double stranded DNA molecule of a eukaryotic cell. However, US10711285B2 teaches that a 5’ overhang of 26-100 nucleotides can efficiently facilitate HDR by the repair template polynucleotide.
Cho et al (Genome Research, vol. 24, no. 1 , 19.11 .2013) relates to an analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Cho et al discloses that paired nickases induce chromosomal deletions in a targeted manner without causing unwanted translocations, highlighting the importance of choosing unique target sequences and optimizing guide RNA and Cas9 nickases to avoid or reduce RNA-guided endonuclease-induced off-target mutations. Cas9 D10A nickases with a pair of sgRNAs at a distance of <170 bp in combination with DNA donor templates lead to NHEJ and HDR events. Cho et al does not disclose exogenous DNA donor templates for DNA repair. Relating to the analysis of off-target mutations and translocation induced by RNA-guided endonucleases and nickases, it was concluded that paired nickases with a distance of 26 bp induce chromosomal deletions in a targeted manner without causing unwanted translocations detected by PCR. This document however only shows that paired Cas9 nickases can induce large deletions of up to 1-kbp chromosomal segments in human cells, the experiments disclosed therein did not employ gene repair.
Tran Ngoc Tung et al (Molecular Therapy, vol. 28, no. 12, 01 .12.2020) describes a CRISPR- Cas9-AAV6-based ELANE gene repair system, wherein a single guide RNA-directed Cas9 nuclease introduces specific DSBs at the targeted sequence, achieves high rates of HDR and restores neutrophil elastase function. However, the unwanted NHEJ-mediated on-target mutations was efficiently generated as well.
Despite recent developments with respect to the use of CRISPR/Cas9 nucleases for genetic engineering, further improvements are required to provide a gene correction approach for therapeutic applications that ideally retains high HDR efficiency whilst minimizing adverse effects, such as the NHEJ repair pathway, generation of off-target effects as well as on-target effects, in particular indels (off-target and on-target mutations). SUMMARY OF THE INVENTION
In light of the prior art the technical problem underlying the present invention is to provide an in vitro method for modifying a double-strand DNA (ds DNA) molecule through single-strand break (SSB)-mediated homology-directed repair (HDR) in a eukaryotic cell that has a high HDR efficiency and minimizes NHEJ, and the generation of unwanted mutations (on-target and off- target).
Another objective of the invention is to provide an isolated eukaryotic cell modified by an in vitro method for modifying ds DNA molecule through SSB-mediated homology-directed repair preferably for use in the treatment of a medical condition.
A further objective of the invention is to provide a kit for use in an in vitro method for modifying dsDNA molecule through SSB-mediated homology-directed repair in a eukaryotic cell.
This problem is solved by the features of the independent claims. Preferred embodiments of the present invention are provided by dependent claims.
In one aspect, the present invention relates to an in vitro method for modifying a double-stranded DNA (dsDNA) molecule in a eukaryotic cell, the method comprising: introducing into the cell a guide RNA-guided DNA nickase or a nucleic acid molecule encoding a guide RNA- guided DNA nickase, at least two guide RNAs, comprising a first guide RNA capable of hybridizing to a first target sequence in the dsDNA, and a second guide RNA capable of hybridizing to a second target sequence in the dsDNA, and an exogenous DNA donor template comprising a DNA template sequence, generating at least two single strand breaks (SSB) in the opposing strands of the dsDNA molecule to be modified, wherein a first guide RNA/DNA nickase complex, comprising the first guide RNA, introduces a nick cleavage of one strand of the dsDNA within the first target sequence, and a second guide RNA/DNA nickase complex, comprising the second guide RNA, introduces a nick cleavage of the opposite strand of the dsDNA within the second target sequence, wherein the distance (spacer) between two single strand breaks (SSB) on the dsDNA molecule to be modified is 202 base pair (bp) or greater, and replacing a DNA sequence of the dsDNA, positioned in proximity to the two single strand breaks (SSB), with the template sequence.
Preferably, the present invention relates to an in vitro method for modifying a double stranded DNA (dsDNA) molecule through single-strand break (SSB)-mediated homology-directed repair (HDR) in a eukaryotic cell, such as a haematopoietic stem stem and progenitor cell (HSPC) or T cell, the method comprising: introducing into the cell a guide RNA-guided DNA nickase or a nucleic acid molecule encoding a guide RNA- guided DNA nickase, at least two guide RNAs, comprising a first guide RNA capable of hybridizing to a first target sequence in the dsDNA, and a second guide RNA capable of hybridizing to a second target sequence in the dsDNA, and an exogenous DNA donor template comprising a DNA template sequence, generating at least two single strand breaks (SSB) in the opposing strands of the dsDNA molecule to be modified, wherein a first guide RNA/DNA nickase complex, comprising the first guide RNA, introduces a nick cleavage of one strand of the dsDNA within the first target sequence, and a second guide RNA/DNA nickase complex, comprising the second guide RNA, introduces a nick cleavage of the opposite strand of the dsDNA within the second target sequence, thereby producing a 5’ or 3’ overhang, wherein the distance (spacer) between two single strand breaks (SSB) on the dsDNA molecule to be modified is 202 base pair (bp) or greater, and replacing a DNA sequence of the dsDNA, positioned in proximity to, adjacent to and/or between the two single strand breaks (SSB), with the template sequence.
The present invention is based on the entirely surprising finding that a distance between two SSB induced by guide RNA-guided nickases of 202 bp or more in the presence of a DNA donor template leads to the induction of HDR with a replacement of the original DNA sequence of the dsDNA molecule to be modified in the region around the SSBs (which can be the region between the SSBs but also a region that extends beyond the SSBs, such as a region in proximity, adjacent to and/or between the two SSBs).
It was found that the design of the method with a spacer between the SSBs that is unexpectedly large, namely at least 202 bp, shifts the repair in the presence of a donor template almost completely towards HDR mediated repair resulting in replacement of the original DNA sequence of the dsDNA molecule in the region of the SSBs, whereas the use of shorter spacers, which has been used in the state of the art, results in a high percentage of NHEJ events which can result in nonsense-mediated decay of resulting mutant mRNA or unwanted dysfunctional protein.
For instance, Ran et al (Cell 154, 1380-1389, 2013) teaches adjusting the offset distance and 5’ overhang distance of sgRNA pairs for reducing INDEL frequency. A set of sgRNA pairs in which the cleavage site of at least one sgRNA is situated near the site of recombination was tested in HEK 293T cells to characterize how offset sgRNA spacing affects the efficiency of HDR. Although an effective offset window greater than 100 bp allows for flexibility in the selection of sgRNA pairs, the author concludes that a pair of sgRNAs with offset distance from 26 bp to 100 bp together with Cas9 nickases and DNA donor templates leads to efficient NHEJ events and facilitates HDR efficiency. In addition, the efficiency of HDR using Cas9n with a pair of sgRNA with an offset window greater than 100 bp has not been measured.
Of further note is that Ran et al shows the efficiency of double-nicking-induced NHEJ in human genes EMX1, DYRK1A and GRIN2B. As disclosed therein, the offset distance between sgRNAs from approx. -200 to 200 bp represents a 5’ overhang (negative number) or a 3’ overhang (positive number). Furthermore, the maximum distance between the single strand breaks amounts to 201 bp, which is distinct from the optimized SSB distance of the present invention.
The advantage of the present invention is based on an optimized distance between two SSB induced by guide RNA-guided nickases. In some embodiments, the distance (spacer) between two single strand breaks (SSB) on the dsDNA molecule to be modified is at least 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230, 231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242,
243, 244, 245, 246, 247, 248, 249, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305,
310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400,
405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, or
500 bp.
The novel technical effect underlying the present invention enables a surprisingly improved method for gene editing and gene repair, due to a high ratio of HDR:NHEJ, for example from 5:1 to 50:1 , as described in more detail below.
When using the longer spacer regions in the method of the present invention, it was found that repair of the dsDNA molecule in the region of the SSBs occurs almost exclusively by HDR. Also, INDEL frequency decreases with increasing spacer length, representing another advantage of the present invention. Furthermore, it was unexpectedly observed that the method of the invention results in a high percentage of desired sequences in the region of the SSBs, which means that the sequence in this region resulting from performing the method of the invention matches the predetermined desired sequence without creation of INDELs.
This is an important advancement in comparison to the state of the art, where replacement event are often associated with creation of INDELs in the region of the dsDNA to be modified, since applications of DNA modification, for examples for gene correction in therapeutic approaches aiming to repair mutated, preferably disease-causing mutations, require reliable and safe repair that avoids the creation undesired DNA sequences, which often occurs in case of NHEJ- mediated repair. Accordingly, the present invention is a promising method for gene repair in cells that reduces the risk of creation of undesired sequences.
Additionally, it was surprisingly found that the method of the invention is highly safe in terms of so-called off-target effects. In genome engineering methods employing CRISPR/Cas technology, a major concern for therapeutic applications is the potential creation of DNA modifications/mutations by the nucleases used in such methods at different sites of the genome than the regions to be modified. Such DNA modifications in undesired regions are referred to as off-target effects or off-target mutations. Methods for gene correction in humans should ideally ensure that off-target effects are avoided. As shown in the example, the method of the invention avoids the generation of off-target mutations almost completely. In most cases, no off-target mutations could be identified, whereas known approaches of the state of the art using Cas9 (inducing double strand breaks) instead of a nickase result in a much higher number of off-target mutations. Furthermore, it could be surprisingly shown that also the number of on-target mutations (which is the introduction of mutations in the dsDNA region to be modified (the target region)) is 40-fold lower when using the method of the present invention.
Accordingly, the method of the present invention not only shifts the percentage of repair events almost completely to HDR repair avoiding NHEJ but is also almost completely devoid of introduction of unwanted mutations, both on-target and off-target. Therefore, it is highly advantageous in comparison to known methods of the state of the art since it is much safer in avoiding unwanted on- and off-target mutations while ensuring repair of the induced strand breaks by the desired HDR mechanism resulting in sequence replacement in the dsDNA molecule to be modified with the DNA template sequence in the region to be modified.
The method of the invention has the additional advantage that the CRISPR/Cas9 system requires only a single Cas9 nickase which can be guided by a short RNA molecule to recognize a specific DNA target instead of generating customized proteins to target specific sequences.
It is a surprising finding that a gene editing in a eukaryotic cell, such as a hematopoietic stem and progenitor cell, can be achieved in a high-fidelity manner through single strand break-mediated homology-directed repair. For this purpose, a guide RNA-guided DNA nickase is introduced into a cell, together with at least two guide RNAs and an exogenous DNA donor template. The method induces the generation of at least two single strand breaks in the dsDNA, wherein a 5’ or 3’ overhang of the processed dsDNA molecule is formed in the region to be modified. Most importantly, the distance between two single strand breaks on the dsDNA molecule to be modified equal to or greater than 202 base pair. As a result of the induction of the two SSBs in presence of the DNA donor template, a DNA sequence of dsDNA, positioned in proximity to, adjacent to and/or between the two single strand breaks is replaced with the template sequence.
One reason why sequence replacement according to the invention is achieved with high specificity in the region to be modified with little to no off- and on-target mutations is the use of a nickase which generates a single nick in one strand of the dsDNA instead of generating double strand break with a blunt end.
Preferably, the DNA nickase of the invention is a Cas9 nickase, such as a Cas9 nickase derived from Streptococcus pyogenes Cas9 (SpCas9). More preferably, the nickase is a modified SpCas9 comprising a mutation at D10A. Preferably, the nickase is the SpCas9 nickase according to the SEQ ID NO 1 ).
SEQ ID NO 1 :
>sp|Q99ZW2|CAS9_STRP1 CRISPR-associated endonuclease Cas9/Csn1 OS=Streptococcus pyogenes serotype M1 OX=301447 GN=cas9 PE=1 SV=1
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKR TARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEK YPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEE NPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQ LSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDL TLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDL LRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAW MTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKY VTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTY HDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWG RLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIAN LAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKEL GSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKV LTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQ LVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHD AYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLA NGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDK LIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEA KGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPE DNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNL GAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD
Surprisingly, it has been found that use of the method of the invention suppresses NHEJ and promotes HDR through generating a 5’ or 3’ overhang in the nicked dsDNA. The induction of the SSBs in the opposing strands of the dsDNA to be modified at an unexpectedly large distance of at least 202 bp resulting in the overhangs may be a critical feature of the present invention. This large spacer may avoid the formation of a DSB (at least in some or most processing events), since the strands may still remain connected through base pairing of the resulting overhangs. This may be a reason why NHEJ repair can be almost completely avoided by the method of the invention. However, the induction of the two SSBs in the required distance from each other still leads to HDR in the presence of the template sequence.
According to the invention, the design of the two guide RNAs ensures that the guide RNA/DNA nickase complexes involving the two respective guide RNAs are positioned on opposing strands of the dsDNA to be modified. In other words, the first guide RNA directs the nickase-complex to the plus-strand, and the second guide RNA direct the nickase complex to the minus-strand. Consequently, the strand-nicks are introduced on opposing strands, resulting in the formation of an overhang on each end of a resulting DNA fragment.
In the context of the present invention, the different components of the method, namely a first guide RNA, a second guide RNA, a guide RNA-guided DNA nickase or a nucleic acid molecule encoding a guide RNA-guided DNA nickase, and an exogenous DNA donor template comprising a DNA template sequence, may be added sequentially to a cell or to a cell culture comprising the cell, or at the same time, for example using a premixed stock solution comprising all or some of the components.
In the context of the method of the invention, it is preferred that 5’ overhangs are produced. It could be shown that HDR efficiency is higher in case of 5’ overhangs as compared to 3’ overhangs. An overhang in the sense of the present invention relates to the portion of ssDNA that would be generated if the two fragments of the dsDNA to be modified would be separated after generation of the two SSBs. Each of the resulting dsDNA molecules has an overhang, which corresponds to the length of the spacer between the two SSBs. Depending on the strand-positioning of the two guide RNAs mediating the positioning of the guide RNA/DNA nickase complex, a 3’ or 5’ overhang on the end of each resulting fragment of the dsDNA molecule can be produced.
In one embodiment, the method as described herein wherein each target sequence comprises or is adjacent to a protospacer adjacent motif facing outwards (PAM-out) at a 3’-end of the target sequence.
It is understood that “facing outwards” in the sense of the invention relates to the PAM facing outwards/to the outside of the region located between the target sequences of the two guide RNAs of the method of the invention. Accordingly, when using for example a SpCas9 D10A, the PAMs of the two target sites are not comprised by the spacer sequences located between the two SSBs, since the PAM is located at the 3’ end of the target sequence and the cut occurs three bases upstream from the PAM. Accordingly, in a preferred embodiment using a PAM-out configuration 5’ overhangs are generated.
A skilled person is able to modify these configurations best suited in case of other nickases, such as Cas9 H840A or others, which may involve other configurations.
In the present invention, it is a surprising finding that under the condition that the distance between two single strand breaks on the dsDNA to be modified equal to or greater than 202 bp, the Cas9 nickase combined with a pair of advantageously distanced guide RNAs together with delivery of an exogenous DNA template, for example by means of a viral vector, leads to efficient homology-directed repair while minimizes on/off-target effects. The single strand break-mediated homology-directed repair according to the invention offers universal gene corrections to repair the known mutations in a variety of diseases.
In one embodiment, the method as described herein wherein replacing a DNA sequence of the dsDNA molecule to be modified with the template sequence occurs primarily by homology- directed repair (HDR), preferably wherein replacing a DNA sequence with the template sequence occurs more frequently by homology-directed repair (HDR) compared to non-homologous end joining (NHEJ), more preferably wherein the ratio of homology-directed repair (HDR) events to non-homologous end joining (NHEJ) events is from 5:1 to 50:1 , preferably from 10:1 to 20:1.
Surprisingly, the increased distance between the two SSBs leads to a remarkable reduction of NHEJ events. As shown in the examples herein, in B2M locus spacer-nick, a spacer of 220 bp generates 42.8% HDR event in comparison to 2.6% NHEJ. The fraction of NHEJ can be further reduced by increasing the spacer. For example, a spacer of 346 resulted in 19.2% HDR, 0.7% NHEJ, and a spacer of 459 bp in 8.4% HDR and non-detectable NHEJ.
In embodiments, the replacing occurs by homologous recombination repair.
The skilled person is aware of various common methods of determining the occurrence of certain DNA repair mechanisms. As shown in the examples below, occurrence of HDR and NHEJ can be detected by standard laboratory techniques for analysing the DNA of a manipulated cell. For example, the repair of double-strand breaks by HDR or NHEJ can be easily detected at any genomic target sequence by PCR amplification of the target region followed by the sequencing of PCR products and sequence comparison to the genomic wildtype locus. Alternatively reporter constructs for DSB repair detection can be designed and inserted int the genome of mammalian cells such that different fluorescent proteins are expressed upon repair of a target size by NHEJ or HDR.
In a preferred embodiment, the distance (spacer) between the at least two single strand breaks (SSB) on the dsDNA molecule to be modified is 202 to 500 bp, preferably 202 to 350 bp.
In embodiments, the distance (spacer) between the at least two single strand breaks (SSB) on the dsDNA molecule to be modified is at least 202, 203, 204, 205, 206, 207, 208, 209, 210, 211 , 212, 213, 214, 215, 216, 217, 218, 219, 220, 221 , 222, 223, 224, 225, 226, 227, 228, 229, 230,
231 , 232, 233, 234, 235, 236, 237, 238, 239, 240, 241 , 242, 243, 244, 245, 246, 247, 248, 249,
250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340,
345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435,
440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, 500 bp.
Surprisingly, the distance of 202 to 500 bp between the at least two single strand breaks on the dsDNA molecule to be modified allows not only HDR dominant gene corrections with less off- target effect but also a high degree of flexibility in the selection of sgRNA pairs.
In embodiments, the method of the invention induces fewer unwanted on- and/or off-target effects, such as micro-insertions and/or deletions (Indels) and/or off-target mutations, compared to a method in which the distance (spacer) between two single strand breaks (SSB) on the dsDNA molecule to be modified is less than 202 base pair (bp) or a method in which a doublestrand break (DSB) inducing guide RNA-guided DNA endonuclease, such as CRISPR/Cas9, is employed, wherein preferably the number and/or frequency of off-target effects is determined using a genome-wide method for detecting off-target mutations, such as a GUIDE-seq method.
On- and off-target effects can be measured by sequencing based techniques with subsequent sequence analysis and comparison, as known to a person skilled in the art. For example, on- off- target effects can be determined by techniques like GUIDE-seq or LAM-HTGTS.
On-target mutations (in particular INDELs) are determined by PCR, following PCR amplicons are sequenced by Sanger or next generation sequencing. Frequencies of on-target mutations are analyzed and compared with reference sequences (wild-type). Genome-wide off-target effects induced by CRISPR/Cas are determined by deep sequencing techniques such as for example GUIDE-seq, AAV-seq and LAM-HTGTS.
It is a surprising finding that the method as described herein significantly reduces the off-target effect in the genomic DNA. The genome-wide off-target mutation induced by the method of the invention in human HSPCs are almost undetectable, as shown in the examples below.
Additionally, the frequencies of indel events in all tested off-target sites of the spacer-nick treated HSPCs were at a very low level and clearly reduced as compared to previous methods of the state of the art. This surprising finding renders the method of the invention a robust and reliable method for gene correction. In another embodiment, the method as described herein wherein the DNA template sequence is 500 bp to 5000 bp in length, preferably 1000 to 5000 bp, more preferably 2000 to 4000 bp, more preferably about 3000 bp in length.
In embodiments, the DNA template sequence is about 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2050, 2100, 2150, 2200, 2250, 2300, 2350, 2400,
2450, 2500, 2550, 2600, 2650, 2700, 2750, 2800, 2850, 2900, 2950, 3000, 3050, 3100, 3150,
3200, 3250, 3300, 3350, 3400, 3450, 3500, 3550, 3600, 3650, 3700, 3750, 3800, 3850, 3900,
3950, 4000, 4050, 4100, 4150, 4200, 4250, 4300, 4350, 4400, 4450, 4500, 4550, 4600, 4650,
4700, 4750, 4800, 4850, 4900, 4950, or 5000 bp in length.
As used herein, it is understood that replacing a DNA sequence of the dsDNA, positioned in proximity to the two single strand breaks (SSB) means replacing a DNA sequence of the dsDNA positioned in proximity to, adjacent to and/or between the two single strand breaks (SSB).
The sequence to be replace by in the context of the present invention can be located between the SSBs induced by the nickase, but it can also span the location of one or both SSBs and therefore extend beyond the spacer sequence. It is understood that a DNA sequence positioned in proximity to the SSBs comprises any sequence that is located between the two SSBs, including the entire spacer sequence, but can also be a sequence that extends beyond the spacer on one or both sides.
It is understood that a sequence “adjacent” to an SSB is a sequence that starts with a nucleotide directly next to the SSB/cutting site of the nickase. For example, the spacer sequence located between the two SSBs is on each end “adjacent” to one of the SBBs.
However, a sequence that is “in proximity” to the SSBs can also comprise sequences whose distal ends start up to 1500 bp outside the spacer sequence, meaning that the distal end of the sequence (as regarded from the next SSB induced by the nickase in the context of the method of the invention) starts in a distance of 1500 bp from the SSB. Accordingly, in embodiments, the sequence to be replaced can span the spacer sequence and up to 1500 bp outside from each of the two SBBs. Preferably, a sequence that is “in proximity” to the SSBs can also comprise sequences whose distal ends start about 1400, 1300, 1200, 1100, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 180, 150, 100, 50, 40, 30, 20, 10, 5, 4, 3, 2, 1 , 0 (= adjacent to the SSB) bp outside the spacer sequence. Furthermore, sequences that are in proximity to the SSBs comprise all sequences that consist of or are comprised by the spacer region.
In embodiments, the sequence to be replaced is defined by the design of the DNA template sequence. To induce HDR mediated sequence replacement, the DNA template sequence requires at least one homology arm, which is understood to be a sequence of at least 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, preferably 500 bp, more preferably 700 bp, or 1000 bp or more, with a sequence identity to a region within the DNA sequence sufficient to mediated HDR, such as at least 90%, preferably 95%, 96%, 97%, 98%, 99%, most preferably 100% sequence identity to a region within the DNA sequence to be replaced. In embodiments, the homology arm has a sequence identity sufficient to enable hybridization of the homology arm to the corresponding region of the sequence to be replace. Preferably, the homology arms share this sequence identity with a region located at the end of the end sequence to be replaced.
Preferably, the DNA template has two homology arms, each of which is homologous/sufficiently identical to enable HDR-mediated sequence replacement. In embodiments, the DNA template sequence comprises a sequence unrelated to the replacement sequence located between (flanked by) two homology arms with sufficient sequence identity to a region of the sequence to be replaced to enable HDR mediated sequence replacement.
As used herein, the terms sequence identity and sequence homology are used interchangeably. Herein, it is understood that a “homologous sequence” has a sufficient sequence identity to a region of the sequence to be replaced to enable replacement through HDR.
In some embodiment, the DNA template sequence comprises at least one sequence (homology arm) of at least 100 bp, preferably 500 bp, with at least 90%, preferably 95%, more preferably 100%, sequence identity to a region within the DNA sequence to be replaced, wherein said homology arm is positioned to hybridize to the dsDNA molecule to be modified in proximity to the two single strand breaks (SSB), preferably wherein the DNA template comprises at least two homology arms, each comprising at least 100 bp, preferably 500 bp, more preferably 700 bp, or 1000 bp or more, with at least 90%, preferably 95%, more preferably 100%, sequence identity to a region within the DNA sequence to be replaced.
In some embodiment, the one or two homology arms are positioned to hybridize to the dsDNA molecule to be modified in proximity to, adjacent to and/or between the two single strand breaks (SSB).
In a preferred embodiment, a homology arm of the DNA template is positioned to hybridize to the dsDNA molecule to be modified, such that the distance between a single strand break (SSB) induced by the RNA guided DNA nickase and the end of the homology arm distal to the SSB is up to 1500 bp, preferably up to 1000 bp, more preferably up to 500 bp. In embodiments, the distance between a single strand break (SSB) induced by the RNA guided DNA nickase and the end of the homology arm distal to the SSB is about 1400, 1300, 1200, 1100, 1000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 300, 250, 200, 180, 150, 100, 50, 40, 30, 20, 10, 5, 4, 3, 2, 1 , 0.
It is a great advantage of the method of the invention that the DNA template sequence can be adjusted as necessary and suited, which can be individually judged by a skilled person depending on the DNA sequence to be replaced and its location within the dsDNA molecule to be modified. There is no strict rule with respect to the designs of the homology arms, which may enable hybridization of a region of the DNA sequence to be replace, which can be located outside or inside the spacer region or which can be comprise the location of one or both SSBs induced in the context of the present invention. Various examples of suitable DNA template designs and positioning of homology arms are shown in the examples below and a skilled person can deduct suitable design rules for the DNA template sequence form these examples and the published state of the art concerning HDR meditated sequence replacement. In embodiments, the DNA template sequence is in the form of and/or comprised by an viral vector, such as an adeno-associated virus (AAV), or is a circular or linear DNA molecule, preferably a plasmid or mini-circle, or a single stranded DNA molecule.
Depending on the kind of sequence replacement that should be performed by the inventive method a skilled person can select a suitable kind of exogenous DNA donor template comprising the DNA template sequence. Depending on the sequence length to be introduced and/or depending on the properties or length of the homology arms, it can be advantageous to select a specific kind of donor template comprising the template sequence. This may also depend on other factors, such as the type of target cell comprising the dsDNA to be modified and the preferred way of introducing exogenous DNA into the respective cell type.
Frequently used dsDNA templates are inserts into bactarial plasmids that can be transfected into mammalian cell lines. Since primary cells can be sensitive to dsDNA it is often preferred to introduce the donor template packaged into the capsid of Adeno associated virus (AAV). Upon infection of cells with AAV particles its genome enters the nucleus as single-stranded DNA that is suitable for HDR.
In a preferred embodiment, the method of the present invention is for modifying an ELANE gene (encoding Neutrophil Elastase), wherein the at least two guide RNAs are capable of hybridizing to sequences in or adjacent to, preferably flanking, a sequence of the ELANE gene to be modified, wherein preferably the sequence of the ELANE gene to be modified comprises one or more pathological gene mutations associated with reduced or aberrant Neutrophil Elastase function, such as in subjects with Severe Congenital Neutropenia (SCN), and the DNA template sequence comprises an ELANE gene sequence without said mutations, wherein more preferably the DNA template sequence comprises exons 4 and/or 5, or parts thereof, of an ELANE gene sequence without said mutations.
In a more preferred embodiment, the at least two guide RNAs comprise a sequence according to SEQ ID NO 26 and SEQ ID NO 34. (e.g. sgELANE-ex4-1 , GAGTGCAGACGTTGCTGCGA, sgELANE-ex5-2, ATTGCCTCCTTCGTCCGGGG). Further preferred combinations of specific guide RNAs (sequence disclosed in table 1 ) for use in the context of the present invention for gene correction for ELANE mutations are disclosed in the table 2.
As shown herein, the ELANE gene repair system and method of the present invention using CRISPR/Cas9 nickase and spacer of at least 202 bp has achieved high HDR replacement rates and lower off-target effect than conventional CRISPR/Cas9-based methods.
The conventional treatment of children suffering from SCN with G-CSF reduces the risk of sepsis and infections by increasing the ANC in the majority of SCN patients. However, SCN patients treated with G-CSF are at high risk of MDS/AML development, especially in the case of G-CSF poor responders. For SCN patients who do not respond to G-CSF or develop MDS/AML, the only available treatment option is allogeneic hematopoietic stem cell transplantation with frequent adverse effects and high mortality (17%).
Nasri et al. have shown that knock out (KO) of the ELANE gene in SCN patient-derived hematopoietic stem and progenitor cells (HSPCs) using CRISPR-Cas9 can rescue neutrophil differentiation. ELANE KO removed the deleterious effects of misfolded NE and restored mature neutrophil formation in vitro. However, while an ELANE KO strategy provides a potentially promising treatment for G-CSF poor responders, it seems to favor the generation, selection, or outgrowth of edited cells that lack NE expression altogether. This causes concern for human patients, since ELANE KO models in mice have established a non-redundant role for NE in the innate immune defense against certain microbial infections. It therefore seems warranted to explore additional gene-repair approaches that can fully restore NE and neutrophil function.
Thus it is advantageous that the method as described herein can successfully repair mutations causing several monogenic blood disorders in patient-derived HSPCs with high rate of HDR and low off-target effect.
In another preferred embodiment, the method of the invention can be used for modifying an HBB gene (encoding Hemoglobin Subunit Beta), wherein the at least two guide RNAs are capable of hybridizing to target sequences in or adjacent to, preferably flanking, a sequence of the HBB gene to be modified, wherein preferably the sequence of the HBB gene to be modified comprises one or more pathological gene mutations associated with reduced or aberrant Hemoglobin Subunit Beta function, such as in subjects with Beta-Thalassemia, and the DNA template sequence comprises an HBB gene sequence without said mutations, wherein more preferably the DNA template sequence comprises exons 1 and/or 2, or parts thereof, of an HBB gene sequence without said mutations.
In a more preferred embodiment, the at least two guide RNAs comprise a sequence according to SEQ ID NO 14 and SEQ ID NO 21 (e.g. sgHBB-ex1-1 , CTTGCCCCACAGGGCAGTAA, sgHBB- ex2-1 , TCCACTCCTGATGCTGTTAT). Further preferred combinations of specific guide RNAs (sequence disclosed in table 1 ) for use in the context of the present invention for gene correction for HBB mutations are disclosed in the table 2.
It is advantageous that the mutation of HBB locus can be corrected with high fidelity by means of the method of the present invention using suitable sgRNAs. It is preferably to use a Cas9 nickase combined with spacer nick sgRNAs ensuring a spacer of at least 202 by and a viral vector for delivering the DNA doner template to HSPCs. The method of the invention is preferable to the state-of-the-art treatment of allogenic hematopoietic stem-cell transplantation to cure for example beta-hemoglobinopathies or conventional CRISPR/Cas9 gene editing based on HDR on double strand breaks.
The known transplantation treatment is limited because of graft-versus-host disease and a lack of immunologically matched donors. An alternative to using allogeneic HSCs to cure the £ - haemoglobinopathies is to use homologous recombination to modify the HBB gene directly in autologous HSCs. In 1985, Smithies and colleagues were able to modify the human HBB gene by homologous recombination in a human embryonic carcinoma cell line, albeit at an extremely low frequency. The subsequent discoveries that a site-specific DNA double-strand break (DSB) could stimulate homologous-recombination-mediated correction of a reporter gene and that engineered nucleases could be used to induce this DSB, formed the foundation of using homologous-recombination-mediated genome editing using engineered nucleases to modify the HBB gene directly. However, this method still does not generate an ideal ration of NHEJ and HDR. Moreover, the off-target effects are still too intensive. The ease of engineering as well as the robust activity of method of the invention makes it a promising tool to apply to the continuing challenge of developing effective and safe homologous-recombination-mediated genome editing to cure p - haemoglobinopathies.
In embodiments, the method of the invention is used for modifying a Perforin gene (preferably PRF1 , encoding a Perforin protein), wherein the at least two guide RNAs are capable of hybridizing to target sequences in or adjacent to, preferably flanking, a modified cDNA sequence of the Perforin gene to be inserted, wherein preferably the sequence of the Perforin gene to be modified comprises one or more pathological gene mutations associated with reduced or aberrant Perforin protein function, such as in subjects with familial hemophagocytic lymphohistiocytosis type 2 (FHL2), a rare life-threatening immune deficiency disorder, and the DNA template sequence comprises a Perforin gene sequence without said mutations.
As shown in the example, in the case of PRF1 correction, it is preferred to use a pair of PAM-out sgRNAs, targeting exon 2 and intron 2 of the PRF1 locus, preferably with a spacer distance 223 bp. In order to correct all known mutations distributed randomly in coding and intronic sequences of the PRF1 gene, it is preferred to insert modified cDNA sequence into exon 2 of the PRF1 locus.
In some embodiment, the method as described herein wherein the guide RNAs comprise a sequence according to SEQ ID NO: 46 and 49, (e.g. sgPRF1-ex2-1 , TGGCCCTGGTTACATGGCGC, sgPRF1-in2-1 , CGGTGGAGTGCCGCTTCTAC). Further preferred combinations of specific guide RNAs (sequence disclosed in table 1 ) for use in the context of the present invention for gene correction for PRF1 mutations are disclosed in the table 2.
In further embodiments, the method of the invention can be used for modifying an lnterleukin-7 receptor (IL7R) gene (encoding an lnterleukin-7 receptor subunit alpha protein), wherein the at least two guide RNAs are capable of hybridizing to target sequences in or adjacent to, preferably flanking, a sequence of the IL7R gene to be modified, wherein preferably the sequence of the IL7R gene to be modified comprises one or more pathological gene mutations associated with reduced or aberrant IL7R protein function, such as in subjects with severe combined immunodeficiency or inflammatory diseases, such as multiple sclerosis or rheumatoid arthritis, and the DNA template sequence comprises a IL7R gene sequence without said mutations.
In the case of the IL7R correction, it is preferred to use a pair of PAM-out sgRNAs targeting 5’ upstream region and exon 1 of the IL7R locus, preferably with a spacer distance 273 bp (termed sglL7R-5’-2 and sglL7R-e1-2, respectively, in the example below). To repair all mutations randomly distributed in the IL7R gene, it can be useful to insert modified cDNA sequence into exon 1 of the IL7R locus.
In some embodiment, the method as described hrein wherein the guide RNAs comprise a sequence according to SEQ ID NO: 39 and 44 (e.g. sglL7R-5’-2, AGTATTGCTGCTGTAAGCAG, sglL7R-e1-2, CAAGTCGTTTCTGGAGAAAG). Further preferred combinations of specific guide RNAs (sequence disclosed in table 1 ) for use in the context of the present invention for gene correction for IL7R mutations are disclosed in the table 2. In aspects, the present invention relates to at lest one guide RNA as disclosed in table 1 , which may be used in combination with another guide RNA that may not be disclosed herein or that is disclosed herein.
In another aspect, the invention relates to an isolated eukaryotic cell, such as a haematopoietic stem and progenitor cell or T cell, modified by a method according to any of the preceding claims, preferably for use in the treatment of a medical condition, wherein the medical condition is associated with the reduced function of a gene product induced by pathological gene mutations and said mutations have been replaced with a DNA template sequence without said mutations.
It is an important advantage of the invention that it is possible to engineer isolated cells in vitro, for example by performing a gene correction using the method of the invention, and subsequently analyse and verify the resulting sequence. Subsequently, the cells of the invention can be used for in vitro analysis and for therapeutic purposes, for example by transplanting the cells to a patient. Preferably, the cells are autologous to the patient, i.e. the cells have initially been isolated from the patient, subsequently underwent the method of the invention, for example for gene correction, and the modified cells are thereafter administered to the same subject.
In a third aspect, the invention relates to a kit for use in an in vitro method for modifying a double stranded DNA (dsDNA) molecule through single-stranded break (SSB)-mediated homology- directed repair (HDR) in a eukaryotic cell, such as a haematopoietic stem and progenitor cell (HSPC) or T cell, the kit comprising: at least two guide RNAs, comprising a first guide RNA capable of hybridizing to a first target sequence in the dsDNA, and a second guide RNA capable of hybridizing to a second target sequence in the dsDNA, wherein the first guide RNA is configured to introduce a nick cleavage of one strand of the dsDNA within the first target sequence and the second guide RNA is configured to introduces a nick cleavage of the opposite strand of the dsDNA within the second target sequence, thereby producing a 5’ or 3’ overhang configured for repair primarily by singlestrand break-mediated homology-directed repair (HDR), and wherein the at least two guide RNAs are configured such that the distance (spacer) between the at least two single strand breaks (SSB) on the dsDNA molecule to be modified is 202 base pair (bp) or greater, preferably 202 to 500 bp, more preferably 202 to 350 bp, preferably comprising the guide RNAs according to SEQ ID NO 26 and 34, SEQ ID NO 14 and 21 , SEQ ID NO: 46 and 49 or SEQ ID NO: 39 and 44, further preferred combinations for correction of corresponding gene mutations are disclosed in table 2. and optionally a guide RNA-guided DNA nickase or a nucleic acid molecule encoding a guide RNA- guided DNA nickase, and/or an exogenous DNA donor template comprising a DNA template sequence. In embodiments of the kit of the invention, the at least two guide RNAs are comprised in separate containers, one for each kind of guide RNA. Furthermore, the guide RNA-guided DNA nickase or a nucleic acid molecule encoding a guide RNA-guided DNA nickase, and/or the exogenous DNA donor template comprising a DNA template sequence may be provided in a further separate container.
The various aspects of the invention are unified by, benefit from, are based on and/or are linked by the common and surprising finding that induction of two SBBs which are separated by a spacer sequence of at least 202 bp in the presence of a suitable DNA template sequence results in highly efficient HDR with minimal to no on- and off-target effects while NHEJ is almost completely suppressed. All advantageous and features of the invention disclosed in the context of the method of the invention also apply to the cells of the invention and the kit of the invention, and vice versa.
DETAILED DESCRIPTION OF THE INVENTION
All cited documents of the patent and non-patent literature are hereby incorporated by reference in their entirety.
In the context of the present invention, the term “modifying” a double stranded DNA refers to any kind of alteration, modification or change of a dsDNA molecule. In particular, modifying relates to deleting, inserting, replacing, substituting or translocating one or one or more nucleotides or pairs of nucleotides or nucleotide sequences from a dsDNA molecule. In the context of the invention, a dsDNA molecule to be modified is the dsDNA molecule, on which one or more of these modifications is introduced by the method of the invention.
As used herein the term "hybridizing" refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single selfhybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme. A sequence capable of hybridizing with a given sequence may be referred to as the "complement" of the given sequence, even if sequence complementarity is only partial.
Stringent conditions for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent and vary depending on several factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology-Hybridization With Nucleic Acid Probes Part I, Second Chapter "Overview of principles of hybridization and the strategy of nucleic acid probe assay", Elsevier, N.Y . Where reference is made to a polynucleotide sequence, then complementary or partially complementary sequences are also envisaged. These are preferably capable of hybridising to the reference sequence under highly stringent conditions. Generally, in order to maximize the hybridization rate, relatively low-stringency hybridization conditions are selected: about 20 to 25° C. lower than the thermal melting point (Tm). The Tm is the temperature at which 50% of specific target sequence hybridizes to a perfectly complementary probe in solution at a defined ionic strength and pH. Generally, in order to require at least about 85% nucleotide complementarity of hybridized sequences, highly stringent washing conditions are selected to be about 5 to 15° C. lower than the Tm. In order to require at least about 70% nucleotide complementarity of hybridized sequences, moderately-stringent washing conditions are selected to be about 15 to 30° C. lower than the Tm. Highly permissive (very low stringency) washing conditions may be as low as 50° C. below the Tm, allowing a high level of mis-matching between hybridized sequences. Those skilled in the art will recognize that other physical and chemical parameters in the hybridization and wash stages can also be altered to affect the outcome of a detectable hybridization signal from a specific level of homology between target and probe sequences. Preferred highly stringent conditions comprise incubation in 50% formamide, 5xSSC, and 1 % SDS at 42° C., or incubation in 5xSSC and 1 % SDS at 65° C., with wash in 0.2xSSC and 0.1 % SDS at 65° C.
As used herein, the term “capable of hybridizing” refers to a guide RNA molecule that is designed in a way that enables hybridization to a selected target sequence under the respective experimental conditions, under which the guide RNA is used. A skilled person is aware of the design criteria for such guide RNAs.
The term “replacement” or "substitution" in the sense of “replacing a DNA sequence of as dsDNA molecule”, as used herein, is defined in accordance with the pertinent art and refers to the replacement of one or more (such as a sequence) of nucleotides with other nucleotides. The term includes for example the replacement of single nucleotides resulting in point mutations. Said point mutations can lead to an amino acid exchange in the resulting protein product but may also not be reflected in the amino acid level (i.e. silent mutations). Also encompassed by the term "substitution" are mutations resulting in the replacement of multiple nucleotides (a nucleotide sequence), such as for example parts of genes, such as parts of exons or introns as well as the replacement of entire genes. The number of nucleotides that replace the originally present nucleotides may be the same or different (i.e. more or less) as compared to the number of nucleotides removed. Preferably, the number of replacement nucleotides corresponds to the number of originally present nucleotides that are substituted.
The term “insertion", in accordance with the present invention, is defined in accordance with the pertinent art and refers to the incorporation of one or more nucleotides into a nucleic acid molecule. Insertion of parts of genes, such as parts of exons or introns as well as insertion of entire genes is also encompassed by the term "insertion". When the number of inserted nucleotides is not dividable by three, the insertion can result in a frameshift mutation within a coding sequence of a gene. Such frameshift mutations will alter the amino acids encoded by a gene following the mutation. In some cases, such a mutation will cause the active translation of the gene to encounter a premature stop codon, resulting in an end to translation and the production of a truncated protein. When the number of inserted nucleotides is instead dividable by three, the resulting insertion is an "in-frame insertion". In this case, the reading frame remains intact after the insertion and translation will most likely run to completion if the inserted nucleotides do not code for a stop codon. However, because of the inserted nucleotides, the finished protein will contain, depending on the size of the insertion, one or multiple new amino acids that may affect the function of the protein.
The term "deletion", as used in accordance with the present invention, is defined in accordance with the pertinent art and refers to the loss of nucleotides or larger parts of genes, such as exons or introns as well as entire genes. As defined with regard to the term "insertion", the deletion of a number of nucleotides that is not evenly dividable by three, in particular in a coding sequence, will lead to a frameshift mutation, causing all of the codons occurring after the deletion to be read incorrectly during translation, potentially producing a severely altered and most likely nonfunctional protein. If a deletion does not result in a frameshift mutation, i.e. because the number of nucleotides deleted is dividable by three, the resulting protein is nonetheless altered as the finished protein will lack, depending on the size of the deletion, one or several amino acids that may affect the function of the protein.
The term double stranded DNA (dsDNA) molecule relates to two deoxyribonucleic acid polynucleotide strands that are bound together or hybridizes through pairing of the bases or nucleotides of the two strands through hydrogen bonds resulting in double-stranded DNA. The method of the present invention can be performed using any kind of dsDNA molecule, including, without limitation, genomic dsDNA of any origin or organism, chromosomal DNA, synthetic dsDNA, amplified or isolated dsDNA.
In preferred embodiments of the invention, the dsDNA molecule to be modified is the genomic or chromosomal dsDNA of a eucaryotic cell, preferably of a mammalian cell, such as a human cell.
As used herein the term "variant" should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs predominantly in nature.
A “cell” in the sense of the present invention refers to, without limitation, any biological cell, which might be derived from any kind of organism, comprising unicellular organisms as well as multicellular organisms, such as for example any kind of plant or animal, including mammals, fish, amphibians, reptiles, birds, molluscs, arthropods, annelids, nematodes, flatworms, cnidarians, ctenophores and sponges. Cells of the present invention further comprise prokaryotic cells, bacteria, eukaryotic cells, blood cells, stem cells, hematopoietic stem cells, hematopoietic stem and progenitor cell (HSPC), immune cells (such as B-cells, dendritic cells, granulocytes, innate lymphoid cells (ILCs), megakaryocytes, monocytes, macrophages, myeloid-derived Suppressor Cells (MDSC), natural killer (NK) cells, platelets, red blood cells (RBCs), T-cells or thymocytes), cancer cells, tumor cells and circulating tumor cells. In some embodiments of the invention, the cell in which the dsDNA is to be modified is a vertebrate cell, more preferably a mammalian cell, such as a human cell. In some embodiments, the cell is not a rice cell. In some embodiments, the cell is not a plant cell.
The term "introducing into the cell", as used herein, relates to any known method of bringing a protein or a nucleic acid molecule into a cell. Non-limiting examples include microinjection, infection with viral vectors, electroporation, transfection, such as transfection using formulations with cationic lipids. Suitable methods for introducing the components of the present invention into a cell are known to the skilled person. CRISPR is an abbreviation of Clustered Regularly Interspaced Short Palindromic Repeats and is a family of DNA sequences in bacteria. The sequences contain snippets of DNA from viruses that have attacked the bacterium. These snippets are used by the bacterium to detect and destroy DNA from further attacks by similar viruses. These sequences play a key role in a bacterial defense system, and form the basis of a technology known as CRISPR/Cas that effectively and specifically changes genes within organisms.
Sequences of the CRISPR loci are transcribed and processed into CRISPR RNAs (crRNAs) which, together with a trans-activating crRNAs (tracrRNAs), complex with CRISPR-associated (Cas) proteins to dictate specificity of DNA cleavage by Cas nucleases through Watson-Crick base pairing between nucleic acids (Wiedenheft, B et al (2012). Nature 482: 331-338; Horvath, P et al (2010). Science 327: 167-170; Fineran, PC et a. (2012). Virology 434: 202-209).
It was shown that the three components required for the type II CRISPR nuclease system are the Cas9 protein, the mature crRNA and the tracrRNA, which can be reduced to two components by fusion of the crRNA and tracrRNA into a single guide RNA (sgRNA) and that re-targeting of the Cas9/sgRNA complex to new sites could be accomplished by altering the sequence of a short portion of the gRNA (Garneau, JE et al (2010). Nature 468: 67-71 ; Deltcheva, E et al. (2011 ). Nature 471 : 602-607, Jinek, M et al (2012) Science 337: 816-821 ).
CRISPR-Cas systems are RNA-guided adaptive immune systems of bacteria and archaea that provide sequence-specific resistance against viruses or other invading genetic material. This immune-like response has been divided into two classes on the basis of the architecture of the effector module responsible for target recognition and the cleavage of the invading nucleic acid (Makarova KS et al. Nat Rev Microbiol. 2015 Nov; 13(11 ):722-36.). Class 1 comprises multisubunit Cas protein effectors and Class 2 consists of a single large effector protein. Both Class 1 and 2 use CRISPR RNAs (crRNAs) to guide a Cas nuclease component to its target site where it cleaves the invading nucleic acids. Due to their simplicity, Class 2 CRISPR-Cas systems are the most studied and widely applied for genome editing. The most widely used CRISPR-Cas system is CRISPR-Cas9.
It was demonstrated that the CRISPR/Cas9 system could be engineered for efficient genetic modification in mammalian cells. The only sequence limitation of the CRISPR/Cas system appears to derive from the necessity of a protospacer-adjacent motif (PAM) located immediately 3’ to the target site. The PAM sequence is specific to the species of Cas9. For example, the PAM sequence 5’-NGG-3’ is necessary for binding and cleavage of DNA by the commonly used Cas9 from Streptococcus pyogenes. However, Cas9 variants with novel PAMs have been and may be engineered by directed evolution, thus dramatically expanding the number of potential target sequences. Cas9 complexed with the crRNA and tracrRNA undergoes a conformational change and associates with PAM motifs throughout the genome interrogating the sequence directly upstream to determine sequence complementarity with the gRNA. The formation of a DNA-RNA heteroduplex at a matched target site allows for cleavage of the target DNA by the Cas9-RNA complex. These methods and mechanisms are well known in the art. Upon binding of the Cas9- RNA complex (preferably comprising a sgRNA) to the target site/target sequence of a dsDNA to be modified, a RNA/DNA Cas9 complex is formed. As known in the art, CRISPR/Cas9 has been exploited to develop potent tools for genome manipulation in animals, plants and microorganisms. The RNA-guided Cas9 endonuclease first recognizes a 2- to 4-base-pair conserved sequence named the protospacer-adjacent motif (PAM), which flanks a target DNA site (target sequence). Upon binding to the PAM, Cas9 interrogates the flanking DNA sequences for base-pairing complementarity to a guide RNA. If there is complementarity between the first 12 base pairs (the ‘seed’ sequence) of the guide RNA and the target DNA strand, RNA strand invasion accompanies local DNA unwinding to form an R- loop. Precise cleavage of each DNA strand by the RuvC and HNH domains of Cas9 generates a blunt double-strand DNA (dsDNA) break (DSB) at a position three base pairs upstream of the 3' edge of the protospacer sequence, measuring from the PAM.
CRISPR/Cas9 genome-editing experiments have been exploiting the host cell machinery to repair the genome precisely at the site of the Cas9-generated DSB. Mutations can arise either by non- homologous end joining (NHEJ) or homology-directed repair (HDR) of DSBs. NHEJ can produce small insertions or deletions (INDELs) at the cleavage site, whereas HDR uses a native (or engineered) DNA template to replace the targeted allele with an alternative sequence by recombination. Additional DNA repair pathways such as single-strand annealing, alternative end joining, microhomology-mediated joining, mismatch and base- and nucleotide-excision repair can also produce genome edits.
As used herein, the term “INDEL” relates to insertion or deletion of bases in the genome of an organism generated upon repair after a dsDNA break. A microindel is preferably defined as an indel that results in a net change of 1 to 50 nucleotides.
Cas9, also named Csn1 is a large protein that participates in both crRNA biogenesis and in the destruction of invading DNA. Cas9 has been described in different bacterial species such as S. thermophilus (Sapranauskas, Gasiunas et al. 2011 ), listeria innocua (Gasiunas, Barrangou et al. 2012; Jinek, Chylinski et al. 2012) and S. Pyogenes (Deltcheva, Chylinski et al. 2011 ). The large Cas9 protein (>1200 amino acids) contains two predicted nuclease domains, namely HNH (McrA- like) nuclease domain that is located in the middle of the protein and a splitted RuvC-like nuclease domain (RNase H fold) (Haft, Selengut et al. 2005; Makarova, Grishin et al. 2006). In wild-type Cas9, these two domains result in blunt cleavage of the invasive DNA within the same target sequence (proto-spacer) in the immediate vicinity of the PAM (Jinek, Chylinski et al. 2012).
Cas9 variants derived from the Streptococcus pyogenes Cas9 (SpCas9) have been generated for use as nickases, dual nickases or Fokl fusion variants. More recently, Cas9 orthologs, and other nucleases derived from class 2 CRISPR-Cas systems including Cpf1 and C2c1 , have been added to the CRISPR toolbox. These ongoing efforts to mine the abundant bacterial and archaeal CRISPR-Cas systems should increase the range of molecular tools available to researchers.
A nickase (or nicking enzyme or nicking endonuclease) is an enzyme that cuts one strand of a double-stranded DNA at a specific recognition nucleotide sequences known as a restriction site. Such enzymes hydrolyse (cut) only one strand of the DNA duplex, to produce DNA molecules that are “nicked”, rather than cleaved. Over 200 nicking enzymes have been studied, and 13 of these are available commercially and are routinely used for research and in commercial products. Nickases can be and have been generated by mutating the nuclease domains of Cas9 independently of each other to create DNA nickase capable of introducing a single-strand cut with the same specificity as a regular CRISPR/Cas9 nuclease (Gasiunas G, Barrangou R, Horvath P, Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl Acad. Sci. USA 109, E2579-E2586 (2012). The use of Cas9 nickases is essentially the same as the use of the fully functional enzyme.
Cas9 nickase is created by mutating one of two Cas9 nuclease domains. Cas9 nickase creates a single-strand rather than a double-strand break. For example, the D10A mutation inactivates the RuvC domain, so this nickase cleaves only the target strand. Conversely, the H840 mutation in the HNH domain creates a non-target strand-cleaving nickase. Instead of cutting both strands bluntly with WT Cas9 and one gRNA, a staggered cut using a Cas9 nickase and two gRNAs.
In other words, in RNA guided DNA nickases the naturally occurring endonucleases function of cleaving both strands of a double-stranded target DNA, is altered into an endonuclease that cleaves (i.e. nicks) only one of the strands. Means and methods of modifying RNA guided DNA endonuclease such as Cas9 accordingly are well known in the art, and include for example the introduction of amino acid replacements into Cas9 that render one of the nuclease domains inactive. More specifically, aspartate can be replaced against alanine at position 10 of the Streptococcus pyogenes Cas9 (SpCas9 D10A; Cong et al. (2013) Science 339:819-823). Further examples are known in the art, for example the H840A replacement in SpCas9 (Mali P et al. Nat Biotechnol. 2013 Sep; 31 (9):833-8; Ran FA et al. Cell. 2013 Sep 12; 154(6): 1380-9).
The catalytic residues of the compact SpCas9 protein are those corresponding to amino acids D10, D31 , H840, H868, N882 and N891 or aligned positions using CLUSTALW method on homologues of Cas Family members. Any of these residues can be replaced by any other amino acids, preferably by alanine residue. Mutation in the catalytic residues means either substitution by another amino acids, or deletion or addition of amino acids that induce the inactivation of at least one of the catalytic domain of Cas9. (Sapranauskas, Gasiunas et al. 2011 ; Jinek, Chylinski et al. 2012). In a particular embodiment, Cas9 may comprise one or several of the above mutations. In another particular embodiment, Cas9 may comprise only one of the two RuvC and HNH catalytic domains. In the present invention, Cas9 nickases of different species, Cas9 homologues, Cas9 engineered and functional variant thereof can be used.
In the context of the present invention, the term “RNA guided DNA endonuclease” refers to DNA endonucleases that interact with at least one RNA-Molecule. In the context of the present invention the terms RNA guided DNA endonuclease and RNA guided endonuclease are used interchangeably. DNA endonucleases are enzymes that cleave the phosphodiester bond within a DNA polynucleotide chain. In case of RNA guided DNA endonuclease the interacting RNA- Molecule may guide the RNA guided DNA endonuclease to the site or location in a DNA where the endonuclease becomes active. In particular, the term RNA guided DNA endonuclease refers to naturally occurring or genetically modified Cas nuclease components or CRISPR-Cas systems, which include, without limitation, multi-subunit Cas protein effectors of class 1 CRISPR-Cas systems as well as single large effector Cas proteins of class 2 systems. In the context of the present invention, DNA endonuclease functioning as nickases are used. Accordingly, in the context of the present invention the RNA guided DNA endonuclease is an RNA guided DNA nickase. In the context of the invention, the RNA guiding the DNA nickase to the target site/sequence is a guide RNA.
Details of the technical application of CRISPR/Cas systems and suitable RNA guided endonuclease are known to the skilled person and have been described in detail in the literature, as for example by Barrangou R et al. (Nat Biotechnol. 2016 Sep 8;34(9):933-941 ), Maeder ML et al. (Mol Ther. 2016 Mar;24(3):430-46) and Cebrian-Serrano A et al. (Mamm Genome. 2017; 28(7): 247-261 ). The present invention uses guide RNA-guided DNA nickases, but is not limited to the use of a specific RNA guided nickase and therefore comprises the use of any given RNA guided nickase in the sense of the present invention suitable for use in the method described herein.
Any RNA guided DNA nickase known in the art may be employed in accordance with the present invention. A suitable nickase may be constructed based on an RNA guided DNA endonuclease selected from the group comprising, without limitation, Cas proteins of class 1 CRISPR-Cas systems, such as Cas3, Cas8a, Cas5, Cas8b, Cas8c, Cas10d, Cse1 , Cse2, Csy1 , Csy2, Csy3, GSU0054, Cas10, Csm2, Cmr5, Csx11 , Csx10 and Csf1 ; Cas proteins of class 2 CRISPR-Cas systems, such as Cas9, Csn2, Cas4, Cpf1 , C2c1 , C2c3 and C2c2; corresponding orthologous enzymes/CRISPR effectors from various bacterial and archeal species; engineered CRISPR effectors with for example novel PAM specificities, increased fidelity, such as SpCas9- HF1/eSpCas9, or altered functions. Particularly preferred are nickases based on RNA guided DNA endonuclease selected from the group comprising Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9, Streptococcus thermophilus Cas9, Neisseria meningitidis Cas9 (NmCas9), Francisella novicida Cas9 (FnCas9), Campylobacter jejuni Cas9 (CjCas9), Cas12a (Cpf1 ) and Cas13a (C2C2) (Makarova KS et al. (November 2015). Nature Reviews Microbiology. 13 (11 ): 722-36). IN the context of the invention, the use of a SpCas9 nickase (SpCas9n) is particularly preferred, especially SpCas9nD10A.
The definition and explanations provided herein are mainly focused on the SpCas9 Crispr/Cas system. However, the person skilled in the art is aware of how to use alternative Crispr/Cas systems as well as tools and methods that provide or allow the gain of information on the details of such alternative systems.
In accordance with the method of the invention, the RNA guided DNA nickase may be introduced as a protein, but alternatively the RNA guided DNA nickase may also be introduced in form of a nucleic acid molecule encoding said protein. It will be appreciated that the nucleic acid molecule encodes said RNA guided DNA nickase in expressible form such that expression in the cell results in a functional RNA guided DNA nickase protein such as SpCas9n protein. Means and methods to ensure expression of a functional polypeptide are well known in the art.
For example, the coding sequences for the nickase may be comprised in a vector, such as for example a plasmid, cosmid, virus, bacteriophage or another vector used conventionally e.g. in genetic engineering. The coding sequences inserted in the vector can e.g. be synthesized by standard methods, or isolated from natural sources. The coding sequences may further be ligated to transcriptional regulatory elements and/or to other amino acid encoding sequences. Such regulatory sequences are well known to those skilled in the art and include, without being limiting, regulatory sequences ensuring the initiation of transcription, internal ribosomal entry sites (IRES) and optionally regulatory elements ensuring termination of transcription and stabilization of the transcript. Non-limiting examples for regulatory elements ensuring the initiation of transcription comprise a translation initiation codon, transcriptional enhancers such as e.g. the SV40- enhancer, insulators and/or promoters, such as for example the cytomegalovirus (CMV) promoter, SV40-promoter, RSV-promoter (Rous sarcome virus), the lacZ promoter, chicken betaactin promoter, CAG-promoter (a combination of chicken beta-actin promoter and cytomegalovirus immediate-early enhancer), the gai10 promoter, human elongation factor la- promoter, A0X1 promoter, GAL1 promoter CaM-kinase promoter, the lac, trp or tac promoter, the lacUV5 promoter, the autographa californica multiple nuclear polyhedrosis virus (AcMNPV) polyhedral promoter or a globin intron in mammalian and other animal cells. Non-limiting examples for regulatory elements ensuring transcription termination include the V40-poly-A site, the tk-poly-A site or the SV40, lacZ or AcMNPV polyhedral polyadenylation signals, which are to be included downstream of the nucleic acid sequence of the invention. Additional regulatory elements may include translational enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Moreover, elements such as origin of replication, drug resistance gene or regulators (as part of an inducible promoter) may also be included.
Nucleic acid molecules encoding said RNA guided DNA nickase include DNA, such as cDNA or genomic DNA, as well as RNA and in particular mRNA. It will be readily appreciated by the skilled person that more than one nucleic acid molecule may encode an RNA guided DNA nickase in accordance with the present invention due to the degeneracy of the genetic code. Degeneracy results because a triplet code designates 20 amino acids and a stop codon. Because four bases exist which are utilized to encode genetic information, triplet codons are required to produce at least 21 different codes. The possible e possibilities for bases in triplets give 64 possible codons, meaning that some degeneracy must exist. As result, some amino acids are encoded by more than one triplet, i.e. by up to six. The degeneracy mostly arises from alterations in the third position in a triplet. This means that nucleic acid molecules having different sequences, but still encoding the same RNA guided DNA endonuclease, can be employed in accordance with the present invention.
The nucleic acid molecules used in accordance with the present invention may be of natural as well as of (semi) synthetic origin. Thus, the nucleic acid molecules may, for example, be nucleic acid molecules that have been synthesized according to conventional protocols of organic chemistry. The person skilled in the art is familiar with the preparation and the use of said probes (see, e.g., Sambrook and Russel "Molecular Cloning, A Laboratory Manual", Cold Spring Harbor Laboratory, N.Y. (2001 )).
As used herein “nucleic acid” shall mean any nucleic acid molecule, including, without limitation, DNA, RNA and hybrids or modified variants thereof. An “exogenous nucleic acid” or “exogenous genetic element” relates to any nucleic acid introduced into the cell, which is not a component of the cells “original” or “natural” genome. Exogenous nucleic acids may be integrated or nonintegrated in the genetic material of the target cell or relate to stably transduced nucleic acids.
The nucleic acid molecules used in accordance with the invention may be nucleic acid mimicking molecules known in the art such as synthetic or semi-synthetic derivatives of nucleic acid molecules and mixed polymers. They may contain additional non-natural or derivatized nucleotide bases, as will be readily appreciated by those skilled in the art. Nucleic acid mimicking molecules or nucleic acid derivatives according to the invention include, without being limiting, phosphorothioate nucleic acid, phosphoramidate nucleic acid, morpholino nucleic acid, hexitol nucleic acid (HNA), peptide nucleic acid (PNA) and locked nucleic acid (LNA).
Furthermore, the method of the present invention comprises introducing into the cell at least two guide RNAs. In the context of the present invention, a “guide RNA” refers to RNA molecules interacting with RNA guided DNA endonuclease, such as a nickase, leading to the recognition of the target sequence to be cleaved by the RNA guided DNA endonuclease. According to the present invention, the term “guide RNA” therefore comprises, without limitation, target sequence specific CRISPR RNAs (crRNA), trans-activating crRNAs (tracrRNA) and chimeric single guide RNAs (sgRNA). crRNAs differ depending on the RNA guided endonuclease and the CRISPR/Cas system but typically contain a target specific sequence of between 20 to 72 nucleotides in length, flanked by two direct repeats (DR) of a length of between 21 to 46 nucleotides. In the case of S. pyogenes, the DRs are 36 nucleotides long and the target sequence is 30 nucleotides long. The 3' located DR of the crRNA is complementary to and hybridizes with the corresponding tracr RNA, which in turn binds to the Cas9 protein.
As used herein, the term "trans-activating crRNA (tracr RNA)" refers to a small RNA, that is complementary to and base pairs with a pre-crRNA (3' located DR of the crRNA), thereby forming an RNA duplex. This pre-crRNA is then cleaved by an RNA-specific ribonuclease, to form a crRNA/tracrRNA hybrid, which subsequently acts as a guide for the endonuclease Cas9, which cleaves the invading nucleic acid.
As described herein, the genes encoding the elements of a CRISPR/Cas system, such as for example Cas9, tracrRNA and crRNA, are typically organized in operon(s). DR sequences functioning together with RNA guided endonuclease such as Cas9 proteins of other bacterial species may be identified by bioinformatic analysis of sequence repeats occurring in the respective Crispr/Cas operons and by experimental binding studies of Cas9 protein and tracrRNA together with putative DR sequence flanked target sequences.
Alternatively, a chimeric single guide RNA sequence comprising such a target sequence specific crRNA and tracrRNA may be employed. Such a chimeric (ch) RNA may be designed by the fusion of a target specific sequence of 20 or more nucleotides (nt) with a part or the entire DR sequence (defined as part of a crRNA) with the entire or part of a tracrRNA, as shown by (Jinek et al. Science 337:816-821 ). Within the chimeric RNA a segment of the DR and the tracrRNA sequence are complementary able to hybridize and to form a hairpin structure. IN the context of the invention, the use of sgRNAs as guide RNAs is preferred.
Moreover, the at least two guide RNA of the present invention differing at least with respect to their target specific sequence may also be encoded by a nucleic acid molecule, which is introduced into the cell. The definitions and preferred embodiments recited above with regard to the nucleic acid molecule encoding the nickase equally apply to the nucleic acid molecule encoding these RNAs. Regulatory elements for expressing RNAs are known to one skilled in the art, for example a U6 promoter.
The present invention relates to the generation of single strand beaks of the dsDNA molecule to be modified, wherein the dsDNA molecule comprises at least two target sequences, which are targeted by the at least two guide RNAs. Therein, the first guide RNA targets/hybridizes to a fist target sequence in the dsDNA to be modified, and the second guide RNA hybridizes to a second target sequence in the dsDNA to be modified, preferably with the target specific sequence of the guide RNA.
In accordance with the present invention, a "target sequence" is a nucleotide sequence in the dsDNA molecule that is recognized by the guide RNA that is associated with the RNA guided nickase due to the target specific sequence comprised by the guide RNA. The target sequence is at least partially complementary to the target specific sequence of the guide RNA and is associated with a so-called protospacer adjacent motif (PAM). The PAM is a 2-6 base pair DNA sequence located adjacent to the target sequence and can be located either at the 5’-end (for example for the Crispr/Cpf1 system) or at the 3’-end of the target sequence (for example for the Crispr/Cas9 system), depending on the Crispr/Cas system employed. An RNA guided nickase, such as a Cas9 or Cpf1 based nickase, will not successfully bind to and cleave the targeted dsDNA molecule if the recognized target sequence is not associated with a PAM sequence. The formation of a DNA-RNA heteroduplex between the target sequence and the target specific sequence of the guide RNA with the bound nickase allows for cleavage of the target DNA by this guide RNA/DNA nickase complex. Cleavage of the targeted dsDNA molecule occurs within the target sequence or at a site in close proximity or adjacent to the target sequence, depending on the function of the used RNA guided nickase and CRISPR/Cas system.
For example, in case of SpCas9 the DNA target sequence of at least 20 nucleotides is located directly upstream/at the 5’-end of an invariant 5’-NGG-3’ PAM. Correct pairing of the guide RNA to the DNA target sequence leads to the generation of a cut in the dsDNA molecule (“cleavage” of the dsDNA molecule by SpCas9) 3 base-pairs (bp) upstream of the PAM within the target sequence.
In case of the CRISPR/Cpf1 system, the Cpf1-crRNA complex cleaves target DNA by identification of a target sequence that may be located downstream/at the 3’ end of a protospacer adjacent motif (for example 5'-YTN-3' (where "Y" is a pyrimidine and "N" is any nucleobase) or 5'- TTN-3'). Cpf1 can introduce a sticky end/staggered end DNA double strand breaks. In case of AsCpfl and LbCpfl a double strand break with a 4 nucleotides overhang can be generated, which can occur 19 bp downstream of the PAM on the targeted (+)-strand and 23 bp downstream of the PAM on the (-)-strand. Corresponding nickases function analogously.
As illustrated by the above examples, the exact site of the cut depends on the Crispr/Cas system or the RNA guided endonuclease/nickase employed in the method of the invention and can therefore be determined by the person skilled in the art upon selection of the RNA guided nickase.
In the context of the present invention, a cleavage site in close proximity to the target sequence is located within 100 nucleotides or base pairs upstream or downstream from the 5’- or 3’-end of the target sequence. Preferably, the double strand break is generated within 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2 or 1 nucleotides/base pairs upstream or downstream from the 5’- or 3’-end of the target sequence or within the target sequence.
In the context of the present invention, the target sequence may also be called “protospacer". The term “target site” may refer to a location or sequence in the dsDNA molecule comprising the target sequence and an associated PAM.
In the context of the present invention, the term “double strand break” or “DSB” refers to interruption of both strands of a dsDNA molecule leading to the separation of the parts of the dsDNA molecule that lie upstream and downstream of the side of the double strand break. In contrast, a “single strand break” or “SSB” refers to the interruption of only one of the two DNA strands and will not lead to a separation of the parts of the dsDNA molecule that lie upstream and downstream of the side of the double strand break.
In the context of the present invention, at least two SSBs can occur due to cleavage of both strands of a dsDNA to be modified by an RNA guided nickase. In embodiments, a DSB may occur due to cleavage (introduction of SSBs) by the nickase at the two target sites on the opposing strands of the dsDNA to be modified, i.e. the (+)- and the (-)-strand, despite the spacer/distance between the SSBs.
In the context of the invention, two SSBs are produced intentionally by RNA guided nucleases to achieve modification of the dsDNA through homology directed repair. Furthermore, NHEJ repair should be avoided.
Cellular DNA repair mechanisms that are generally thought to be involved in DSB repair include homology-directed repair (HDR) a-NHEJ and c-NHEJ. While the c-NHEJ pathway operates throughout all phases of the cell cycle, HDR is restricted to the S and G2 phases, seeking for homologous sequences. During mitosis, DSB repair is entirely shut down to guard the chromosomes against the fusion of telomeres. In the G1 (and GO) phase and in resting cells, c- NHEJ repair is dominant as HDR is silenced. All pathways are active and competing in the S/G2 phases. DSB induction in a population of cycling cells leads to a variety of edited alleles, with c- NHEJ as the dominant outcome.
Homology directed repair (HDR) is a mechanism in cells to repair double-strand DNA lesions. The most common form of HDR is homologous recombination. The HDR mechanism can only be used by the cell when there is a homologous piece of DNA present in the nucleus, mostly in G2 and S phase of the cell cycle. Other examples of homology-directed repair include single-strand annealing and breakage-induced replication. When the homologous DNA is absent, non- homologous end joining (NHEJ) takes place instead.
In proliferating cells, HDR is mediated by the homologous recombination (HR) pathway, wherein the native pathway uses the intact homologous sequences of sister chromatids as template for the repair of DSB sites and leads to the reconstitution of the wild-type allele. It has been shown in the art that it is possible to achieve precise sequence modifications at targeted DSBs. Therefore, the HR pathway can be co-opted by providing an exogenous DNA donor template (may alsob e referred to as a repair templates) containing sequence regions homologous the DSB ends. The sequence between the homologous ends, either an insertion or replacement, is then transferred into the targeted locus during HR, enabling the generation of precisely modified ‘knockin’ alleles, e.g., for codon replacements or the insertion of reporter genes. Large sequence insertions can require the use of double-stranded DNA donor templates, such as plasmid-based gene-targeting vectors with homology regions of at least 100 bp, preferably 500 or more bp.
Homology directed repair (HDR) of DNA double strand breaks (DSBs) requires resection of DNA DSB ends. Resection creates 3'-ssDNA overhangs which then anneal with a homologous DNA sequence. This homologous sequence can then be used as a template for DNA repair synthesis that bridges the DSB. HDR preferably occurs through the error-free homologous recombination repair (HRR), but can also occur through the error-prone single strand annealing (SSA), or the least accurate microhomology-mediated end joining (MMEJ). HRR and SSA share the initial steps that involve ATM signalling, formation of the so-called ionizing radiation-induced foci (IRIF), extensive resection of DNA DSB ends and activation of ATR signalling. In homologous recombination, 3'-ssDNA overhangs anneal with complementary sister chromatid strands. In SSA, 3'-ssDNA overhangs anneal with each other through homologous direct repeats contained in each overhang, resulting in deletions of one of the repeats and the DNA sequence in between the repeats during DNA repair synthesis. Contrary to HRR and SSA, which both involve annealing of long stretches of highly homologous DNA sequences, MMEJ entails annealing of short regions of two 3'-ssDNA overhangs (up to 20 nucleotides) and is therefore more promiscuous and more likely to join unrelated DNA molecules. The error rate of MMEJ is additionally increased by the low fidelity of the DNA polymerase theta (POLQ), which performs DNA repair synthesis in MMEJ. For reviews of this topic, please refer to Khanna KK, Nat Genet 2001 ; Thompson LH and Schild D, Mutat Res 2001 ; Thompson LH and Schild D, Mutat Res 2002; Ciccia A and Elledge SJ, Mol. Cell 2010.
By using the method of the present invention, NHEJ comprising c-NHEJ and a-NHEJ, can be avoided.
NHEJ- and HDR-mediated DSB repair are well established processes or mechanisms that are known to the person skilled in the art and have been described by Danner et al. (Mamm Genome 28, 262-274 -). The initiation of DSB repair is identical for both c-NHEJ and HDR. The ATM (ataxia telangiectasia mutated) protein kinase is a key initial regulator of the DNA damage response and coordinates DSB repair. ATM is activated by the MRN (Mre11-Rad50-Nibrin) complex and other factors at DNA breaks. Upon monomerization and autophosphorylation, ATM phosphorylates Serine 139 of histone H2AX, forming yH2AX. The phosphorylated residue on yH2AX is recognized by MDC1 , which in turn recruits more MRN complexes. These further activate ATM and creates a positive feedback loop driving the expansion of yH2AX chromatin domains into yH2AX foci. MDC1 becomes phosphorylated by ATM at its TQXF repeats and initiates downstream signaling by recruiting the E3 ubiquitin ligase RNF8. RNF8 and its E2 enzyme partner UBC13 polyubiquitinate the H1 linker histone. This further promotes the recruitment of the E3 ubiquitin ligase RNF168 that ubiquitinates histone H2A at Lysine 13 and 15. H2A-K15Ub together with dimethylated Lysine 20 of histone H4 (H4K20me2) are chromatin marks for the recruitment of the checkpoint protein 53BP1 . The control of accumulation of 53BP1 determines if the DSB event is repaired by c-NHEJ, or through resection and subsequent HDR. The classical c-NHEJ pathway initiates with the localization of 53BP1 to a DSB and blocks 5' resectioning. 53BP1 blocks CtIP-based resectioning and recruits Rif1 , which further blocks resectioning and inhibits BRCA1 accumulation. Unresected ends allow Ku70/80 to bind, further inhibiting resection. Ku proteins form a scaffold and recruit DNA-PKcs (catalytical subunit) to form the complete DNA-PK, which then recruits endprocessing factors (like Artemis) and the XRCC4/XLF/DNA Ligase-IV complex. The XRCC4/XLF factors stabilize and align the DNA fibers and DNA Ligase IV ligates the two strands. Repair of chemically or irradiation-induced DSBs is greatly complicated by the need to excise and repair damaged bases. However, this will be left out of this review as DSBs from Cas9 nucleases form blunt ends with 5' phosphorylated DNA, the substrate for DNA Ligase IV. The ability to excise damaged bases and then ligate non- complementary strands has resulted in c-NHEJ being often thought of as a mutagenic process. However given a complimentary cut, such as created by restriction enzymes, mutation-free ligation events can be 75% or higher. Error-prone mutations that have previously been attributed to c-NHEJ are often a result of DSB resectioning and annealing through the similarly named but mechanistically distinct a-NHEJ.
Alternative non-homologous end joining pathways (a-)NHEJ encompass microhomology- mediated end joining (MMEJ) and single-strand annealing (SSA). Once thought to only be backup pathways, a-NHEJ events can in some cases occur up to 10% of the frequency of c- NHEJ. These repair events can result in deletions of various sizes, and only sometimes anneal and ligate through microhomologies. However, they always begin with the same resection steps as in homologous recombination, involving the MRE11 complex and CtIP. Resection can be <20 bp for microhomology or up to thousands of bps for SSA. The choice between a-NHEJ and HR comes from the inability of RPA to be replaced by Rad51 by Rad52/BRCA2. This limits the ssDNA to proceed through the HR pathway. Importantly, the extensive resection, when repaired by a-NHEJ, results in an increased chromosomal translocation frequency, a major driver of human cancer.
The homologous recombination pathway requires the exclusion of 53BP1 and resection in order to be initiated. H2A is de-ubiquitinated upon mitotic entry so 53BP1 is excluded from the chromatin. During the S/G2 phase, BRCA1 excludes Rif1 from the foci, and recruits CtIP and the MRN complex. This complex initiates a cleavage step which is then further 5'-resected by Exo1 . The resection extends 2-4 kb on each side of the DSB. The exposed single-stranded DNA (ssDNA) is quickly bound by RPA for protection. RPA is replaced by Rad51 through the action of BRCA2 and Rad52 to form a nucleofilament competent for homology search. The Rad51 filaments maintain the ssDNA in a B-form which has triplets open for Watson-Crick pairing with complementary triplets in homologous duplex DNA. It should be noted that this review highlights only some of the key factors of the HR pathway and more complete reviews are available.
In embodiments of the invention, the DNA template sequence comprises at least one sequence (homology arm), preferably two homology arms, of at least 100 bp, preferably 500 bp, more preferably 700 bp, or 1000 bp or more, with at least 90%, preferably 95%, more preferably 100%, sequence identity to a region within the DNA sequence to be replaced, wherein said homology arm is positioned to hybridize to the dsDNA molecule to be modified in proximity to, adjacent to and/or between the two single strand breaks (SSB). In the context of the present invention, the terms “homology arms” or “homology arms that are targeted to the dsDNA molecule to be modified” refer to regions or sequences of the exogenous nucleic acid molecule that are homologous to the sequences in proximity to the two double strand break ends of the dsDNA molecule to be modified.
Homology arms may have 90 %, preferably 95 %, 97 %, 98 %, 99 % or 100 % sequence identity to the corresponding sequences of the dsDNA molecule to be modified. Homology arms have sufficient sequence identity to ensure specific binding to the target sequence. Methods to evaluate the identity level between two nucleic acid sequences are well known in the art. For example, the sequences can be aligned electronically using suitable computer programs known in the art. Such programs comprise BLAST (Altschul et al. (1990) J. Mol. Biol. 215, 403), variants thereof such as WU-BLAST (Altschul and Gish (1996) Methods Enzymol. 266, 460), FASTA (Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85, 2444) or implementations of the Smith-Waterman algorithm (SSEARCH, Smith and Waterman (1981 ) J. Mol. Biol., 147, 195). These programs, in addition to providing a pairwise sequence alignment, also report the sequence identity level (usually in percent identity) and the probability for the occurrence of the alignment by chance (P-value). In accordance with the present invention, it is preferred that BLAST is used to determine the identify level between two nucleic acid sequences.
As used herein the terms “exogenous DNA donor template comprising a DNA template sequence” refers to an exogenous DNA molecule that is introduced into the cell and that comprises a sequence to be integrated into the dsDNA molecule to be modified (the DNA template sequence). In the context of the present invention, the template sequence replaces a DNA sequence of the dsDNA molecule to be modified that is in proximity to, adjacent to and/or between the two SSB introduced into the dsDNA molecule. In preferred embodiments, the sequence to be replaced is a mutated sequence resulting in a functionally impaired or inactive gene product encoded by the dsDNA molecule to be modified, and the DNA template sequence comprises a corrected/functional version of the respective sequence, so that replacement of the mutated sequence by the template sequence leads to expression of a functional gene product by the modified dsDNA molecule. In embodiments, the DNA template sequence can comprise a sequence endogenous and/or exogenous to the cell. Examples of a sequence to be integrated include DNA sequences encoding a protein or a non-coding RNA (e.g., a microRNA). In embodiments, the template sequence can comprise a coding sequence that is operably linked to a regulatory sequence for controlling gene expression, such as a promoter or promoter/enhancer sequence sequences. In embodiments, a reporter gene or reporter cassette can be integrated into the dsDNA to be modified by replacing a DNA sequence in proximity to, adjacent to and/or between the two SSBs.
Sequences that are homologous to the homology arms of the DNA donor template can be sequence located upstream and downstream of the sequence to be replaced. It can be understood that the homologous sequences are comprised by the DNA sequence to be replaced. In embodiments, the upstream homology arm is a nucleic acid sequence that shares sequence similarity with the genome sequence (sequence of the dsDNA to be modified) upstream of the targeted site for integration or the replacement sequence. Similarly, the downstream sequence is a nucleic acid sequence that shares sequence similarity with the chromosomal sequence downstream of the targeted site of integration. For example, in case of replacement of an exon comprising one or more mutations, the upstream and downstream sequences homologous to the homology arms of the donor template can be the flanking intron sequences.
In embodiments, the homology arms in the exogenous polynucleotide template can have 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the upstream and downstream sequences of the targeted genome sequence. Preferably, the sequence identity is about 95%, 96%, 97%, 98%, 99%, or 100%.
In embodiments, the homology arms may comprise from about 20 bp to about 2500 bp, for example, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 bp. In some preferred embodiments, the homology arms are about 200 bp to about 2000 bp, about 600 bp to about 1000 bp, or more particularly about 700 bp to about 1000 bp.
In some methods, the exogenous DNA template sequence may further comprise a marker. Such a marker may make it easy to screen for targeted integrations. Examples of suitable markers include restriction sites, fluorescent proteins, or selectable markers, such as (antibiotic) resistance genes. The exogenous DNA donor template of the invention can be constructed using recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996). In contrast to HDR, the c-NHEJ pathway mediates the re-ligation of DSB ends without the involvement of a repair template. Although some fraction of c-NHEJ repair events result in precisely reconstituted wild-type sequences, a fraction of cleaved sequences gains a random insertion or deletion of one or more nucleotides (INDELs). Therefore, DSB repair by the supposedly error-prone c-NHEJ pathway is frequently used to generate INDELs within coding regions, which often will cause a frameshift knockout mutation.
The method of the invention comprises introducing into the cell an exogenous DNA donor template comprising a DNA substitute sequence. The exogenous DNA donor template is an exogenous DNA molecule, which is not a component of the cells “original” or “natural” DNA composition, in particular its genome. The DNA donor template comprises or consists of the DNA template sequence.
In the context of the present invention, the “DNA template sequence”, which may also be called “DNA substitute sequence” or “replacement sequence”, is a DNA sequence that is introduced into the dsDNA molecule to be modified replacing a DNA sequence of the dsDNA to be modified that is positioned in proximity to, such as adjacent to and/or between the two single strand breaks (SSB).
The exogenous DNA donor template comprising the template sequence can be introduced into the cell in various forms. In embodiments, the exogenous DNA donor template is comprised by a viral vector, such as an adeno-associated virus (AAV), that is introduced into the cell. In embodiments, the exogenous DNA donor template is comprised by or consists of a circular or linear DNA molecule, preferably a plasmid or a mini-circle, or a single stranded DNA molecule.
In preferred embodiments, the exogenous DNA donor template consists of the DNA substitute sequence. This is possible, for example, if the exogenous DNA donor template is a linear dsDNA molecule, such as for example a PCR amplification product, or a DNA mini-circle. A mini-circle is a plasmid like circular DNA that has had all other parts except the sequence of interest removed. Thus, a single cut can linearize a fragment that is then ready to integrate.
In certain embodiments, the exogenous DNA donor template can be introduced into the cell through delivery of a nucleic acid encoding for the DNA substitute sequence, for example by means of viral delivery through Adeno-associated viruses (AAV), retroviruses, non-integrating or integrating lentiviruses. Genetically modified viruses have been widely applied for the delivery of genes into cells. A viral vector may be employed in the context of the present invention.
Non-viral methods may also be employed, such as alternative strategies that include conventional plasmid and nucleic acid transfer and delivery. Physical methods to introduce vectors and nucleic acid molecules or proteins into cells are known to a skilled person. One example relates to electroporation, which relies on the use of brief, high voltage electric pulses, which create transient pores in the membrane by overcoming its capacitance. One advantage of this method is that it can be utilized for both stable and transient gene expression in most cell types. Furthermore, gold nanoparticles for delivery of PCR-like double strand DNA products attached to the gold nanoparticle can be used. Alternative methods relate to the use of liposomes or protein transduction domains. Appropriate methods are known to a skilled person and are not intended as limiting embodiments of the present invention.
The ability to manipulate any genomic sequence by gene editing has created diverse opportunities to treating many different diseases and disorders (For a review see Maeder et al, Molecular Therapy, 24:3, 2016, 430-446). Of relevance to the present invention is the opportunity of correcting deficient gene sequences in vivo. The present invention enables gene therapy for correcting pathogenic gene sequences by DNA template sequence according to the method described herein. For example, hematologic disorders associated with genetic defects could be treated by gene correction in hematopoetic stem cells or hematopoetic stem and progenitor cells. Of further relevance is the treatment of liver disease, by liver-targeted gene editing and the treatment of muscle disease by gene correction in muscle stem cells. Respiratory disorders may be treated, for example, cystic fibrosis, which is caused by mutations to the CFTR chloride channel. Gene editing according to the present invention may be employed to repair the CFTR mutations in patient lung cells. Antimicrobials are another potential therapeutic target of the present invention, i.e. by targeting the genomes of other organisms, for example bacterial genes could be replaced by the method described herein.
In embodiments, the present invention encompasses administration of the cells of the present invention to a subject. As used herein, "administration" or "administering" shall include, without limitation, introducing the cells into the blood circulation of a subject, for example by intravenous administration. Such administering can also be performed, for example, once, a plurality of times, and/or over one or more extended periods. A single administration is preferred, but repeated administrations over time (e.g., hourly, daily, weekly, monthly, quarterly, half-yearly or yearly) may be necessary in some instances. Such administering is also preferably performed using an admixture and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known to those skilled in the art.
In embodiments, the eucaryotic cell comprising the dsDNA to be modified is a hematopoietic stem cell (HSCs). As used herein, the term HSC comprises hematopoietic stem and progenitor cells (HSPCs), including not terminally differentiated progenitor cells originating from HSCs. Hematopoietic stem cells (HSCs) have the ability to repopulate an entire hematopoietic system, and several genetic and acquired diseases of the blood could potentially be cured by genome editing of HSCs. It has been demonstrated efficient targeted integration in HSCs by combining zinc-finger nuclease (ZFN) expression with exogenous homologous recombination donors delivered via single-stranded oligonucleotides, integrase-defective lentiviral vectors, or recombinant adeno-associated viral vectors of serotype 6 (rAAV6). Targeting HSCs by homologous recombination at disease-causing loci is difficult in clinically relevant HSPCs.
In embodiments, the method of the invention can be used to modify the HBB gene. “HBB” which is hemoglobin subunit beta and is involved in oxygen transport from the lung to various peripheral tissues. Approximate 80% of known mutations are located in exon 1 and 2 of the HBB gene locus which cause beta-thalassemia, methemoglobinemia, sickle cell disease, etc. Nearly 400 mutations in the HBB gene have been found to cause beta thalassemia. Most of the mutations involve a change in a single DNA building block (nucleotide) within or near the HBB gene. Other mutations insert or delete a small number of nucleotides in the HBB gene. HBB gene mutations that decrease beta-globin production result in a condition called beta-plus (P+) thalassemia. Mutations that prevent cells from producing any beta-globin result in beta-zero (£°) thalassemia. Problems with the subunits that make up hemoglobin, including low levels of beta-globin, reduce or eliminate the production of this molecule. A lack of hemoglobin disrupts the normal development of red blood cells. A shortage of mature red blood cells can reduce the amount of oxygen that is delivered to tissues to below what is needed to satisfy the body's energy needs. A lack of oxygen in the body's tissues can lead to poor growth, organ damage, and other health problems associated with beta thalassemia.
In embodiments, the method of the invention can be used to modify the ELANE gene. “ELANE” is the gene coding for neutrophil elastase, which modifies the functions of the natural killer cells, monocytes and granulocytes. Mutations in ELANE locus cause Severe Congenital Neutropenia (SCN). Approximate 60% known mutations are located in exon 4 and 5 of ELANE locus.
As used herein “off-target effect” is used synonymously with “off-target mutation” and shall mean a nonspecific and unintended genetic modification that arise through Crispr-Cas9 endonuclease complex, in the present case involving a nickase. If the complex binds to other sequences than the target sequence, which is often a result of homologous sequences and/or mismatch tolerance, it will cleave at these non-target sites (=off-target site) and cause non-specific genetic modifications. Off-target effects comprise unintended point mutations, deletions, insertions, inversion and translocations.
As used herein an “unwanted on-target effect” is used synonymously with “unwanted on-target mutation” and shall mean a nonspecific and unintended genetic modification that arise through Crispr-Cas9 endonuclease (here nickase) complex at the target site (comprising the target sequence and the intended SSB) and that is unintended and not due to the DNA template sequence.
As used herein “unwanted” shall mean the effect of an off-target or on-target was unforeseen and not predicted and is potentially damaging. The effect was or does not fulfill the aim of planed gene editing. As used herein “GUIDE-seq method” refers to an approach to find off-target mutations due to nuclease activity. GUIDE-seq or Genome Wide Unbiased Identification of DSBs Enabled by Sequencing is based on the incorporation of double stranded oligodeoxynucleotides (dsODN) into DSBs via NHEJ. Its amplification is followed by sequencing. Since two primers will be used to sequence the dsODNs, the regions flanking the DSB along with the DSB will be amplified, allowing mapping the off-target mutation. This technique has been applied to identify all previously known off-target sites as well as new ones with frequencies as low as 0.03%. Just like BLESS, however, GUIDE-seq can only detect DSBs present at the time of study.
As used herein “single-stranded DNA oligodeoxynucleotides (ssODN)” shall be delivered to the cell wherein ssODN acts as a template for homology directed repair of the double strand break and integrated into the locus of interest.
As used herein “sg RNA offset” is defined as distance between two guides sequence which is defined as the distance between the PAM-distal (5’) ends of a given sgRNA pair.
As used herein “spacer-nick sgRNAs” shall mean a pair of sgRNAs involved in the CRISPR/Cas9 nickase complex which generate the distance equal to or greater than 202 bp between two single strand breaks.
FIGURES
The following figures are presented to describe particular embodiments of the invention, without being limiting in scope.
Brief description of the Figures:
Figure 1. Gene insertion in human HSPCs and T cells.
Figure 2. Spacer-nick-mediated gene correction at the targeted HBB, ELANE, IL7R and PRF1 loci in human HSPCs.
Figure 3. Genome-wide off-target mutations identified by GUIDE-seq and Amplicon-seq.
Figure 4. AAV integrations and genetic alterations captured by LAM-HTGTS.
Figure 5. Gene targeting at the B2M and CD48 loci in human HPSCs and T cells.
Figure 6. Efficiencies of gene correction at the targeted loci in human HSPCs.
Figure 7. HBB gene correction in human T cells.
Figure 8. Sanger sequencing data of the targeted loci in human HSPCs.
Figure 9. Nextera Tn5-mediated GUIDE-seq and AAV-seq.
Figure 10. Pipelines analyze GUIDE-seq, AAV-seq and LAM-HTGTS data.
Figure 11 . On/off-target sites were mapped on human genome.
Figure 12. Genome-wide AAV integration sites. Figure 13. The spacer-nick system led to precise and efficient HDR with minimal NHEJ in longterm HSCs.
Figure 14. Gene insertion into the B2M locus in long-term HSCs.
Detailed description of the Figures:
Figure 1. Gene insertion in human HSPCs and T cells.
Targeting strategy to insert T2A-mCherry into human B2M (a) and CD48 (b) loci. Common sgB2M-1 and sgCD48-2 are indicated in red arrows; PAM-out sgB2M-47 and sgCD48-59 are indicated in dark blue arrows; and spacer-nick sgB2M-220, -346 and sg-459, and sgCD48-200, - 229, -264, -346 and -427 are in light blue arrows. Spacer distances of double-nick or spacer-nick sgRNAs are indicated. Primers used to amplify the WT/NHEJ and HDR sequences are indicated. FACS analysis shows the percentages of mCherry+ HSPCs and T cells targeting the B2M (c) and CD48 (d) loci 3 days post editing. The bar graphs summarize the frequencies of mCherry+ HSPCs and T cells targeting the B2M (e) and CD48 (f) loci from three independent experiments. Data are shown as means ± SD. The pie charts show frequencies of WT (grey), NHEJ (blue) and HDR (orange) sequences in the targeted HSPCs that received the indicated RNPs and AAV donor vectors, targeting the B2M (g) and CD48 (f) loci. Data are shown as means ± SD from three independent experiments.
Figure 2. Spacer-nick-mediated gene correction at the targeted HBB, ELANE, IL7R and PRF1 loci in human HSPCs.
(a) Strategy to repair known mutations occurred in the exons 1 and 2 of the HBB, the exons 4 and 5 of the ELANE, IL7R (modified cDNA), and PRF1 (modified cDNA) genes using the spacernick approach. A pair of spacer-nick sgRNAs targeting the HBB (sg-e1-1 and sg-e2-1 ), ELANE (sg-e4-1 and sg-e5-2), IL7R (sg-5’-2 and sg-e1-2) and PRF1 (sg-e2-1 and sg-ln2-1 ) loci, and their spacer distances are indicated. Donor templates with indicated length of homology arms (HAs) include a Sall recognition site in the cases of HBB and ELANE or modified cDNA sequences in the cases of IL7R and PRF1. Primers used to amplify the targeted alleles are depicted, (b) The bar graphs show gene correction efficiencies (HDR) quantified by Sall-mediated RFLP (in the cases of HBB and ELANE) or correct integration PCR (in the cases of IL7R and PRF1) assays in the targeted HSPCs treated with the indicated sgRNAs/Cas9 or sgRNAs/Cas9n and AAV donor vectors, (c) The bar graphs show frequencies of WT (grey), NHEJ (blue) and HDR (orange) events by Sanger sequencing in the targeted HBB, ELANE, IL7R and PRF1 genes in human HSPCs that received either sgRNAs/Cas9 (Cas9) or spacer-nick (Cas9n) RNPs and AAV donor vectors. Data are shown as means ± SD and based on three independent experiments, (d) The bar graphs show the ratio of HDR:NHEJ events shown in (c), at the targeted HBB, ELANE, IL7R and PRF1, respectiviely, in human HSPCs treated with either sgRNAs/Cas9 (Cas9) or sgRNAs/Cas9n (Cas9n) RNPs and AAV donor vectors. Data are shown as means ± SD and based on three independent experiments, *** P<0.001.
Figure 3. Genome-wide off-target mutations identified by GUIDE-seq and Amplicon-seq. The potential protospacer sequence is shown in the top line with mismatches to the on-target site shown and termed (OT-1 to -15). The on-target site is marked with a green square. The number of GUIDE-seq reads are shown on the right of each site. Data are shown for the sgRNAs/Cas9 (Cas9) and spacer-nick (Cas9n) targeting at the following HBB (a) (sgRNAs according to SEQ ID NO: 14 and 21 ), ELANE (b)(sgRNAs according to SEQ ID NO: 26 and 34), IL7R (c)(sgRNAs according to SEQ ID NO: 38 and 44) and PRF1 (d)(sgRNAs according to SEQ ID NO: 46 and 49) sites, (e) The bar graphs show frequencies of Indel reads quantified by Amplicon-seq at on-/off- target sites identified by GUIDE-seq in untreated HSPCs (Ctrl-1 , black), HSPCs treated with dsODN only (Ctrl-2, grey), sgRNAs/Cas9 (Cas9, red) or sgRNAs/Cas9n (Cas9n, blue) RNPs. Data are shown as means ± SD and based on three independent experiments, (f) Heatmaps show number of dsODN tag integrations on on-/off-target sites in treated HSPCs as indicated. Data are represented from three independent experiments
Figure 4. AAV integrations and genetic alterations captured by LAM-HTGTS.
(a) AAV-seq read counts and number of AAV intergration sites are identified and mapped on human chromosomes of HSPCs treated infected with sgRNAs/Cas9 (upper) or sgRNAs/Cas9n (below) RNPs targeting the HBB locus, and AAV-HBB donor vectors. The top hits of AAV integrations are indicated in red including the on-target site of sgHBB-1 and sgHBB-2 and off- target sites (OT-1 , OT-2 and OT-6) identified by GUIDE-seq. Random AAV integration sites (in black) are detected and mapped throughout human chromosomes, (b) The scheme of LAM- HTGTS assay. Red circle depicts biotin, orange rectangle - streptavidin, (c) Scheme shows gene repair approach including spacer distance of spacer-nick sgRNAs, DNA template, length of HAs. All possible outcomes of genetic alterations induced by gene editing apporaches including small indels, deletion, AAV integration, inversion, HDR, and translocation are indicated. LAM-PCR were amplified using an external biotinylated primer annealing outside of 5’ or 3’ HAs and subsequently used a nested primer nearby the first cleavage site (5’-1 or 3’-1 ). (d) Schematic representation of sgRNA cleavage sites and HDR donor for LAM-HTGTS-profiled genes: HBB, ELANE, and PRF1. (e) Dotplot showing HDR efficiency in AAV-only (grey), Cas9 (red), and Cas9n (blue) treated HSPCs. (f) Dotplot showing the relative fidelity of the methods: the ratio between an intact sequence (WT) and any mutated sequence in the HSPCs treated with AAV only (grey), Cas9 (red) and Cas9n (blue) in combination with AAV donor vectors, (g) Heatmaps demonstrating the distribution between all outcomes outlined in (c) for both 5’ and 3’-enrichtments at the targeted HBB and ELANE loci. 5’-enrichment in PRF1 is invalid for the analysis of cleavage outcomes due to the distance to the closest sgRNA target site.
Figure 5. Gene targeting at the B2M and CD48 loci in human HPSCs and T cells.
(a) Experimental scheme of gene insertion in human HSPCs and T cells which were electroporated with RNPs and subsequently infected with AAV donor vectors. Correct integration PCR showing HDR and WT/NHEJ sequences at the targeted B2M (b) and CD48 (c) loci in human HSPCs (upper) and T cells (below) that were treated as indicated, (d) The bar graphs show frequencies of NHEJ (blue) and wild-type (WT, grey) events at the targeted B2M (left) and CD48 (right) loci in human HSPCs treated with Cas9, double (d)-nick and spacer-nick RNPs. Data are shown as means ± SD from three independent experiments.
Figure 6. Efficiencies of gene correction at the targeted loci in human HSPCs. Sall-mediated RFLP assays showing the gene correction efficiency of the targeted HBB (a) and ELANE (b) loci in the HSPCs treated as indicated. Asterisks indicate the Sall-cleaved bands. Correct integration PCR showing WT/NHEJ and HDR events at the targeted IL7R (c) and PRF1 (d) loci in the HSPCs treated as indicated.
Figure 7. HBB gene correction in human T cells.
(a) Sall-mediated RFLP assay showing efficiency of HBB gene correction in human T cells treated with two sgRNAs and either Cas9 or Cas9n RNPs, and AAV-HBB donor vector. Asterisks indicate the Sall-cleaved bands, (b) Quantification of HDR efficiency in T cells shown in (a), (c) The bar graph shows frequencies of WT (grey), NHEJ (blue) and HDR (orange) sequences at the targeted HBB gene in human T cells, (d) The bar graph shows ratio of HDR/NHEJ in the targeted HBB locus. Data represent means ± SD from three independent experiments.
Figure 8. Sanger sequencing data of the targeted loci in human HSPCs.
Representative sequences of WT (black), NHEJ (blue) and HDR (orange) at the targeted HBB (a), ELANE (b), IL7R (c) and PRF1 (d) in human HSPCs that received either sgRNA(s)/Cas9 or sgRNAs/Cas9n RNPs, and AAV donor vectors.
Figure 9. Nextera Tn5-mediated GUIDE-seq and AAV-seq.
(a) Experimental scheme of GUIDE-seq and Amplicon-seq in the targeted HSPCs. (b) Workflow of Tn5-mediated GUIDE-seq and AAV-seq methods, (c) Optimization of DNA tagmentation with different amounts of Nextera Tn5 transposase (TDE1 ) in 20 pl reaction with 50 or 100 ng of gDNA. The expected size of tagmented DNA fragments is 0.5-1 .5 kb (white lines), (d) Mapping of sequencing reads identified on and off-target DSB positions. Sequencing reads on both sides (5’ and 3’) of DSBs were mapped within 5.0 kb distance.
Figure 10. Pipelines analyze GUIDE-seq, AAV-seq and LAM-HTGTS data.
Briefly, for GUIDE-seq and AAV-seq analysis samples were demultiplexed, reads were checked for correct priming and the sequences of AAV inverted terminal repeats (ITRs) and dsODN were trimmed to adjacent genomic sequences. The adjacent genomic sequences are globally mapped to human genome reference (hg38) using Bowtie2. Mapped reads that were overlapped the off- target sites, predicted by CRISPOR and CrisprRgold programs were quantified. For LAM-HTGTS analysis, samples were demultiplexed, checked for correct priming and trimmed to genome interface. The trimmed reads were aligned to sequences of the AAV ITRs for quantifying AAV integrations. The reads that were not aligned to AAV ITRs, were globally aligned to human genome for quantifying wild-type, indel, deletion and inversion, HDR and translocation events.
Figure 11. On/off-target sites were mapped on human genome.
Chromosome ideograms show the distribution of on/off-target sites identified by GUIDE-seq for CRISPR/Cas9 (left) or spacer-nick (right) that target human HBB. On-target sites are marked in green, off-target sites are in red and red triangles indicate off-target sites predicted by CrispRGold.
Figure 12. Genome-wide AAV integration sites. (a) Experimental scheme of AAV-seq and LAM-HTGTS methods. Genome-wide distribution of AAV integration sites in the HSPCs received only AAV-HBB donor vectors (b), AAV donor vectors and, either sg-e1-1/sg-e2-1/Cas9 (c) or sg-e1-1/sg-e2-1/Cas9n (d) RNPs that target the human HBB locus. AAV integrations mapped on HBB on/off-target sites (on-target, OT-1 , OT-2 and OT- 6) identified GUIDE-seq are highlighted in red.
Figure 13. The spacer-nick system leads to precise and efficient HDR with minimal NHEJ in long-term HSCs.
(A and B) Similar percentages of the HSC, MPP1 and MPP2 subsets in control (Ctrl, no RNPs), Cas9, double-nick or spacer-nick-treated cells were detected three days post targeting the B2M locus. (C and D) Based on mCherry expression, Cas9, the double-nick and spacer-nick approaches led to similar HDR efficiencies (-40%) within the HSC, MPP1 and MPP2 subsets. (E, F, G, H and I) the Cas9 and double-nick approaches led to NHEJ frequencies of -42% (B2M) and 36% (HBB), whereas the spacer-nick gene correction approach led to a more than 20-fold decrease in NHEJ frequencies (-1.6%).
Figure 14. Gene insertion into the B2M locus in long-term HSCs.
(A) Gating strategy for CD34+ CD38- population after three days targeting the B2M locus in human CD34+ cells. (B) Frequencies of CD34+ CD38- HSPCs treated with control (Ctrl, no RNPs), Cas9, double-nick or spacer-nick RNPs, and AAV donor vectors. Data are shown as means ± SD from three independent experiments. (C) FACS analysis, pre-gated on CD34+ CD38- cells, showing the co-expression of CD90 and EPCR markers in long-term HSCs. (D) Correct integration PCR showing WT/NHEJ and HDR events at the targeted B2M locus in the sorted CD34+ CD38- CD45RA-CD90+ EPCR+ HSCs.
EXAMPLES
An ideal gene correction approach for therapeutic applications preserves HDR efficiency and, event more important, minimizes adverse effects, such as on- and off-target mutations. To achieve this goal, we developed an approach that combines Cas9n with a pair of PAM-out sgRNAs at a long spacer distance > 200 bps (termed spacer-nick sgRNAs). Cas9n is guided by 2 sgRNAs to two regions on opposite strands and nicks both DNA strands at distances > 200 bps, termed spacer-nick approach hereafter. Here, we optimize crucial parameters for the spacer-nick approach that facilitates efficient HDR events while minimizing NHEJ events together with AAV6 as a donor template, demonstrate an universal gene correction at the targeted HBB, ELANE, IL7R and PRF1 loci using spacer-nick system, and compare the specificity of classical CRISPR/Cas9 and spacer-nick approaches in primary human HSPCs and T cells.
Figure imgf000039_0001
CRISPR/Cas9-mediated gene correction in autologous hematopoietic stem and progenitor cells (HSPCs) and T cells is an excellent tool for gene therapy. However, the potential off-target activity of this system is a major concern for therapeutic applications 1’9. Here, we develop a high- fidelity “spacer-nick” gene correction approach that combines the Cas9D10A nickase with a pair of PAM-out sgRNAs at a distance of 200-350bp. Together with adeno-associated virus (AAV) 6 to deliver donor template, this approach leads to efficient HDR, while robustly minimizing on- and off-target effects. Using this approach, we achieve efficient HDR and minimal NHEJ events at the B2M and CD48 loci in human HSPCs and T cells. Furthermore, we develop spacer-nick-based universal gene correction methods to repair known mutations occurring in the HBB, ELANE, IL7R and PRF1 genes. We achieve gene correction efficiencies of 20-50% at the targeted loci with minimal unintended on-target mutations in human HSPCs and T cells. Within the scope of the off- target assessment, we discover unintended genetic alterations induced by classical CRISPR/Cas9 that are significantly reduced in the spacer-nick-treated HSPCs. Thus, the spacernick gene correction approach is safer than the classical CRISPR/Cas9-based methods and suitable for gene therapy.
Example 1:
To quantitatively determine HDR and NHEJ efficiencies in primary human HSPCs and T cells, we designed a targeting reporter system by inserting the coding sequences of a self-cleavage peptide coupled to the fluorescent marker mCherry in-frame into the last exon of the human B2M and CD48 loci. We then designed sgRNAs targeting the B2M or CD48 genes nearby the STOP codon, termed sgB2M-1 or sgCD48-2 hereafter, respectively (Fig. 1a, b). For the double-nick approach, we designed a PAM-out sgRNA with a distance of 47 bp to sgB2M-1 (sgB2M-47) or of 59 bp to sgCD48-2 (sgCD48-59), respectively. To determine the optimal spacer distances of a pair of spacer-nick sgRNAs in the spacer-nick approach for inducing efficient HDR while minimizing NHEJ events, we designed several PAM-out sgRNAs with a spacer distance of 220, 346 and 459 bp to sgB2M-1 , or of 200, 229, 264, 346 and 427 bp to sgCD48-2, respectively (Fig. 1a, b). We then assessed the editing activities of these sgRNAs by electroporating RNPs containing Cas9 nuclease and sgRNAs into activated human HSPCs (Fig. 5a). ICE sequencing analysis and T7EI assays of the targeted sequences showed that all sgRNAs led to efficient gene editing of up to -80% (Table 1 ).
To quantify HDR efficiencies, we generated B2M and CD48 donor templates including T2A- mCherry fragment flanked by 1 .3 kb of 5’ and 0.7 kb of 3’ homology arms (HAs) (Fig 1 a, b). Next, human activated CD34+ HSPCs and T cells were electroporated with RNPs containing Cas9 and 2 sgRNAs, or Cas9n with a pair of double-nick sgRNAs, or Cas9n with a pair of spacer-nick sgRNAs. Subsequently, the electroporated cells were infected with AAV6 carrying the B2M (AAV6-B2M-mCherry) or CD48 (AAV6-CD48-mCherry) donor templates (Fig. 5a). Three days post targeting, the edited HSPCs and T cells were analyzed by flow cytometry to measure HDR efficiencies. The double-nick approach led to 61 % and 66%, or 12% and 20% of HDR events at the B2M or CD48 locus in human HSPCs and T cells, respectively. Similar HDR frequencies were present in the cells treated with Cas9, 2 sgRNAs and AAV donor vectors (Fig. 1c-f). In combination with AAV6 donor vectors, the spacer-nick approach created 44% and 31 % (B2M, sgB2M-1 and sgB2M-220), or 13% and 20% (CD48, sgCD48-2 and sgCD48-200) of HDR events in in both human HSPCs and T cells, respectively. Thus, increasing spacer distances of spacernick sgRNAs resulted in functional HDR with reduced efficiency at the B2M and CD48 loci in human HSPCs and T cells (Fig. 1 c-f).
To genetically confirm HDR and NHEJ sequences at the targeted loci, we amplified and sequenced the targeted sequences from genomic DNA of the HSPCs and T cells treated with either RNPs only or RNPs and AAV6 donor vectors, using external primers annealing to a genomic sequence outside of the 5’ and 3’ HAs (Fig. 1a, b, Fig. 5b, c and Table 3). The doublenick and Cas9 approaches led to efficient NHEJ of ~90 (B2M) and 40% (CD48), whereas the spacer-nick approach led to low NHEJ frequencies (< 3% at the B2M and < 1 % at the CD48) in the treated HSPCs (Fig. 1g, h and Fig. 5d). Sanger sequencing analysis confirmed that approximately 40% and 56% (in the B2M) or 16% and 13% (in the CD48) of HDR sequences were detected in the HSPCs that had received either Cas9 with both sgRNAs or Cas9n with a pair of double-nick sgRNAs, and AAV6 donor vectors, respectively. Together with AAV6 donor vectors, the spacer-nick approach mediated efficient HDR -43% (in the case of B2M, with sgB2M-1 and sgB2M-220) and 14% (in the case of CD48, with sgCD48-2 and sgCD48-200) in the targeted HSPCs. Increasing spacer distances of spacer-nick sgRNAs led to a significant reduction of HDR in both the human B2M and CD48 loci (Fig. 1g, h). Thus, the spacer-nick system with a spacer distance of 200-350 bps led to efficient HDR while minimizing NHEJ frequency in human HSPCs and T cells.
Example 2
Next, we developed the spacer-nick approach to repair known mutations at the HBB, ELANE, IL7R and PRF1 loci that cause severe beta-thalassemia, severe congenital neutropenia, severe combined immune deficiency and familial hemophagocytic lymphohistiocytosis, respectively. Approximately 80% of known mutations are located in exons 1 and 2 of the HBB locus, while -60% of known mutations occurred in exons 4 and 5 of the ELANE locus 29’30. Using the spacernick approach, we developed a universal gene correction method to repair these mutation hotspots (Fig. 2a). In the cases of IL7R and PRF1 loci, mutations are randomly distributed in coding and intronic sequences 31 32. We therefore decided to insert modified cDNA sequences into the IL7R and PRF1 loci using the spacer-nick approach (Fig. 2a). Next, we designed several sgRNAs targeting both strands of the HBB, ELANE, IL7R, and PRF1 loci. ICE analysis and T7EI assays showed that these sgRNAs led to efficient gene editing in human HSPCs (Table 1 ). For HBB gene correction, we here used a pair of spacer-nick sgRNAs including a previously published sgRNA (sg-e1 -1 , targeting exon 1 ) 13 and a spacer-nick sgRNA (sg-e2-1 ), targeting exon 2 with a spacer distance of 257 bp (Fig. 2a). For the ELANE gene correction, we selected a spacer-nick sgRNA (sg-e5-2), targeting exon 5, with a spacer distance of 274 bp to our previously published sgRNA targeting exon 4 (sg-e4-1 ) 33 (Fig. 2a). In the case of the IL7R gene correction, we selected a pair of spacer-nick sgRNAs (sg-5’-2 targeting 5’ upstream region and sg-e1-2 targeting exon 1 ), with a spacer distance of 273 bp (Fig. 2a). In the case of the PRF1 gene correction, we used a pair of spacer-nick sgRNAs (sg-e2-1 targeting exon 2 and sg-in2-1 targeting intron 2), with a spacer distance of 223 bp (Fig. 2a). To correct HBB and ELANE mutation hotspots, we generated DNA donor templates including silent mutations and a diagnostic Sall restriction enzyme that enabled us to quantify HDR efficiencies (Fig. 2a). To repair the known mutations occurring in coding and intronic sequences of the IL7R or PRF1 gene, we generated donor templates haboring modified cDNA and poly-A sequences (Fig. 2a).
To assess the gene correction efficiencies using the spacer-nick system in human HSPCs and T cells, the activated HSPCs and T cells were electroporated with RNPs containing Cas9n or Cas9 with a pair of spacer-nick sgRNAs and subsequently infected with AAV6 carrying the donor templates. In the case of Cas9 and ELANE, only one sgRNA was used (sg-e4-1 ). Three days post targeting, gene correction efficiencies were assessed by Sall-mediated restriction fragment length polymorphism (RFLP) assay (HBB and ELANE) or by correct integration PCR (IL7R and PRF1), using primer sets binding to external HAs and modified cDNA sequences as shown in Fig. 2a (Table 3). The spacer-nick system led to -20-50% of gene correction efficiencies at the targeted HBB, ELANE, IL7R, and PRF1 loci in both human HSPCs and T cells, comparable to the efficiencies obtained by classical CRISPR/Cas9 mutagenesis (Fig. 2b, Fig. 6a-d and Fig. 7a, b).
To quantify NHEJ and HDR events on the targeted sequences, the target PCR products were cloned and sequenced. Sanger sequencing analysis revealed that both the spacer-nick and CRISPR/Cas9 systems led to efficient HDR at the targeted HBB (-40%), ELANE (-20%), IL7R (-30%), and PRF1 (-50%) loci in human HSPCs and T cells (Fig. 2c and Fig. 7c). While the classical CRISPR/Cas9 system caused frequent NHEJ events at high frequency at the targeted HBB (-60%), ELANE (-30%), IL7R (-68%) and PRF1 (-20%) loci, the spacer-nick approach created significant fewer NHEJ events (< 5%) at all targeted loci (Fig. 2c and Fig. 7c). As consequence, the HDR:NHEJ ratio at all targeted loci was significant higher with the spacer-nick system than the classical CRISPR/Cas9 approach in both HSPCs and T cells (Fig. 2d and Fig. 7d and 8a-d). We also achieved efficient HDR with minimal NHEJ events when we combined different pairs of spacer-nick sgRNAs targeting at the HBB, ELANE, IL7R, and PRF1 loci in human HSPCs (Table 2). Thus, we provide an efficient, precise, and universal gene editing approach to repair known mutations in exons 1 and 2 of the HBB (-80%), exons 4 and 5 of the ELANE (-60%), and exon/and intron sequences of the IL7R and PRF1 genes in human HSPCs and T cells, while minimizing undesirable on-target NHEJ events.
Example 3:
To determine genome-wide off-target mutations that might be induced by sgRNA-guided nucleases in human HSPCs, we adapted the GUIDE-seq protocol that is based on the integration of a blunt-end double-stranded oligodeoxynucleotide (dsODN, 34 bps) into the nuclease- introduced DSBs 9. We performed GUIDE-seq with Cas9 or Cas9n, and a pair of spacer-nick sgRNAs targeted the HBB, ELANE, IL7R and PRF1 genes in human HSPCs that were electroporated with dsODN only, dsODN and Cas9 RNPs, or dsODN and Cas9n RNPs. Ten days post editing, genomic DNA was harvested (Fig. 9a). We modified the original GUIDE-seq method by using Nextera Tn5 transposases that fragment genomic DNA and add universal adapters to both ends of tagmented DNA fragments (Fig. 9b, c). PCR amplification with primers that annealed to each of the dsODN strands and Illumina adapters allowed next generation sequencing and analysis through our pipeline that closely follows the original GUIDE-seq software 9 (Fig. 10 and Table 3). By mapping GUIDE-seq reads to human genome, we achieved reads of adjacent gemomic sequence on both sides of each integrated dsODN tag that enabled identification of DSB position in the genome (Fig. 9d). Reads that overlapped on- and predicted off-target sites, using CRISPOR 34 and CrispRgold 35 programs, were counted. GUIDE-seq revealed that numbers of dsODN insertions at on-target sites of HBB, ELANE, IL7R and PRF1 loci in the HSPC that had treated with Cas9 and 2 sgRNAs, were up to ~263-fold higher than those in the spacer- nick-treated HSPCs (Fig. 3a-d). By analyzing dsODN integration sites, we were able to identify several high-risk off-target sites induced by Cas9 and sgRNAs targeting HBB (OT-1 to -6), ELANE (OT-7 and -8), IL7R (OT-9 to -14) and PRF1 (OT-15) loci in the HSPCs, mapped to the nucleotide level (Fig. 3a-d). Off-target sequences, induced by sgRNA-guided Cas9 nuclease, were found dispered throughout the genome (Fig. 11 ). Strikingly, such off-target sites were completely absent in the spacer-nick-treated HSPCs (Fig. 3a-d).
While SSBs introduced by sgRNA-guided Cas9n prevent integration of the blunt-end dsODN, it has been shown that sgRNA-guided Cas9 nickases may still introduce point mutations in off- target sites 36. As a consequence, GUIDE-seq may underestimate the load of mutations in on/off- target sites. To address this point, we performed amplicon deep sequencing on on- and off-target sites, identified by GUIDE-seq, of genomic DNA of the targeted HSPCs (Fig. 9a).
On- and off-target sites were amplified from gDNA isolated from the targeted HSPCs, received dsODN tag and either sgRNAs/Cas9 or sgRNAs/Cas9n RNPs. PCR products were indexed, pooled and sequenced using Miniseq. Indel profiles show read count of intact, deletion/insertion (1+ nts) and dsODN tag integration events at the IL7R on-target site 1 in HSPCs received Cas9 (left) or Cas9n (right) RNPs and dsODN tag.
Consistent with the GUIDE-seq data, the frequencies of indel reads at all on- and off-target sites were significantly higher in the HSPCs treated with sgRNAs/Cas9 RNPs than the HSPCs treated with spacer-nick RNPs (Fig. 3e). The frequencies of indel reads in off-target sites amplified from genomic DNA of the spacer-nick-treated HSPCs were at the level of a background, i.e. similar to control groups (Ctrl-1 : no editing and Ctrl-2: dsODN only) (Fig. 3e). Moreover, dsODN tag integrations were exclusively detected in on/ and off-target sites amplified from genomic DNA of HSPCs that were treated with dsODN and sgRNAs/Cas9 RNPs (Fig. 3f). Thus, based on GUIDE- seq and Amplicon-seq, the spacer-nick approach significantly reduces unwanted on- and off- target activities.
Example 4:
It has been shown that the unintended integration of portions of the AAV vector itself into the genome 3738. We investigated the unintended integration of AAV donor vectors into our HBB- corrected HSPCs targeted with spacer-nick and the classical CRISPR/Cas9 approaches by adapting AAV-seq 39. The AAV-seq preparation and computational analysis is similar with GUIDE-seq (Fig. 9b and Fig. 10). The activated HSPCs were electroporated with RNPs containing either Cas9 or Cas9n with a pair of spacer-nick sgRNAs (sg-e1 -1 and sg-e2-1 ) targeting HBB and subsequently infected with AAV-HBB donor vectors. Eighteen days post targeting, genomic DNA was harvested, fragmented with Tn5 transposase and AAV-seq libraries were deep sequenced (Fig. 12a and Table 3). AAV-seq revealed that AAV integration sites were identified in the HSPCs infected with AAV only (Fig. 12b). We identified numbers of AAV integrations at on-target (HBB) and 3 high-risk off-target sites (OT-1 , OT-2, and OT-6), identified by GUIDE-seq, in the sgRNAs/Cas9/AAV-treated HSPCs. In contrast, AAV integrations were mainly detected at the target sites (HBB) and were low/or non-detectable at off-target sites in the spacer-nick/AAV-treated HSPCs (Fig. 4a and Fig. 12c-d). The limitation of the AAV-seq method is that this PCR-based AAV-seq method is not quantitative with respect to the frequencies of AAV integrations at the on-target sites.
To comprehensively map all possible gene editing outcomes induced by the gene editing approaches at the HBB, ELANE, PRF1 loci in human HSPCs, we used the unbiased LAM- HTGTS sequencing method 440 (Fig. 4b). To exclude any contaminitation by remaining AAV donor vectors, we performed a linear amplification using an external primer annealing outside of HA for each strand of the targeted site and consequently an internal primer nearby (-50-100 bp) the cleavage sites for enrichtment. This method allows to quantify wild-type, small indels, deletion, AAV integration, inversion, HDR-mediated events and translocations (Fig. 4c and d). The activated HSPCs were treated as above (Fig. 12a). In the case of ELANE gene repair system, we used one sgRNA (sg-e4-1 ) in combination with Cas9 nuclease. The LAM-HTGTS data were analyzed using our pipeline depicted in Fig. 10. With this analysis, we detected similar gene correction efficiencies (-10-60%) at all targeted loci in the HSPCs treated with either the CRISPR/Cas9 or spacer-nick approaches, and AAV donor vectors (Fig. 4e and 4g). Moreover, LAM-HTGTS showed that the spacer-nick system robustly minimized on-target mutations as indicated in the fold change of wild-type vs mutated reads (~3- to 60-fold) at the targeted loci (Fig. 4f). As a consequence, frequencies of small indel, deletion and invsertion outcomes at the targeted HBB and PRF1 loci in the spacer-nick/AAV-treated HSPCs was significantly lower than those in the sgRNAs/Cas9/AAV-treated cells (Fig. 4g). In the case of ELANE targeting, although small indel events were dectected at lower levels in the spacer-nick/AAV-treated HSPCs, however the deletion outcome measured with 3’ enriching primer sets, was low, but higher in the spacer-nick/AAV-treated HSPCs than the sgRNAs/Cas9/AAV-treated HSPCs as the Cas9-treated cells used only one sgRNA and could not produce such a deletion (Fig. 4g). We detected a low prevalence (<0.8%) of AAV insertions at on-target sites and no difference between the CRISPR/Cas9 and spacer-nick treatments (Fig. 4g). Importantly, no chromosomal translocations were detected in this experiment (limit of detection <0.01 %). Thus, our data show that the spacernick gene correction approach retain HDR efifficiency, while minimizing on- and off-target effects in human HSPCs.
Example 5:
To address whether spacer-nick leads to efficient HDR and minimal on-target NHEJ events in human long-term HSCs, we targeted the B2M and HBB loci in human CD34+ cells. As controls, Cas9 nucleases were combined with one sgRNA (B2M and HBB) or two sgRNAs-based doublenick approach (B2M). Long-term engrafting HSCs are enriched in the CD34+CD38-CD45RA- CD90+EPCR+ population, whereas multipotent progenitor (MPP) subsets were defined as CD34+CD38-CD45RA-CD90- (MPP1 ) and CD34+CD38-CD45RA+CD90- (MPP2), respectively (33-36). We detected similar percentages of the HSC, MPP1 and MPP2 subsets in controls (Ctrl, no RNPs), Cas9, double-nick or spacer-nick-treated cells three days post targeting the B2M locus (Fig. 13, A and B, and Fig. 14). Based on mCherry expression, Cas9, the double-nick and spacernick approaches led to similar HDR efficiencies (-40%) within the HSC, MPP1 and MPP2 subsets (Fig. 13, C and D). To genetically quantify HDR and NHEJ events in long-term HSCs, we sorted the B2M and HBB-targeted CD34+CD38-CD45RA-CD90+EPCR+ HSCs and analyzed the loci by Sanger sequencing. Consistent with the mCherry expression data, we detected similar HDR rates in Cas9, double-nick (B2M) and spacer-nick-treated cells. Importantly, however, the Cas9 and double-nick approaches led to NHEJ frequencies of -42% (B2M) and 36% (HBB), whereas the spacer-nick gene correction approach led to a more than 20-fold decrease in NHEJ frequencies (-1 .6%) (Fig. 13, E to I, and Fig. 14D). Thus, the spacer-nick system led to precise and efficient HDR with minimal unwanted on-target mutations in human long-term engrafting HSCs. Conclusion of the examples
In summary, we provide a high-fidelity genome-editing approach using Cas9n with a pair of spacer-nick sgRNAs at a spacer distance of 200-350 bp, together with AAV6 as a donor template, that permits high HDR efficiency. Using the spacer-nick system, we developed the universal gene correction to repair known mutations the HBB, ELANE, IL7R, and PRF1 genes in human HSPCs and T cells. Furthermore, we show that in human HSPCs the classical CRISPR/Cas9 system exhibits high off-target activities that these can be significantly reduced by using the spacer-nick gene correction approach. Taken together, the high-fidelity spacer-nick system holds promise for therapeutic applications to cure severe genetic diseases such as betathalassemia, severe congenital neutropenia, severe combined immune deficiency and familial hemophagocytic lymphohistiocytosis, and other monogenic blood disorders.
Materials and Methods
Isolation and culture human HSPCs and T cells
Human CD34+ HSPCs were isolated from mobilized peripheral blood of healthy donors using Ficoll-Paque PLUS (GE Healthcare) and human CD34 microbead kit according to the manufacturer’s protocol (Milteny Biotec). 2x105 CD34+ HSPCs were cultured in 1 ml of serum-free StemSpan™ SFEM II medium (Stemcell technologies) supplied with human SCF (100 ng/ml), human TPO (100 ng/ml), human FLT3L (100 ng/ml), human IL-6 (100 ng/ml) (Peprotech), UM171 (35 nM, Stemcell technologies) and SR1 (0.75 uM, Stemcell technologies). Human CD3+ T cells were isolated from peripheral blood of healthy donors using Pan T cell isolation kit according to the manufacturer’s protocol (Milteny Biotec). 2x105 CD3+ T cells were cultured in 1 ml of RPMI1640 (Gibco) supplied with 10% FCS, 1x Glutamax and human IL-2 (100 U/ml, Peprotech).
AAV donor template cloning
To generate pAAV-B2M/ or CD48-T2A-mCherry donor vectors, the left and right homology arms were amplified from human genomic DNA and cloned into Xhol/EcoRI and AsiSI/Kpnl sites of the pTV-T2A-mCherry vectors. The Notl-flanked B2M/or CD48-T2A-mCherry fragments were cloned into the pAAV vector (Cell Biolabs). To generate pAAV-HBB, pAAV-IL7R or pAAV-PRF1 donor vector, HBB (exon 1 and 2), IL7R (modified cDNA), or PRF1 (modified cDNA) donor template including homology arms, silent mutations and Sall recognition site were synthesized by GeneArt Gene Synthesis (Thermo Scientific) and subsequently cloned into pAAV vector (Cell Biolabs). The pAAV-ELANE donor vector was previously generated 33.
AAV production
To produce rAAV6 viruses, HEK293T cells were co-transfected with pAAV-donor template, pAAV-6-Rep/Cap, and pAAV-Helper plasmids using the PEI transfection protocol. 12 h later, the medium was replaced by DMEM+/+ supplemented with 10% FCS, 25mM HEPES (Gibco) and 10 pg/ml Gentamycin (Lonza). Three days later, the cell pellet was collected and lysed by 3 cycles of thaw-freeze in dry-ice/ethanol bath (10 min per cycle). Then the cell lysate was cleared by spinning at 3500 rpm for 15 min. The cleared supernatant was transferred into new falcon tubes and treated with DNA endonuclease Benzonase (Millipore) for 1 h at 37°C. The cell lysate was spun down and the supernatant was loaded into lodixanol gradient tubes (Beckman) and fractionated by ultra-centrifugation at 58000 rpm for 130 min at 18°C, using a type 70Ti rotor (Beckman). The 40% iodixanol layer was collected using an 18-gauge needle and syringe. The supernatant was filtered through a 0.2 pm PES filter and dialyzed overnight with PBS at 4°C in dialysis cassettes (Thermo Scientific). Finally, the rAAV6 supernatant was concentrated using Amicon® Ultra-15 Centrifugal Filter Units (Millipore). The concentration of rAAV6 viruses was measured by real-time PCR using TaqMan probes specific for the AAV ITR sequence (Thermo Scientific). sciRNA. dsODN and RNP electroporation and AA V infection
SgRNAs (Table 1 ) were purchased from Synthego or IDT. SpCasO, SpCasOn (Cas9D10A) and dsODN (34 nt) were purchased from IDT. To generate the sgRNA complexes, crRNA (100 pmol, 1 .2 pg) and tracrRNA (100 pmol, 1 .2 pg) were mixed at a 1 :1 ratio, incubated at 95°C for 5 min, and ramped down to room temperature. To generate the RNP complexes, Cas9 or Cas9n (50 pmol, 8.2 pg) were mixed with sgRNAs (100 pmol, 3.3 pg) at a 1 :2 molarity ratio and incubated at 25°C for 10 min. 2x105 human CD34+ HSPCs and T cells were suspended into 20 pl of P3 electroporation buffer (Lonza) containing RNPs. After electroporation with RNPs, human HSPCs (DZ-100) and T cells (EH-100) were transferred to a pre-warmed medium supplied with cytokines and placed into an incubator at 37°C and 5% CO2. 15-30 minutes later, the rAAV6 donor particles were added to electroporated cells at a MOI 1x105 GC/cell for HSPCs and a MOI 1x106 GC/cell for human T cells. For GUIDE-seq experiments, HSPCs and T cells were electroporated with RNPs and 25 pmol of dsODN (34 nt). The medium was changed the next day. The targeted cells were harvested at different time points. The dead cells were removed by using a dead cell removal kit according to the manufacturer’s protocol (Milteny Biotec). The living cells were analyzed or harvested for genomic DNA extraction for further analysis.
FACS analysis
Three days post targeting, the edited cells were collected and blocked with FcyR antibodies (BioLegend) for 10 min at 4oc. Subsequently, these cells were stained with fluorescent- conjugated antibodies for 15 min at 4°C. The cells were finally washed with FACS buffer (PBS/1 % BSA) and analyzed by BD Fortessa. The data were analyzed using FlowJo®.
PCR, T7EI and RFLP assays
Genomic DNA from the targeted HSPCs and T cells was extracted using the QuickExtract DNA extraction kit (Epicentre) following the manufacturer’s protocol. To access gene editing efficiencies of sgRNAs, the targeted sequences were amplified from genomic DNA by PCR (30 cycles) using the Herculase II Fusion DNA Polymerase (Agilent Technology) with gene-specific primers (Table 2). PCR products were cleaned using AMPure XP beads (Beckman Coulter). The purified PCR products were subjected to T7EI assay as described previously 12. For ICE analysis, the purified PCR products were sequenced using Sanger sequencing and then analyzed by ICE tool (Synthego). Percentage of Indels was calculated based on decomposition algothirm and represented as ICE-score 41. For correct integration PCR and RFLP assays, the targeted fragments were amplified using the KOD hot-start DNA polymerase (Millipore) with gene-specific primers (Table 2). PCR products were purified using AMPure XP beads (Beckman Coulter) and digested with Sall restriction enzyme for 1 h at 37°C. Cleaved DNA fragments were separated on 1.2% agarose gel. DNA concentration of each band was quantified using Imaged software (NIH). Percentages of indels and HDR were calculated as described 10. For quantifying HDR and NHEJ events on the targeted loci, the purified PCR products were cloned into the sequencing plasmids using CloneJET PCR cloning kit (Thermo Scientific) following the manufacturer’s protocol.
Colonies were picked into 96-well LB agar plates and plasmids were purified using NucleoSpin® 96 Plasmid Core Kit (Macherey-Nagel) and sequenced by the Sanger method (LGC genomics, Berlin, Germany).
DNA Sanger sequencing and ICE analysis
For tracking of indels using ICE sequening analysis (Synthego), the targeted sequences were amplified from gemonic DNA by PCR using the Herculase II Fusion DNA Polymerase (Agilent Technology). PCR products were cleaned using AMPure XP beads (Beckman Coulter). The cleaned PCR products were sequenced using Sanger sequencing and then analyzed by ICE sequencing tool (Synthego). Percentage of Indels was calculated based on decomposition agothirm and represented as ICE-score 37. For quantifying HDR and NHEJ events on the targeted loci, PCR products were cloned into the sequencing plasmids using CloneJET PCR cloning kit (Thermo Scientific) following the manufacturer’s protocol. Colonies were picked into 96-well LB agar plates and plasmids were purified using NucleoSpin® 96 Plasmid Core Kit (Macherey-Nagel) and sequenced by the Sanger method (LGC genomics, Berlin, Germany).
Tn5-mediated GUIDE-seq and AAV-seq
Genomic DNA was isolated from the targeted cells (~2-5x105) 10 days for GUIDE-seq and 18 days for AAV-seq after electroporation and AAV infection using a GenFind V3 Reagent Kit according to the manufacturer’s protocol (Beckman Coulter). 100 ng of genomic DNA were tagmented by using Tn5 transposase (Illumina) in 20 pl reaction at 55°C for 7 min. Tagmented DNA fragments were purified by using Zymo DNA clean and concentrator-5 according to the manufacturer’s protocol (Zymo research). To verify the tagmentation (-0.3-1.5 kb), 1/10 Zymo elutes were loaded on 1 .2% agarose gel. Preparations of GUIDE-seq and AAV-seq libraries were followed previous protocols 9 39. Finally, the GUIDE-seq and AAV-seq libaries were loaded onto llumina Miniseq for deep sequencing.
PCR amplicon-seq
To perform amplicon deep sequencing for on/off-target analysis, on- and off-target sites, identified by GUIDE-seq were amplified from genomic DNA by PCR using Platinum SuperFi PCR Master Mix (Thermo Scientific) with gene specific primers including overhang adapter sequences that are comparable to Illumina Nextera XT index adapters (Table 2) and following PCR conditions: 98°C for 2 min; 20 cycles (98°C for 10s, 60°C for 30s, 72°C for 30s) and 72°C for 5 min. PCR products were purified using AMPure XP beads (Beckman Coulter), quantified using a Qubit dsDNA HS assay kit (Invitrogen) and normalized to 1 ng/pl. For multiplexing sequencing libraries these PCR products were indexed through a second PCR with Nextera XT DNA Library Preparation Kit v2 set A (Illumina) using Platinum SuperFi PCR Master Mix (Thermo Scientific) and following PCR conditions: 98°C for 2 min; 10 cycles (98°C for 10s, 60°C for 30s, 72°C for 30s) and 72°C for 5 min. Indexed PCR products were cleaned using AMPure XP beads (Beckman Coulter), quantified using a Qubit dsDNA HS assay kit (Invitrogen), normalized to 10 ng/ul, and pooled. The amplicon libraries were loaded onto llumina Miniseq for deep sequencing.
LAM-HTGTS
To detect all potential genome-editing outcomes, we modified linear amplification-mediated high throughput genome-wide translocation sequencing (LAM-HTGTS) method 4’40. Briefly, we performed a Linear PCR from 330ng genomic DNA (-50.000 genomes) using PrimeSTAR GXL polymerase (TaKaRa) with an external biotinylated primer that anneals to a genomic sequence outside of the 5’ or 3’ HA that excludes the contamination by the remaining AAV donor vectors (Table 2). Linear PCR was performed with the following PCR conditions: 98°C for 5 min; 80 cycles (98°C for 30s, 65°C for 30s, 68°C for 90s) and 68°C for 2 min. Biotinylated PCR products were then captured by Streptavidin Dynabeads MyONE C1 (Life Technologies). Captured products were cleared off the genomic DNA by washing. Products were ligated with a doublestranded adapter harboring a degenerate hexanucleotide overhang and protected from selfligation by amino-C3 linkers on 5’-ends. Illumina adapters were introduced via nested PCR on the bead-captured products. Internal primers annealed in close vicinity to the cleavage sites (-50-100 bp) and harbored Illumina Nextera adapter sequences on their 5’-ends. Q5 polymerase (NEB) was used for the nested PCR. The PCR products were purified using ProNex beads (Promega) and quantified using a Qubit dsDNA HS assay kit (Invitrogen). For multiplexing sequencing libraries, these PCR products were indexed through a PCR with Illumina indices using Q5 polymerase (NEB). The LAM-HTGTS libraries were loaded onto Illumina Miseq (300 + 300 cycles) or MiniSeq (150 + 150 cycles) for deep sequencing.
Figure imgf000048_0001
The piplines for analyzing the GUIDE-seq, AAV-seq and LAM-HTGTS were written based on the built off of code in previous publications 4243 and shown in Fig. 10. The Scripts and Jupyter notebooks are available (github.com/Eric Danner). For GUIDE-seq and AAV-seq, samples were demultiplexed, reads were checked for correct priming and the sequences of AAV inverted terminal repeats (ITRs) and dsODN were trimmed to adjacent genomic sequences using Cutadapt (github.com/Marcel Martin). The adjacent genomic sequences are globally mapped to human genome reference (hg38) using Bowtie2 44. Perfect mapped reads are checked for overlap of regions within 5.000 bps of predicted off-target sites using CRISPOR 34 and CrisprRgold 35, programs. For LAM-HTGTS, samples were demultiplexed, checked for correct priming and reads were trimmed to the genome interface. Trimmed sequences were aligned first locally to the AAV ITRs. Reads without alignment to AAV ITRs were mapped on in-silico generated amplicons of expected outcomes, following the previous publication 42, using Bowtie2. Perfect mapped reads are then quantified for HDR count. Reads that cover the sgRNA break (targeting??) site are analyzed and quantified for indel, deletion and inversion outcomes. To determine nuclease-driven translocations, reads that did not align to to this point were trimmed with Cutadapt to the proximal gRNA site, and globally aligned to human genome reference (hg38) using Bowtie2. Well aligned reads that overlapped within 5.000 bps of predicted off-targets sites (CrispRgold) were recognized as nuclease-driven translocations. Plotting was done with homemade R scripts.
Statistical Statistical tests were performed using Prism 7.0 (GraphPad) using a Mann Whitney test or two- way ANOVA. *** pcO.001 , ** p<0.01 and * p<0.05.
Tables of the example
Table 1 . List of sqRNAs and gene editing efficiencies
Figure imgf000049_0001
Figure imgf000050_0001
Gene editing efficiencies of sgRNAs were measured by ICE analysis and T7EI assay. ND; not determined. Table 2. Potential combination of spacer-nick sgRNAs for gene correction.
Figure imgf000050_0002
Figure imgf000051_0001
This table show the potential combination of spacer-nick sgRNAs for gene correction, efficiencies of HDR and NHEJ sequences were analyzed by Topo cloning and Sanger sequencing. ND; not determined. Table 3. Primers were used in this study
Figure imgf000051_0002
Figure imgf000052_0001
Figure imgf000053_0001
Figure imgf000054_0001
Figure imgf000055_0001
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Claims

58 CLAIMS:
1 . An in vitro method for modifying a double stranded DNA (dsDNA) molecule through singlestrand break (SSB)-mediated homology-directed repair (HDR) in a eukaryotic cell, such as a haematopoietic stem and progenitor cell (HSPC) or T cell, the method comprising: introducing into the cell i. a guide RNA-guided DNA nickase or a nucleic acid molecule encoding a guide RNA-guided DNA nickase, ii. at least two guide RNAs, comprising a first guide RNA capable of hybridizing to a first target sequence in the dsDNA, and a second guide RNA capable of hybridizing to a second target sequence in the dsDNA, and
Hi. an exogenous DNA donor template comprising a DNA template sequence, generating at least two single strand breaks (SSB) in the opposing strands of the dsDNA molecule to be modified, i. wherein a first guide RNA/DNA nickase complex, comprising the first guide RNA, introduces a nick cleavage of one strand of the dsDNA within the first target sequence, and a second guide RNA/DNA nickase complex, comprising the second guide RNA, introduces a nick cleavage of the opposite strand of the dsDNA within the second target sequence, thereby producing a 5’ or 3’ overhang, ii. wherein the distance (spacer) between two single strand breaks (SSB) on the dsDNA molecule to be modified is 202 base pair (bp) or greater, and replacing a DNA sequence of the dsDNA, positioned in proximity to the two single strand breaks (SSB), with the template sequence.
2. The method according to claim 1 , wherein replacing a DNA sequence of the dsDNA molecule to be modified with the template sequence occurs primarily by homology-directed repair (HDR), preferably wherein replacing a DNA sequence with the template sequence occurs more frequently by homology-directed repair (HDR) compared to non-homologous end joining (NHEJ), more preferably wherein the ratio of homology-directed repair (HDR) events to non- homologous end joining (NHEJ) events is from 5:1 to 50:1 , preferably from 10:1 to 20:1 .
3. The method according to any of the preceding claims, wherein the distance (spacer) between the at least two single strand breaks (SSB) on the dsDNA molecule to be modified is 202 to 500 bp, preferably 202 to 350 bp.
4. The method according to any of the preceding claims, wherein the method induces fewer unwanted on- and/or off-target effects, such as micro-insertions and/or deletions (Indels) and/or off-target mutations, compared to a method in which the distance (spacer) between 59 two single strand breaks (SSB) on the dsDNA molecule to be modified is less than 202 base pair (bp) or a method in which a double-strand break (DSB) inducing guide RNA- guided DNA endonuclease, such as CRISPR/Cas9, is employed, wherein preferably the number and/or frequency of off-target effects is determined using a genome-wide method for detecting off-target mutations, such as a GUIDE-seq method. The method according to any of the preceding claims, wherein each target sequence comprises or is adjacent to a protospacer adjacent motif facing outwards (PAM-out) at a 3’- end of the target sequence. The method according to any of the preceding claims, wherein the DNA template sequence is 500 bp to 5000 bp in length, preferably 1000 to 5000 bp, more preferably 2000 to 4000 bp, more preferably about 3000 bp in length. The method according to any of the preceding claims, wherein the DNA template sequence comprises at least one sequence (homology arm) of at least 100 bp, preferably 500 bp, with at least 90%, preferably 95%, more preferably 100%, sequence identity to a region within the DNA sequence to be replaced, wherein said homology arm is positioned to hybridize to the dsDNA molecule to be modified in proximity to the two single strand breaks (SSB), preferably wherein the DNA template comprises at least two homology arms, each comprising at least 100 bp, preferably 500 bp, more preferably 700 bp, or 1000 bp or more, with at least 90%, preferably 95%, more preferably 100%, sequence identity to a region within the DNA sequence to be replaced. The method according to the preceding claim, wherein a homology arm of the the DNA template is positioned to hybridize to the dsDNA molecule to be modified, such that the distance between a single strand break (SSB) induced by the RNA guided DNA nickase and the end of the homology arm distal to the SSB is up to 1500 bp, preferably up to 1000 bp, more preferably up to 500 bp. The method according to any of the preceding claims, wherein the DNA template sequence is in the form of and/or comprised by an viral vector, such as an adeno-associated virus (AAV), or is a circular or linear DNA molecule, preferably a plasmid or mini-circle, or a single stranded DNA molecule. The method according to any of the preceding claims, for modifying an ELANE gene (encoding Neutrophil Elastase), wherein the at least two guide RNAs are capable of hybridizing to sequences in or adjacent to, preferably flanking, a sequence of the ELANE gene to be modified, wherein preferably the sequence of the ELANE gene to be modified comprises one or more pathological gene mutations associated with reduced or aberrant Neutrophil Elastase function, such as in subjects with Severe Congenital Neutropenia, and the DNA template sequence comprises an ELANE gene sequence without said mutations, 60 wherein more preferably the DNA template sequence comprises exons 4 and/or 5, or parts thereof, of an ELANE gene sequence without said mutations. The method according to any of claims 1-10, for modifying an HBB gene (encoding Hemoglobin Subunit Beta), wherein the at least two guide RNAs are capable of hybridizing to target sequences in or adjacent to, preferably flanking, a sequence of the HBB gene to be modified, wherein preferably the sequence of the HBB gene to be modified comprises one or more pathological gene mutations associated with reduced or aberrant Hemoglobin Subunit Beta function, such as in subjects with Beta-Thalassemia, and the DNA template sequence comprises an HBB gene sequence without said mutations, wherein more preferably the DNA template sequence comprises exons 1 and/or 2, or parts thereof, of an HBB gene sequence without said mutations. The method according to any of claims 1-10, for modifying a Perforin gene (preferably PRF1 (FHL2), encoding a Perforin-1 protein), wherein the at least two guide RNAs are capable of hybridizing to target sequences in or adjacent to, preferably flanking, a sequence of the Perforin gene to be modified, wherein preferably the sequence of the Perforin gene to be modified comprises one or more pathological gene mutations associated with reduced or aberrant Perforin protein function, such as in subjects with hemophagocytic lymphohistiocytosis (HLH), such as familial hemophagocytic lymphohistiocytosis type 2 (FHL2), and the DNA template sequence comprises a Perforin gene sequence without said mutations. The method according to any of claims 1-10, for modifying an lnterleukin-7 receptor (IL7R) gene (encoding an lnterleukin-7 receptor subunit alpha protein), wherein the at least two guide RNAs are capable of hybridizing to target sequences in or adjacent to, preferably flanking, a sequence of the IL7R gene to be modified, wherein preferably the sequence of the IL7R gene to be modified comprises one or more pathological gene mutations associated with reduced or aberrant IL7R protein function, such as in subjects with immunodeficiency or inflammatory diseases, such as multiple sclerosis or rheumatoid arthritis, and the DNA template sequence comprises a IL7R gene sequence without said mutations. An isolated eukaryotic cell, such as a haematopoietic stem and progenitor cell (HSPC) or T cell, modified by a method according to any of the preceding claims, preferably for use in the treatment of a medical condition, wherein the medical condition is associated with the reduced function of a gene product induced by pathological gene mutations and said mutations have been replaced with a DNA template sequence without said mutations. A kit for use in an in vitro method for modifying a double stranded DNA (dsDNA) molecule through single-stranded break (SSB)-mediated homology-directed repair (HDR) in a eukaryotic cell, such as a haematopoietic and progenitor cell (HSPC) cell or T cell, the kit comprising: 61 i. at least two guide RNAs, comprising a first guide RNA capable of hybridizing to a first target sequence in the dsDNA, and a second guide RNA capable of hybridizing to a second target sequence in the dsDNA, wherein the first guide RNA is configured to introduce a nick cleavage of one strand of the dsDNA within the first target sequence and the second guide RNA is configured to introduces a nick cleavage of the opposite strand of the dsDNA within the second target sequence, thereby producing a 5’ or 3’ overhang configured for repair primarily by single-strand break-mediated homology-directed repair (HDR), and wherein the at least two guide RNAs are configured such that the distance (spacer) between the at least two single strand breaks (SSB) on the dsDNA molecule to be modified is 202 base pair (bp) or greater, preferably 202 to 500 bp, more preferably 202 to 350 bp, preferably comprising the guide RNAs according to SEQ ID NO X-Y, and optionally ii. a guide RNA-guided DNA nickase or a nucleic acid molecule encoding a guide RNA-guided DNA nickase, and/or
Hi. an exogenous DNA donor template comprising a DNA template sequence.
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