Compounds for increasing genome editing efficiency

Abstract

To provide compounds, compositions and kits suitable for increasing precise genome editing efficiency in eukaryotic target cells or target organisms.SOLUTION: Provided herein is in vitro use of: (i) a DNA protein kinase catalytic subunit which is catalytically inactive, but structurally intact, and which comprises at least one mutation compared to the wild-type sequence; (ii) a nucleic acid molecule encoding the DNA protein kinase catalytic subunit of (i) and / or (iii) a eukaryotic cell comprising or capable of expressing the DNA protein kinase catalytic subunit of (i), for genome editing in a eukaryotic target cell, particularly in a mammalian target cell.SELECTED DRAWING: None

Description

[Technical Field]

[0001] statement The present invention relates to compounds, compositions and kits suitable for increasing the efficiency of precise genome editing in eukaryotic target cells or target organisms. [Background technology]

[0002] CRISPR is a bacterial nuclease immune system for viral DNA that has been employed in eukaryotic cells to precisely cut chromosomal DNA sequences, which are then repaired by two competing pathways: non-homologous end joining (NHEJ) or homology-directed repair (HDR).

[0003] In NHEJ, the first proteins to bind to the DNA ends are Ku70 / Ku80, followed by DNA protein kinase catalytic subunit (DNA-PKcs) (Shrivastav et al. 2008). The kinase phosphorylates itself and other downstream effectors at the repair site. Recruitment and phosphorylation of several proteins, such as Artemis, leads to end-processing ligation by Ligase IV (LIG4), X-ray repair cross-complementing protein 4 (XRCC4), and non-homologous end-joining factor 1 (XLF) (Dueva and Iliakis 2013).

[0004] When this canonical NHEJ pathway is inhibited, the alternative NHEJ pathway (A-NHEJ) is activated (Nussenzweig and Nussenzweig 2007). It requires, among other proteins, poly(ADP-ribose)-polymerase 1 (PARP-1), Werner syndrome ATP-dependent (WRN) helicase, and DNA ligase 3 (LIG3) or DNA ligase I (LIG1). HDR is initiated by binding of the MRN-complex (Mre11, Rad50, and Nbs1) complex to double-strand breaks (DSBs) (Shrivastav et al. 2008). Together with other proteins, such as the DNA endonuclease RBBP8 (CtIP), Bloom helicase (BLM), and exonuclease 1 (EXO1), terminal nucleotides are removed at the 5' end, generating long 3' single-stranded DNA (ssDNA) overhangs on either side of the DNA break (Dueva and Iliakis 2013). These tails are then coated and stabilized by the replication protein A (RPA) complex, followed by the generation of Rad51 nucleoprotein filaments assisted by breast cancer 2 (BRCA2) (Shrivastav et al. 2008). Rad52 promotes the displacement of RPA bound to the ssDNA with Rad51, facilitating ssDNA annealing (Grimme et al. 2010). Strand invasion by the donor DNA and subsequent DNA synthesis by the polymerase ultimately results in an accurately repaired DNA. The protein kinase ataxia telangiectasia mutated (ATM) plays a major role in HDR as it phosphorylates at least 12 repair proteins (Shrivastav et al. 2008 ).

[0005] CRISPR Cas9-induced NHEJ of DSBs is error-prone and frequently introduces insertions and deletions (indels) at the break site, making it useful for knocking out targeted genes. In contrast, HDR allows for precise repair of DSBs by using a homologous donor DNA sequence. This donor sequence is provided in the experiment, and if it contains mutations, these are introduced into the genome.

[0006] A requirement for Cas9-induced DSBs is an NGG sequence (PAM site) in DNA. Cas9 targeting is determined by the binding of a guide RNA (gRNA) complementary to the 20 nucleotides adjacent to the PAM site. However, Cas9 nuclease can also cleave the genome at sites with sequence similarity to the sequence targeted by the gRNA (Fu et al. 2013). These off-target double-strand breaks mean that, along with the desired mutation, unwanted mutations can appear elsewhere in the genome.

[0007] One strategy to reduce such off-target cuts is to use a mutant Cas9, such as Cas9 D10A, which introduces single-stranded nicks rather than DSBs (Shen et al. 2014). Using two gRNAs to introduce two nicks on opposing DNA strands close together results in a DSB at the desired locus while reducing the risk of two off-target nicks occurring elsewhere in the genome that are close enough to cause a DSB. Another strategy is to use Cpf1 (Zetsche et al. 2015). This nuclease introduces staggered cuts near T-rich PAM sites and has been shown to produce fewer off-target effects (Kim et al. 2016) (Kleinstiver et al. 2016).

[0008] In current approaches, the efficiency of precise genome editing (PGE) for targeted nucleotide substitutions, especially in stem cells, is usually low, ranging from 0.5 to 15% (Yu et al. 2015) (Gonzalez et al. 2014). Some researchers have addressed the low efficiency of precise genome editing by attempting to promote HDR or reduce NHEJ.

[0009] Cell cycle synchronization to the G2 / M phase has been shown to increase PGE2 produced by single-stranded oligodeoxynucleotide (ssODN) donors in HEK293T cells (from 26% to 38%), primary human neonatal fibroblasts (from undetectable to 0.6%), and human embryonic stem cells (hESCs) (from undetectable to 1.6%) (Lin et al. 2014), and to increase PGE2 produced by double-stranded oligodeoxynucleotide (dsODN) donors in hESCs (from 7 to 41% after sorting) (Yang et al. 2016), because homologous recombination is restricted to this phase and its proteins are upregulated.

[0010] Improved efficiency was also achieved by silencing key proteins such as Ku70 / 80 and ligase IV with siRNA (from 5 to 25%) or by coexpressing adenovirus type 5 proteins E1B55K and E4orf6 (from 5 to 36%) in HEK293 / TLR cells using dsODN donors (Chu et al. 2015). E1B55K and E4orf6 proteins mediate ubiquitination and proteosomal degradation of LIG4, among other targets.

[0011] The use of small molecules has become a common strategy to enhance genome editing. The small molecule SCR7, a ligase IV inhibitor, is claimed to block NHEJ and increase the efficiency of PGE (from 5 to 22.7%) in mouse embryos (Maruyama et al. 2015). Other researchers have described similar enhancements in HEK293 / TLR cells, a small but significant enhancement in HEK293A, or no significant effect in mouse embryos, rabbit embryos, and human stem cells (Chu et al. 2015), (Pinder et al. 2015), (Song et al. 2016), (Yang et al. 2016). al. 2016) (Zhang et al. 2017). Recently, Greco et al. reanalyzed the structure and inhibitory properties of SCR7 (Greco et al. 2016). They concluded that SCR7 and its derivatives are neither selective nor potent inhibitors of human LIG4.

[0012] Pharmacological inhibition of DNA-PK, a key protein complex in the NHEJ pathway, with the small molecules NU7441, KU-0060648, and NU7026 has been shown to reduce the frequency of NHEJ and increase PGE in HEK293 / TLR cells (1.9 to 3.8%), HEK293 (3 to 7.6%) and human induced pluripotent stem cells (hiPSCs) (13 to 16%) (dsODN donors), and mouse embryonic fibroblasts (3 to 10%) (ssODN donors) (Robert et al. 2015; Suzuki et al. 2016; Zhang et al. 2017).

[0013] A single small molecule has also been described that enhances homologous recombination with CRISPR-Cas9. The RAD51-stimulating compound RS-1 increased PGE in rabbit embryos (4.4 to 26.1%), HEK293A cells (3.5 to 21%), and U2OS cells (1.9 to 2.4%) (Song et al. 2016) (Pinder et al. 2015), but not in hiPSCs (all with dsODN donors) (Zhang et al. 2017). In porcine fetal fibroblasts, no effect of RS-1 on PGE efficiency was found using ssODN donors (Wang et al. 2017). al. 2016).

[0014] Furthermore, using a library screen of approximately 4,000 small molecules, Yu et al. found that the β3-adrenergic receptor agonist L755507 increased PGE in human hiPSCs using ssODNs (from 0.35 to 3.13%) and in mouse ESCs using dsODN donors (from 17.7 to 33.3%), although the target of the molecule's repair pathway is unknown (Yu et al. 2015). Others have not found significant stimulation of PGE by L755507 in HEK293A cells or human hiPSCs (Pinder et al. 2015) (Zhang et al. 2017). Pinder et al. compared SCR7, RS-1, and L755507 alone and in combination and found no additive effect when SCR7 and L755507 were added with RS-1 compared to RS-1 alone.

[0015] From this current review of small molecules that enhance CRISPR (CRIPR)-Cas9 genome editing, we find that inhibitors of DNA-PK can increase PGE in CRISPR-Cas9 genome editing, but the effects of SCR7, L755507, and RS-1 were inconsistent across cell lines and loci. We also find that previously tested combinations of small molecules did not show additive effects. Summary of the Invention

[0016] The present inventors have found that certain compounds, especially when applied as a combination of two or more different compounds, can increase the efficiency of precise genome editing.In particular, the present inventors have found that compounds selected from the following: histone deacetylase (HDAC) inhibitors, NEDD8-activating enzyme (NAE) inhibitors, DNA-dependent protein kinase (DNA-PK), particularly its catalytic subunit (DNA-PKcs) inhibitors, and replication protein A (RPA) inhibitors, and combinations of compounds selected from these different classes of inhibitors can increase the efficiency of genome editing.The compounds and their combinations are suitable for both non-medical use (for example, as research tools) or medical use (for example, for in vivo or ex vivo use).

[0017] Furthermore, we found that catalytically inactive but structurally intact DNA-PKcs enhances the efficacy of precise genome editing, regardless of the presence of the above compounds, an effect that is broadly applicable because it is found in multiple different systems for genome editing. [Brief explanation of the drawings]

[0018] [Figure 1] Figure 1: Flowchart of genome editing and analysis. iCRISPR 409-B2 iPSCs are treated with 2 μg / ml doxycycline for at least two days to induce Cas9 or Cas9D10A expression. Reverse transfection with RNAiMAX, gRNA (7.5 nM each), ssODN (10 nM), and the small molecule being evaluated is performed in 96-well plates for one day. The amount of cells used results in up to 80% confluency. Cells are then amplified for three days with regular media changes. After recovery, DNA is extracted, PCR amplification of the target locus is performed, Illumina sequencing is performed, and CRISPResso (Pinello et al. 2016) sequence analysis is performed for indel content and precise genome editing. [Figure 2]Figure 2: Design of gRNAs and ssODNs for precise genome editing (PGE) of human CALD1, KATNA1, and SLITRK1 to the ancestral state of the last common ancestor of humans and Neanderthals. The CALD1, KATNA1, and SLITRK1 loci are shown, along with the gRNAs used for DSB generation and their efficiency scores (sc) (sgRNA scorer 1.0, Chari et al. 2015). PAM sites are gray, target sequences are blue, and modified bases are red. Arrows indicate the location of Cas9D10A (Cas9n)-induced nicks or Cas9-induced DSBs. For Cas9n editing, both guides are used, whereas for Cas9 editing, CALD1 g1, KATNA1 g2, and SLITRK1 g2 are used. The respective ssODNs for editing with both Cas9 variants are also shown. The desired mutation is shown in green, and additional mutations are orange. "Block" indicates a Cas9 blocking mutation to prevent recutting of the locus. All Cas9D10A donors have a 50-nt homology arm after the nick, while all Cas9 donors are 90 nt total, with the desired mutation in the middle. The complete sequences are shown in Table 2. [Figure 3-1] Figure 3: Initial screening of solvent effects and the influence of different small molecule concentrations on iCRISPR Cas9D10A-mediated genome editing of CALD1, KATNA1, and SLITRK1. Precise genome edits (PGEs) and indels are indicated by green circles (triangles) or blue diamonds, respectively. Each symbol represents a technical replicate. The respective average is shown as a black line. Each skull represents up to 20% cell death as measured by phase-contrast light microscopy. All cells were killed by 1 μM and 0.1 μM trichostatin A. Concentrations selected for further experiments are shown in cyan. [Figure 3-2]Figure 3: Initial screening of solvent effects and the influence of different small molecule concentrations on iCRISPR Cas9D10A-mediated genome editing of CALD1, KATNA1, and SLITRK1. Precise genome edits (PGEs) and indels are indicated by green circles (triangles) or blue diamonds, respectively. Each symbol represents a technical replicate. The respective average is shown as a black line. Each skull represents up to 20% cell death as measured by phase-contrast light microscopy. All cells were killed by 1 μM and 0.1 μM trichostatin A. Concentrations selected for further experiments are shown in cyan. [Figure 3-3] Figure 3: Initial screening of solvent effects and the influence of different small molecule concentrations on iCRISPR Cas9D10A-mediated genome editing of CALD1, KATNA1, and SLITRK1. Precise genome edits (PGEs) and indels are indicated by green circles (triangles) or blue diamonds, respectively. Each symbol represents a technical replicate. The respective average is shown as a black line. Each skull represents up to 20% cell death as measured by phase-contrast light microscopy. All cells were killed by 1 μM and 0.1 μM trichostatin A. Concentrations selected for further experiments are shown in cyan. [Figure 3-4] Figure 3: Initial screening of solvent effects and the influence of different small molecule concentrations on iCRISPR Cas9D10A-mediated genome editing of CALD1, KATNA1, and SLITRK1. Precise genome edits (PGEs) and indels are indicated by green circles (triangles) or blue diamonds, respectively. Each symbol represents a technical replicate. The respective average is shown as a black line. Each skull represents up to 20% cell death as measured by phase-contrast light microscopy. All cells were killed by 1 μM and 0.1 μM trichostatin A. Concentrations selected for further experiments are shown in cyan. [Figure 4]Figure 4: Effect of small molecules on the efficiency of precise genome editing (PGE) at CALD1, KATNA1, and SLITRK1 by Cas9D10A and Cas9. PGE efficiency is shown in relative units (RU), with the control mean set to 1 to account for variations in efficiency at different loci. Technical replicates of n independent experiments are shown. Gray and black bars represent the control and mean of each small molecule, respectively. The concentrations used were NU7026 20 μM, trichostatin A (TSA) 0.01 μM, MLN4924 0.5 μM, NSC19630 1 μM, NSC15520 5 μM, AICAR 20 μM, RS-1 1 μM, resveratrol 1 μM, SCR7 1 μM, L755507 5 μM, STL127685 5 μM, and B02 20 μM. [Figure 5-1]Figure 5: Effect of small molecule combinations on the efficiency of precise genome editing (PGE) in CALD1, KATNA1, and SLITRK1 by Cas9D10A and Cas9, and in HPRT and DMNT1 by Cpf1. Small molecules have an additive effect on PGE efficiency in the 409-B2 iCRISPR iPSC line by Cas9D10A (A), but not by Cas9 (B). PGE, PGE + indels, and indels are indicated by green, gray, or blue bars, respectively. Error bars indicate standard deviations of three technical replicates for A, B, and C and two technical replicates for D. The concentrations used were NU7026 20 μM, trichostatin A (TSA) 0.01 μM, MLN4924 0.5 μM, NSC 19630 1 μM, NSC 15520 5 μM, AICAR 20 μM, and RS-1 1 μM. CRISPY mix refers to a small molecule mixture of NU7026, TSA, MLN4924, and NSC 15520. The significance of changes in knock-in efficiency was determined using a two-way ANOVA and Tukey's multiple comparisons pooled across the three genes CALD1, KATNA1, and SLITRK1. Gene and treatment were treated as random and fixed effects, respectively. Three technical replicates were included for each gene in the analysis. P values ​​are adjusted for multiple comparisons (**P<0.01, ***P<0.001). [Figure 5-2]Figure 5: Effect of small molecule combinations on the efficiency of precise genome editing (PGE) in CALD1, KATNA1, and SLITRK1 by Cas9D10A and Cas9, and in HPRT and DMNT1 by Cpf1. Small molecules have an additive effect on PGE efficiency in the 409-B2 iCRISPR iPSC line by Cas9D10A (A), but not by Cas9 (B). PGE, PGE + indels, and indels are indicated by green, gray, or blue bars, respectively. Error bars indicate standard deviations of three technical replicates for A, B, and C and two technical replicates for D. The concentrations used were NU7026 20 μM, trichostatin A (TSA) 0.01 μM, MLN4924 0.5 μM, NSC 19630 1 μM, NSC 15520 5 μM, AICAR 20 μM, and RS-1 1 μM. CRISPY mix refers to a small molecule mixture of NU7026, TSA, MLN4924, and NSC 15520. The significance of changes in knock-in efficiency was determined using a two-way ANOVA and Tukey's multiple comparisons pooled across the three genes CALD1, KATNA1, and SLITRK1. Gene and treatment were treated as random and fixed effects, respectively. Three technical replicates were included for each gene in the analysis. P values ​​are adjusted for multiple comparisons (**P<0.01, ***P<0.001). [Figure 5-3]Figure 5: Effect of small molecule combinations on the efficiency of precise genome editing (PGE) in CALD1, KATNA1, and SLITRK1 by Cas9D10A and Cas9, and in HPRT and DMNT1 by Cpf1. Small molecules have an additive effect on PGE efficiency in the 409-B2 iCRISPR iPSC line by Cas9D10A (A), but not by Cas9 (B). PGE, PGE + indels, and indels are indicated by green, gray, or blue bars, respectively. Error bars indicate standard deviations of three technical replicates for A, B, and C and two technical replicates for D. The concentrations used were NU7026 20 μM, trichostatin A (TSA) 0.01 μM, MLN4924 0.5 μM, NSC 19630 1 μM, NSC 15520 5 μM, AICAR 20 μM, and RS-1 1 μM. CRISPY mix refers to a small molecule mixture of NU7026, TSA, MLN4924, and NSC 15520. The significance of changes in knock-in efficiency was determined using a two-way ANOVA and Tukey's multiple comparisons pooled across the three genes CALD1, KATNA1, and SLITRK1. Gene and treatment were treated as random and fixed effects, respectively. Three technical replicates were included for each gene in the analysis. P values ​​are adjusted for multiple comparisons (**P<0.01, ***P<0.001). [Figure 5-4]Figure 5: Effect of small molecule combinations on the efficiency of precise genome editing (PGE) in CALD1, KATNA1, and SLITRK1 by Cas9D10A and Cas9, and in HPRT and DMNT1 by Cpf1. PGE efficiency of HPRT and DMNT1 by recombinant Cpf1 in 409-B2 hiPSCs was similarly increased with CRISPY Mix (C). With CRISPY Mix, PGE efficiency was also increased by plasmid-delivered Cas9n-2A-GFP (GFP-FACS enriched) in SC102A1 hiPSCs and H9 hESCs, and by recombinant Cpf1 in chimpanzee SandraA ciPSCs (D). PGE, PGE + indels, and indels are indicated by green, gray, or blue bars, respectively. Error bars indicate standard deviations of three technical replicates for A, B, and C and two technical replicates for D. The concentrations used were NU7026 20 μM, trichostatin A (TSA) 0.01 μM, MLN4924 0.5 μM, NSC 19630 1 μM, NSC 15520 5 μM, AICAR 20 μM, and RS-1 1 μM. CRISPY mix refers to a small molecule mixture of NU7026, TSA, MLN4924, and NSC 15520. The significance of changes in knock-in efficiency was determined using a two-way ANOVA and Tukey's multiple comparisons pooled across the three genes CALD1, KATNA1, and SLITRK1. Gene and treatment were treated as random and fixed effects, respectively. Three technical replicates were included for each gene in the analysis. P values ​​are adjusted for multiple comparisons (**P<0.01, ***P<0.001). [Figure 6]Figure 6: Effect of CRISPY mix and small molecule combinations on the efficiency of Cpf1-mediated HPRT precise genome editing (PGE) in non-pluripotent cell types. All possible combinations of CRISPY mix components are shown for HEK293 and K562 cells (A). NU7026 increases PGE efficiency, whereas TSA and NSC15520 have no clear effect. MLN4924 has a clear disruptive effect on PGE efficiency in these cell lines with cancer characteristics. MLN4924 also has a disruptive effect on PGE efficiency in primary cells (B). CRISPY mix without MLN4924 has a greater effect on PGE efficiency than NU7026 alone in CD4+ T and CD34+ progenitor cells. NU7026 and NSC15520 also have a disruptive effect on PGE efficiency in primary human epidermal keratinocytes (HEKa). PGE, PGE + indels, and indels are indicated by green, gray, or blue bars, respectively. Error bars indicate the standard deviation of two technical replicates for each of two independent experiments for A and two independent experiments for B. CRISPY mix represents a mixture of small molecules: 20 μM NU7026, 0.01 μM trichostatin A (TSA), 0.5 μM MLN4924, and 5 μM NSC 15520. [Figure 7]Figure 7: Toxicity of the CRISPY mix and its components. (A) shows a resazurin assay of 409-B2-iCRISPR-Cas9n cells after 24 hours of incubation with the small molecules and combinations shown in Figures 4 and 5, with or without RNAiMax, gRNA, and ssODN. Resazurin is converted to fluorescent resorufin by cellular dehydrogenases, and the resulting fluorescence (excitation: 530-570 nm, emission: 590-620 nm) is a recognized marker for cell viability (O'Brien et al. 2000). The CRISPY mix, highlighted by a black border, is slightly toxic, without additive toxic effects of its components. Error bars indicate the standard deviation of two technical replicates. (B) shows a karyotype analysis after five passages of cells with the CRISPY mix and mock treatment. At least 20 metaphase nuclei from the bulk and five clones of each condition were analyzed using trypsin-induced Giemsa staining. No numerical or large-scale chromosomal aberrations were identified, except for a small subset of metaphase nuclei from two single clones corresponding to CRISPY mix (3 of 25 metaphase nuclei (polyploid)) and mock treatment (4 of 25 metaphase nuclei (polyploid)). [Figure 8-1] Figure 8: Effect of CRISPY mix and its component NU7026 on the efficacy of BFP insertion (ssODN) in human induced pluripotent stem cells. The design of the mtagBFP2 (Subach et al. 2011) ssODN donor and iCRISPR system is shown in (A). We inserted an 871-nt sequence (including a 50-nt homology arm) encoding a 2A self-cleaving peptide directly in front of the blue fluorescent protein (BFP) immediately after the N-terminal nuclear localization signal sequence (NLS) of Cas9n in the heterozygous AAVS1 iCRISPR locus (409-B2 iCRISPR-Cas9n hiPSC). When the sequence was inserted, doxycycline led to the expression of nuclear-translocated BFP. [Figure 8-2]Figure 8: The effect of CRISPY mix and its component NU7026 on the efficacy of BFP insertion (ssODN) in human induced pluripotent stem cells. Representative images of mock, NU7027, and CRISPY mix treatments after 7 days of BFP expression are shown in (B) as phase contrast (PC), propidium iodide nuclear staining (PI), mtagBFP2 expression (BFP), and the combination of PI and BFP. Two images (50x magnification, white size bar 200 µm) from each of three technical replicates for each treatment were used to quantify the percentage of cells with BFP insertion using ImageJ (C). [Figure 9]Figure 9: Catalytically inactive DNA-PKcs (K3753R) promotes homology-directed repair (HDR) and blocks non-homologous end joining (NHEJ). After a double-strand break (DSB) induced, e.g., by CRISPR Cas9 or Cpf1 (light blue with gray gRNA), the DNA ends are covered with Ku70 / 80 (orange), followed by binding of DNA-PKcs (cyan), both of which form a DNA-PK complex at each DSB end (A). Autophosphorylation of DNA-PKcs leads to the recruitment and activation of downstream NHEJ proteins. When DNA-PKcs is catalytically active, NHEJ prevails over HDR. When DNA-PKcs is catalytically inactivated (e.g., by the K3753R mutation), autophosphorylation cannot occur and the NHEJ pathway is blocked. Kinase-inactive DNA-PKcs preferentially leads to HDR repair of DSBs. The organizational structure of DNA-PKcs is shown in (B) along with its phosphorylation clusters, its kinase (purple), and the K3753R mutation (dark blue) (modified from Neal et al. 2014). The 4128 aa (approximately 470 kDa) long enzyme has the following serine / threonine phosphorylation clusters: N (residues 56 and 72), JK (residues 946 and 1003), PQR (five residues between 2023 and 2056), and ABCDE (six residues between 2609 and 2647). Some clusters activate DNA-PKcs upon phosphorylation, whereas others disengage it from NHEJ or even inactivate the kinase (Neal et al. 2014). Phosphorylation of the N cluster and T3950, as well as the K3753R mutation, have been shown to inactivate the kinase activity (Neal et al. 2011; Shrivastav et al. 2008; Douglas et al. 2007). DNA-PKcs and its surrounding sequence at K3753 are evolutionarily conserved in vertebrates (C) (Kent et al. 2002). [Figure 10]Figure 10: Catalytically inactive DNA-PKcs (K3753R) results in nearly complete conversion of DNA double-strand breaks into precise genome edits (PGEs). The KR mutant results in increased PGEs compared to wild-type DNA-PKcs in 409-B2 hiPSCs, regardless of the type of double-strand break introduced. Increased PGEs are shown for CALD1, KATNA1, SLITRK1, and PRKDC (R3753K) by Cas9 double-nicking (A), and for HPRT by CALD1 or Cpf1 by Cas9 (B). PGEs, PGEs + indels, and indels are indicated by green, light green, or blue bars, respectively. Error bars indicate standard deviations for three technical replicates for each of two independent experiments for A and two technical replicates for B. Cells were incubated with doxycycline for 3 days to express Cas9n for double-nicking in A. [Figure 11-1] Figure 11: Efficient multiplex precise genome editing (MPGE) of CALD1, KATNA1, and SLITRK1. Electroporation of gRNA and ssODN DNA donors achieved robust bulk precise genome editing (PGE) efficiency for all three genes, including the DNA-PKcs KR mutation, in 409-B2 hiPSCs (A). PGE, PGE + indel, and indel are indicated by green, light green, or blue bars, respectively. Error bars indicate standard deviations of two technical replicates. For double nicking, cells were incubated with doxycycline for 4 days and Cas9n expression was allowed. The ancient mutation and silent blocking mutation do not always integrate together into the chromosome where the targeted nucleotide substitution (TNS) occurred; a blocking mutation separated from other mutations can integrate as the only TNS (B). [Figure 11-2]Figure 11: Double-nicking guide targets (blue) and mutations (green) for each gene are shown to scale. A heat map of genome editing events (TNS and / or indels) in the chromosomes containing CALD1, KATNA1, and SLITRK1 for the 33 clones analyzed is shown in (C). Most clones remained wild-type or were correctly edited for all three genes. The heat map shows either two chromosomes for a single gene or six chromosomes for all three genes, with the integration of an indel and any TNS (left panel) or at least an ancestral mutation (right panel), whether a block mutation or an ancestral mutation. MPGE clone generation is possible in less than two weeks when single-cell dilution plating is performed using a small portion of freshly electroporated cells (D). Of the 56 colonies picked, 45 survived, 12 of which were derived from more than one cell (based on DNA sequencing read ratios), leaving 33 clones for analysis. [Figure 12] Figure 12: Additive effect of CRISPY mix and DNA-PKcs (K3753R) on the efficiency of precise genome editing (PGE) in CALD1, KATNA1, and SLITRK1 by Cas9D10A. PGE efficiency is significantly increased in the DNA-PKcs KR mutant (KR) compared to the wild type (WT), and is further increased by the addition of CRISPY mix to single genes (A), and even more so for multiple PGEs, which generally have lower efficiency. PGE, PGE + indels, and indels are indicated by green, light green, or blue bars, respectively. Error bars indicate the standard deviation of three technical replicates. CRISPY mix represents a small molecule mixture of 20 μM NU7026, 0.01 μM trichostatin A (TSA), 0.5 μM MLN4924, and 5 μM NSC 15520. For double nicking, cells were incubated with doxycycline for 2 days (4 days for multiplexing) to allow Cas9n expression. [Figure 13]Figure 13: Representative karyogram of 409-B2 iCRISPR hiPSCs containing the DNA-PKcs KR mutation after 3 months in culture. Of 25 metaphase nuclei analyzed by trypsin-induced Giemsa staining, all showed a healthy karyotype (46,XX). No numerical or gross chromosomal aberrations were identified (350 bands, 3 grayscale levels). DETAILED DESCRIPTION OF THE INVENTION

[0019] In a first aspect, the present invention provides a method for genome editing, as hereinbelow referred to as a method for the preparation of a gene encoding a gene for genome editing. The present invention relates to a compound designated Compound (I), which is an inhibitor of histone deacetylase (HDAC).

[0020] HDAC inhibitors are known as cytostatic agents that inhibit tumor cell growth by inducing cell cycle arrest, differentiation and / or apoptosis. HDAC inhibitors usually act by binding to the zinc-containing catalytic domain of HDAC. They can be classified according to the chemical moiety that binds to the zinc ion. Examples of suitable classes of HDAC inhibitors are: (1) Hydroxamate compounds, (2) Cyclic tetrapeptides and depsipeptides that bind to zinc ions via thiol groups; (3) benzamide compounds, (4) electrophilic ketones and (5) Fatty acid compounds is.

[0021] HDAC inhibitors are reviewed, for example, in Khan & La Thangue (Immunol. Cell Biol. 90 (2012), 85-94) and Falkenberg & Johnstone (Nature Rev. Drug Discovery 13 (2014) 673-691), which are incorporated herein by reference.

[0022] According to the present invention, the HDAC inhibitor is preferably selected from synthetic non-nucleoside compounds, eg small molecules having a molecular weight of 1500 Da or less, or 1000 Da or less. Specific examples of HDAC inhibitors include Trichostatin A, Vorinostat, Entinostat, Panobinostat, Mocetinostat, Belinostat, Romidepsin, MC1568, Tubastatin A HCl, Givinostat, LAQ824, CUDC-101, Quisinostat 2HCl, Pracinostat, PCI-34051, Droxinostat, PCI-24781, RGFP966, AR-42, Rocilinostat, Valproic Acid acid, CI994, CUDC-907, Tubacin, M344, Resminostat, RG2833, Divalproex Sodium, Scriptaid, Phenylbutyrate, Tubastatin A, CAY10603, Nexturastat A, BG45, LMK-235, Santacruzamate A, BRD73954, HPOB, TMP269, Tasquinimod and 4SC-202, and salts or solvates thereof, in particular pharmaceutically acceptable salts or solvates thereof.

[0023] A preferred compound (I) is trichostatin A (including its salts and solvates).

[0024] In a second aspect, the present invention relates to a compound that is an inhibitor of NEDD8-activating enzyme (NAE), hereinafter referred to as compound (II), for use in genome editing.

[0025] NAE inhibitors have been reviewed, for example, by Nawrocki et al. (Exp Opin Investing Drugs 21 (2012), 1564-1573) or as anti-viral agents, e.g., as reviewed by Le-Trilling et al. (Sci. Rep. 6 (2016), doi:19977), which are incorporated herein by reference.

[0026] According to the present invention, the NAE inhibitor is preferably selected from synthetic non-nucleoside compounds, e.g., small molecules having a molecular weight of 1500 Da or less, or 1000 Da or less. A preferred NAE inhibitor is MLN4924 (Pevonedistat) or any salt or solvate thereof, in particular any pharmaceutically acceptable salt or solvate thereof.

[0027] In a third aspect, the present invention relates to a compound that is an inhibitor of DNA-dependent protein kinase (DNA-PK), in particular an inhibitor of its catalytic subunit (DNA-PKcs), hereinafter referred to as compound (III), for use in genome editing.

[0028] DNA-PK inhibitors are known chemotherapeutic agents, as reviewed, for example, by Davidson et al. (Front. Pharmacol. 4 (2013), doi:13 3389) (hereby incorporated by reference).

[0029] According to the present invention, the DNA-PK inhibitor is preferably selected from synthetic non-nucleoside compounds, e.g., small molecules having a molecular weight of 1500 Da or less, or 1000 Da or less. Specific examples of DNA-PK inhibitors are NU7026, NU7441, PIK-75, and PI-103, as well as salts or solvates thereof, particularly pharmaceutically acceptable salts and solvates thereof.

[0030] In a preferred embodiment, compound (III) is NU7026 (including salts and solvates thereof).

[0031] In a fourth aspect, the present invention relates to a compound that is an inhibitor of replication protein A (RPA), hereinafter referred to as compound (IV), for use in genome editing.

[0032] RPA inhibitors are known as antitumor agents, as reviewed, for example, by Neher et al. (Mel. Cancer Ther. 10 (2011), 1756-1806) (hereby incorporated by reference).

[0033] According to the present invention, the RPA inhibitor is preferably selected from synthetic non-nucleoside compounds, e.g., small molecules having a molecular weight of 1500 Da or less or 1000 Da or less. Specific examples of RPA inhibitors are NSC15520, TDRL-505, and NSC111847, as well as salts or solvates thereof, particularly pharmaceutically acceptable salts and solvates thereof.

[0034] A preferred embodiment of compound (IV) is NSC15520 (including salts and solvates thereof).

[0035] The present inventors have found that compound (I), compound (II), compound (III), or compound (IV) increases the frequency of precise genome editing in eukaryotic cells, such as cells of animals, e.g., mammals, including humans.

[0036] In particular, the inventors have found that compounds (I), (II), (III), and / or (IV) have additive effects when administered together. In particular, when using a combination of compounds trichostatin A, MLN4924, NSC15520, and NU7026, an increase in precise genome editing of up to 6.7-fold or nearly 50% edited chromosomes was achieved. This is, to the inventors' knowledge, the highest genome editing efficiency ever described in human pluripotent stem cells. Furthermore, when the above-mentioned compound combination was used in pluripotent stem cells containing a catalytically inactive DNA protein kinase catalytic subunit, specifically the K3753R mutant, near-complete and precise genome editing was achieved, with up to 82% edited chromosomes or a 19.2-fold gain. Furthermore, they achieved multiplexed precise genome editing (MPGE) in three genes on both chromosomes in less than two weeks without selection, demonstrating MPGE for the first time in a mammalian system. One-third of the clones analyzed contained targeted nucleotide substitutions in three genes on both chromosomes.

[0037] A particularly powerful additive effect has been found when a combination of two or more of compounds (I), (II), (III), and (IV), in particular a combination of at least one compound (III) and at least one compound (I), and optionally at least one compound (II) and / or at least one compound (IV), is administered together with the use of a nuclease (e.g., Cpf1) or nickase enzyme system (e.g., Cas9D10A) capable of introducing staggered cuts at desired loci into DNA double strands, e.g., chromosomal DNA.

[0038] In further experiments, the potent effect of administering a combination of at least one compound (III) and at least one compound (I), and optionally at least one compound (IV), particularly in the absence of compound (II), together with the use of a nuclease (e.g., Cpf1) or nickase enzyme system (e.g., Cas9D10A) capable of introducing staggered cuts at desired loci in DNA double strands, e.g., chromosomal DNA, was demonstrated in hematopoietic cells, e.g., CD4 + T cells such as T cells or hematopoietic progenitor cells (e.g., CD34 + cells).

[0039] In the human embryonic kidney cell line HEK293 and the leukemia cell line K562, a strong effect was found when at least one compound (III) was administered, optionally together with at least one compound (I) and / or at least one compound (IV), particularly in the absence of compound (II), in conjunction with the use of a nuclease (e.g., Cpf1) or nickase enzyme system (e.g., Cas9D10A) capable of introducing staggered cuts at desired loci in DNA double strands, e.g., chromosomal DNA.

[0040] Thus, one aspect of the present invention relates to a combination, e.g., a composition or kit, comprising at least two of (a) compound (I), (b) compound (II), (c) compound (III), and (d) compound (IV). A preferred embodiment is a combination in which compound (I) is trichostatin A, and / or compound (II) is MLN4924, and / or compound (III) is NU7026, and / or compound (IV) is NSC15520. In particular, the combination of the present invention is intended for use in genome editing, including multiple genome editing on both chromosomes, in both non-medical and medical applications.

[0041] In the context of the present invention, the term "combination" includes a composition comprising at least two compounds as described above, mixed together, optionally with a suitable carrier, e.g., a pharmaceutically acceptable carrier. The term "combination" also includes a kit comprising at least two compounds as described above in separate forms, each optionally with a suitable carrier, e.g., a pharmaceutically acceptable carrier.

[0042] Furthermore, the present invention relates to a combination, e.g., a composition or kit comprising: (i) at least one compound (I) and at least one compound (II); (ii) at least one compound (I) and at least one compound (III); (iii) at least one compound (I) and at least one compound (IV); (iv) at least one compound (II) and at least one compound (III); (v) at least one compound (II) and at least one compound (IV); or (vi) at least one compound (III) and at least one compound (IV). Preferred compounds (I), (II), (III), and / or (IV) are as described above.

[0043] Furthermore, the present invention relates to a combination, e.g., composition or kit comprising: (i) at least one compound (I), at least one compound (II), and at least one compound (III); (ii) at least one compound (I), at least one compound (II), and at least one compound (IV); or (iii) at least one compound (II), at least one compound (III), and at least one compound (IV). Preferred compounds (I), (II), (III), and / or (IV) are as described above.

[0044] Furthermore, the present invention relates to combinations, examples, compositions or kits comprising at least one compound (I), at least one compound (II), at least one compound (III), and at least one compound (IV). Preferred compounds (I), (II), (III), and / or (IV) are as described above.

[0045] In particularly preferred embodiments, the present invention relates to combinations comprising at least one compound (III) and at least one compound (I), and optionally at least one compound (II) and / or at least one compound (IV). In some embodiments, compound (II) is absent.

[0046] In more particularly preferred embodiments, the present invention relates to combinations comprising at least one compound (III) and at least one of compound (I) and compound (IV). In some embodiments, compound (II) is absent.

[0047] The combinations of the invention described may further comprise one or more additional compounds. In one embodiment, the combination may comprise a compound for synchronizing cells in the G2 / M phase, such as nocodazole and ABT-751 (Yang et al., 2016), paclitaxel (Shu et al., Apoptosis 2 (1997), 463-470), or colchicine or vincristine (Blajeski et al., J. Clin. Invest. 110 (2002), 91-95), or a salt or solvate thereof. In a further embodiment, the combination may comprise an Alt-NHEJ inhibitor, such as NSC19630, or a salt or solvate thereof, in particular with a catalytically inactive DNA protein kinase catalytic subunit.

[0048] The combinations, examples, compositions, or kits of the present invention are suitable for use in genome editing of eukaryotic target cells, particularly mammalian target cells, e.g., as described below, including animal target cells, such as target cells from non-human animals such as mice or zebrafish, and including stem cells, e.g., human stem cells (e.g., embryonic stem cells or pluripotent stem cells). In some embodiments, the target cells are stem cells of a eukaryotic target organism, including artificial or embryonic pluripotent stem cells, such as human artificial or embryonic pluripotent stem cells, as well as artificial or embryonic pluripotent stem cells from non-human animals. In other embodiments, the target cells are hematopoietic cells or hematopoietic progenitor cells. In yet other embodiments, the target cells are immortalized cells, such as cancer cells.

[0049] The combinations, examples, compositions, or kits of the present invention are particularly suitable for genome editing procedures that involve introducing staggered cuts, particularly staggered cuts with 5' overhangs, into the genome of target cells. To achieve this result, the target cells may contain a mutant CRISPR / Cas9 nickase or CRISPR / Cpf1 enzyme, such as the CRISPR / Cas9 D10A or CRISPR / Cas9 H840A enzyme, which are mutant CRISPR / Cas9 nickases. Alternatively, other genome editing enzymes, e.g., CRISPR, transcription Enzymes that provide staggered cuts in double-stranded target DNA may be present, such as activator-like effector nucleases (TALENs), zinc finger nuclease proteins, Thermus thermophiles Argonaute (TtAgo), recombinases, or meganucleases, or other enzymes. The present invention is also suitable for use with split-fusion versions of the above enzymes, such as split-fusion versions of Cas9 or Cas9 D10A (Zetsche et al., 2015). The enzyme(s) may be introduced into target cells by themselves, e.g., as proteins or ribonucleoproteins, or as nucleic acid molecules encoding the respective enzyme(s). The nucleic acid molecules may be introduced as expression vectors, such as plasmids, operably linked to appropriate expression control elements for transient or stable expression in target cells. Suitable transfection techniques for introducing proteins or nucleic acids into eukaryotic target cells are well known in the art and include lipofection, electroporation (e.g., nucleofection), calcium phosphate or viral-based methods.

[0050] In a particular embodiment, the present invention relates to the use of a combination comprising at least one compound (III) and at least one compound (I), and optionally at least one compound (II) and / or at least one compound (IV), for genome editing in eukaryotic target cells, which are stem cells, including artificial or embryonic pluripotent stem cells, such as human artificial or embryonic stem cells, wherein the genome editing procedure comprises introducing a staggered cut, in particular a staggered cut with a 5' overhang, into the genome of the target cell. The staggered cut can be introduced into the genome of the target cell by the enzyme as described above.

[0051] In a further specific embodiment, the present invention relates to T cells (e.g., CD4 + Hematopoietic cells such as T cells or CD34 + The present invention relates to the use of a combination comprising at least one compound (III) and at least one compound (I), and optionally at least one compound (IV), particularly in the absence of compound (II), for genome editing in eukaryotic target cells, such as hematopoietic progenitor cells, wherein the genome editing procedure comprises introducing a staggered cut, particularly a staggered cut with a 5' overhang, into the genome of the target cell. The staggered cut can be introduced into the genome of the target cell by the enzyme as described above.

[0052] In a further specific embodiment, the present invention relates to the use of a combination comprising at least one compound (III), optionally together with at least one compound (I) and / or compound (IV), for genome editing in eukaryotic target cells, which are immortalized mammalian cells, e.g., HEK293 or K562, particularly in the absence of compound (II), wherein the genome editing procedure comprises introducing a staggered cut, particularly a staggered cut with a 5' overhang, into the genome of the target cell. The staggered cut can be introduced into the genome of the target cell by an enzyme as described above.

[0053] The combinations, examples, compositions, or kits of the present invention may further comprise (i) a catalytically inactive but structurally intact DNA protein kinase catalytic subunit (DNA-PKcs), (ii) a nucleic acid molecule encoding the DNA protein kinase catalytic subunit of (i) and / or (iii) a eukaryotic cell containing or capable of expressing the DNA protein kinase catalytic subunit of (i) (including split-fusion forms thereof). Preferably, the catalytically inactive but structurally intact variant has at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% amino acid sequence identity to the corresponding wild-type sequence, e.g., human sequence NP_008835.5, and contains at least one mutation that results in reduced kinase activity compared to the wild-type sequence. Suitable structurally intact, catalytically inactive variants and tests for detecting such variants are described, e.g., by Neal et al., 2001 (incorporated herein by reference).

[0054] Catalytically inactive but structurally intact DNA-PKcs subunits can be introduced into target cells as proteins or as nucleic acids encoding the respective subunits for transient or stable expression in the target cells. This approach can be used in combination with knockdown of the endogenous DNA-PKcs gene in the target cells (e.g., by targeted homologous recombination or the use of RNA interference (e.g., siRNA)). Furthermore, this approach can be used in cells that do not have endogenous DNA-PKcs, for example, by using a DNA-PKcs mutant from a different species (e.g., a closely evolutionarily related species).

[0055] In particular, the DNA-PKcs mutant contains at least one mutation in the catalytic loop (amino acids 3919-3927), including the catalytic triad (N3927, D3922, H3924), or in the P loop (amino acids 3729-3735), or in the adjacent region (amino acids 3736-3760), including amino acids F3946, T3950, and especially K3753 (based on NCBI reference sequence NP_008835.5). It contains a truncation (e.g., Y4046) that reduces or inactivates kinase activity. * ) (based on NCBI reference sequence NP_008835.5). The amino acid positions shown may differ in DNA-PKcs or its orthologs in species other than humans.

[0056] Even more specifically, the DNA-PKcs mutants comprise at least one mutation at position K3753 (e.g., mutation K3753R and / or K3753H), at least one mutation at position D3922 (e.g., mutation D3922A), at least one mutation at position T3950 (e.g., mutation T3950D), and / or at least one mutation at position F39460 (e.g., F3946D) (based on NCBI reference sequence NP_008835.5). The indicated amino acid positions may differ in DNA-PKCs or their orthologs in species other than humans.

[0057] Furthermore, DNA-PKcs may contain at least one mutation in a phosphorylation cluster that is normally the target of its autophosphorylation function. These may include, for example, inactivating mutations, such as, but not limited to, alanine, for the PQR cluster (S2023 and / or S2029 and / or S2041 and / or S2053 and / or S2056), and / or activating (phosphomimic) mutations, such as, but not limited to, aspartic acid, for the ABCDE cluster (T2069 and / or S2612 and / or T2620 and / or S2624 and / or T2638 and / or T2647) and / or N cluster (S56 and / or S72) and / or JK cluster (T946 and / or S1003), based on the NCBI reference sequence NP_008835.5. The amino acid positions shown may differ in DNA-PKCs or their orthologues in species other than human.

[0058] The combinations, e.g., compositions, or kits are suitable for use with any type of donor nucleic acid molecule, including, but not limited to, single-stranded or double-stranded DNA molecules amplified in vivo or in vitro or chemically synthesized. The length of the donor nucleic acid molecule typically ranges from about 20 to 2,000 nt or more (e.g., about 80 to 120 nt, 50 to 200 nt, or 500 to 2,000 nt). The donor nucleic acid molecule is designed to contain at least one desired mutation, taking into account the wild-type sequence to be introduced into the genome of the target cell by genome editing. The mutation can be a single nucleotide mutation or a mutation encompassing multiple nucleotides. In this context, the term mutation refers to a substitution, deletion, or insertion of a single nucleotide or multiple nucleotides.

[0059] The above aspects can be used in vivo, e.g., in isolated cells or cell clusters, as well as in vivo. The combinations can be used in vitro or in cells of a target organism. The combinations can be applied in the cell types described above and using genome editing procedures described above, particularly those involving the use of DNA cleavage enzyme systems capable of introducing staggered cuts into double-stranded DNA. This embodiment also includes uses in medicine, including human medicine or veterinary medicine.

[0060] A further aspect of the present invention relates to the use of (i) a catalytically inactive but structurally intact DNA protein kinase catalytic subunit, as described above, in particular the mutant subunit K3753R, (ii) a nucleic acid molecule encoding the DNA protein kinase catalytic subunit of (i), and / or (iii) a eukaryotic cell containing or capable of expressing the DNA protein kinase catalytic subunit of (i), for genome editing in eukaryotic target cells, in particular vertebrate target cells, e.g., rodent, (.) mammalian target cells, including human or non-human target cells, including human stem cells.

[0061] This embodiment includes use in vivo, e.g., in isolated cells or cell clusters, as well as in vitro, in cells of a target organism. The DNA-PKcs variants can be applied in all cell types and with all types of genome editing procedures, including the use of any DNA cleavage enzyme (e.g., an enzyme system capable of introducing staggered cuts into double-stranded DNA, as described above), as well as other enzyme systems (e.g., an enzyme system capable of introducing blunt-ended cuts). This embodiment also includes uses in medicine, including human or veterinary medicine.

[0062] A further aspect of the present invention is the use of a combination, e.g., composition, or kit, comprising at least two different compounds (I), (II), (III), and (IV), in medicine, including human or veterinary medicine. An effective dosage of a compound according to the present invention, or a salt, solvate, or prodrug thereof, is used in addition to a physiologically acceptable carrier, diluent, and / or adjuvant for preparing a pharmaceutical composition. The dosage of the active compound may vary depending on the route of administration, the age and weight of the patient, the nature and severity of the disease being treated, and similar factors. The daily dosage may be given as a single dose, administered at once, or may be subdivided into two or more daily doses, and is generally between 0.001 and 2000 mg. It is particularly preferred to administer a daily dosage of 0.1 to 500 mg, e.g., 0.1 to 100 mg.

[0063] Suitable administration forms are oral, parenteral, intravenous, transdermal, topical, inhalation, nasal, and sublingual formulations. It is particularly preferred to use oral, parenteral (e.g., intravenous or intramuscular, nasal) formulations of the compounds according to the invention, e.g., dry powders, or sublingual formulations. Conventional galenic formulation forms such as tablets, sugar-coated tablets, capsules, dispersible powders, granules, aqueous solutions, alcoholic aqueous solutions, aqueous or oily suspensions, syrups, juices, or drops can be used.

[0064] Solid pharmaceutical forms may contain inactive ingredients and carrier materials such as calcium carbonate, calcium phosphate, sodium phosphate, lactose, starch, mannitol, alginate, gelatin, guar gum, magnesium stearate, aluminum stearate, methylcellulose, talc, highly dispersed silicic acid, silicone oil, high molecular weight fatty acids (such as stearic acid), gelatin, agar, or vegetable or animal fats or oils, or solid high molecular weight polymers (such as polyethylene glycol); formulations suitable for oral administration may contain additional flavorings and / or sweeteners, if desired.

[0065] Liquid pharmaceutical forms may be sterilized and / or may, where appropriate, contain auxiliary substances such as preservatives, stabilizing agents, wetting agents, osmotic agents, emulsifying agents, spreading agents, solubilizing agents, salts for regulating the osmotic pressure or for buffering, sugars or sugar alcohols, and / or viscosity adjusters.

[0066] Preparations for parenteral administration may be in the form of discrete dosage units such as ampoules or vials. Solutions of the active compound, preferably aqueous solutions, and in particular isotonic solutions and suspensions, are also preferably used. These injection forms can be made available as ready-to-use preparations by mixing the active compound, for example, a lyophilized product containing, if appropriate, other solid carrier substances, with the desired solvent or suspension, or can be prepared only immediately before use.

[0067] The formulation for nasal administration may be in the form of an aqueous or oily solution, or an aqueous or oily suspension, or may be in the form of a lyophilized product prepared with a suitable solvent or suspending agent before use.

[0068] Inhalable formulations may be present as powders, solutions or suspensions. Preferably, the inhalable formulation is in the form of a powder, for example as a mixture of the active ingredient with suitable formulation auxiliaries such as lactose.

[0069] The formulations are manufactured, dispensed and sealed under normal antimicrobial and aseptic conditions.

[0070] The compounds of the present invention can be administered alone or in combination therapy with additional active agents.

[0071] The medical use of the combination of the present invention particularly includes targeted gene therapy, e.g., the treatment of disorders associated with an undesirable genotype in a patient in need of treatment. For example, the disorder is a metabolic dysfunction or cancer. According to the present invention, cells derived from a patient can be subjected to a genome editing procedure in the presence of the combination as described above, thereby increasing the efficiency of precise genome editing. This procedure can be performed in vivo, i.e., by administering the combination to the patient, or ex vivo using cells isolated from the patient (which are then reimplanted into the patient after successful genome editing). The patient can be a vertebrate, such as a mammal, preferably a human patient. Finally, the combination of the present invention is also suitable for genome editing in plant cells or plants.

[0072] The present invention will now be explained in more detail by the following figures and examples. [Example]

[0073] method cell culture Stem cell lines cultured for this project included human 409-B2 hiPSCs (female, Riken BioResource Center) and SC102A1 hiPSCs (male, BioCat GmbH), chimpanzee SandraA ciPSCs (female, Mora-Bermudez et al. 201637), and H9 hESCs (female, WiCell Research Institute, ethics permit AZ 3.04.02 / 0118). Stem cell lines were grown on Matrigel Matrix (Corning, 35248). mTeSR1 (StemCell Technologies, 05851) containing mTeSR1 supplement (StemCell Technologies, 05852) was used as the culture medium. The non-pluripotent cell types and their respective media used were: HEK293 (ECACC, 85120602) in DMEM / F-12 (Gibco, 31330-038) supplemented with 10% FBS (SIGMA, F2442) and 1% NEAA (SIGMA, M7145); K562 (ECACC, 89121407) in IMDM (ThermoFisher, 12440053) supplemented with 10% FBS; CD4 + T (HemaCare, PB04C-1) and RPMI 1640 (ThermoFisher, 11875-093) supplemented with 10% FBS (activated with Dynabeads Human T-Activator (CD3 / CD28) (ThermoFisher, 11131D)); CD34 + StemSpan SFEM (Stemcell, 09600) supplemented with progenitor cells (HemaCare, M34C-1) and StemSpan CC110 (StemCell, 02697) and H The media was EKa (Gibco, C0055C), Medium 154 (ThermoFisher, M154500), and Human Keratinocyte Growth Supplement (ThermoFisher, S0015). Cells were grown at 37°C in a humidified incubator gassed with 5% CO2. Medium was changed daily for stem cells and every two days for non-pluripotent cell lines. Cell cultures were maintained for 4–6 days until 80% confluent and then subcultured at a 1:6–1:10 dilution. Adherent cells were dissociated using EDTA (VWR, 437012C). To increase cell survival, 10 μM Rho-associated protein kinase (ROCK) inhibitor Y-27632 (Calbiochem, 688000) was added to the medium after 1 day of cell division.

[0074] Generation and validation of iCRISPR cell lines iCRISPR-Cas9 lines were generated using human 409-B2 iPSCs as described by Gonzalez et al. (Gonzalez et al. 2014). To generate the iCRISPR-Cas9D10A line, the Puro-Cas9 donor was subjected to site-directed mutagenesis using the Q5 mutagenesis kit (New England Biolabs, E0554S). Primers were ordered from IDT (Coralville, USA) (shown in Table 2). Expression of pluripotency markers SOX2, OCT-4, TRA1-60, and SSEA4 in iCRISPR lines indicated the pluripotency of PSCs. Quantitative PCR was used to confirm doxycycline-inducible Cas9 or Cas9D10A expression, and digital PCR was used to rule out off-target integration of the iCRISPR cassette (data not shown).

[0075] small molecule The commercially available small molecules used in this study were NU7026 (Sigma, T8552), TSA (Sigma, T8552), MLN4924 (Adooq BioScience, A11260), NSC 19630 (Calbiochem, 681647), NSC 15520 (ChemBridge, 6048069), AICAR (Sigma, A9978), RS-1 (Calbiochem, 553510), resveratrol (Selleckchem, S1396), SCR7 (XcessBio, M60082-2s), L755507 (TOCRIS, 2197), B02 (Sigma, SML0364), and STL127685 (Vitas-M). STL127685 is a 4-fluorophenyl analog of STL127705, which is not commercially available. Dimethyl sulfoxide (DMSO) (Thermo Scientific, D12345) was used to make a 15 mM (or 10 mM for NU7026) stock. The limiting factor for NU7026 concentration is solubility. Suitable working solutions were made for various concentrations, such that the addition of each small molecule resulted in a final DMSO concentration of 0.08% (or 0.2% for NU7026) in the medium. Addition of all small molecules results in a final DMSO concentration of 0.7%.

[0076] gRNA and ssODN design We introduced a single desired mutation into three genes, CALD1, KATNA1, and SLITRK1, to revert to the state of the last common ancestor of humans and Neanderthals (Pruefer et al. 2013). Pairs of gRNAs for editing with Cas9D10A nickase were selected to efficiently cleave within a short distance from the desired mutation and their respective partner sgRNAs. Efficiency was estimated using the sgRNA scorer 1.0 tool (Chari et al. 2015) as percentile rank scores. Donor ssODNs for nickase editing were designed to carry the desired mutation and Cas9 blocking mutations upstream and downstream of each nick to prevent re-cutting of the locus. The gRNAs of the nickase gRNA pair that cut closer to the desired mutation were used for Cas9 nuclease editing with a 90-nt ssODN centered on the desired mutation and containing a Cas9 blocking mutation (Figure 2). The ssODNs for editing HPRT and DMNT1 using Cpf1 were designed to contain a blocking mutation near the PAM site and an additional mutation near the cleavage site. gRNAs (crRNA and tracR) and ssODNs were ordered from IDT (Coralville, USA). The ssODN and crRNA targets are shown in Table 2.

[0077] Lipofection of oligonucleotides Two days before lipofection, cells were incubated with medium containing 2 μg / ml doxycycline (Clontech, 631311). Lipofection (reverse transfection) was performed using the alt-CRISPR manufacturer's protocol (IDT) with final concentrations of 7.5 nM of each gRNA and 10 nM of each ssODN. Briefly, 0.75 μl of RNAiMAX (Invitrogen, 13778075) and each oligonucleotide were separately diluted with 25 μl of OPTI-MEM (Gibco, 1985-062) and incubated at room temperature for 5 minutes. Both dilutions were mixed to obtain 50 μl of OPTI-MEM containing RNAiMAX, gRNA, and ssODN. The lipofection mixture was incubated at room temperature for 20–30 minutes. During incubation, cells were dissociated with EDTA for 5 minutes and counted using a Countess Automated Cell Counter (Invitrogen). Lipofection mix, 100 μl containing 25,000 dissociated cells in mTeSR1 supplemented with Y-27632, 2 μg / ml doxycycline, and each small molecule(s) to be tested were thoroughly mixed and placed in one well of a 96-well plate coated with Matrigel Matrix (Corning, 35248). After 24 hours, the medium was replaced with regular mTeSR1 medium.

[0078] Electroporation (nucleofection) of oligonucleotides and ribonucleoproteins Recombinant AsCpf1 and SpCas9 proteins and electroporation facilitator were ordered from IDT (Coralville, USA), and nucleofection was performed using the manufacturer's protocol with the following modifications: 1 million cells of each strain, 78 pmol of electroporation facilitator, 160 pmol of each gRNA (crRNA / tracR duplex for Cas9 and crRNA for Cpf1) (320 pmol for double nicking with both gRNAs for one gene), 200 pmol of ssODN donor, and 252 pmol of CRISPR protein in 100 μl of Human Stem Cell nucleofection buffer (Lonza, VVPH-5022) or Human T Cell nucleofection buffer for CD4. + T cells(Lonza,VPA-1002) and Human CD34 Cell nucleofection buffer for CD34 + In a cuvette for progenitor cells (Lonza, VPA-1003), the B-16 program (or CD4 + For T cells, U-14 cells were used. For multiplexing, we used the iCRISPR-Cas9n hiPSC line expressing Cas9n, and only electroporated gRNA and single-stranded DNA donor. Cells were counted using a Countess Automated Cell Counter (Invitrogen).

[0079] FACS sorting 2 μg of plasmid DNA (pSpCas9n(BB)-2A-GFP(PX461), Addgene #48140) that does not inducibly express Cas9. Transfection into new cells was performed using the B-16 program of a Nucleofector 2b Device (Lonza) in a cuvette containing 1 million SC102A1 iPSCs or H9 ESCs in 100 μl of Human Stem Cell nucleofection buffer (Lonza, VVPH-5022). Cells were counted using a Countess Automated Cell Counter (Invitrogen). 24 h after nucleofection, cells were dissociated using Accutase (Sigma, A6964), filtered to obtain a single-cell solution, and subjected to fluorescence-associated cell sorting (FACS) for GFP-expressing cells. During sorting using a BD FACSAria III (Becton-Dickinson), cells were maintained at 4°C in mTeSR1 supplemented with Y-27632. 48 hours after selection, cells were subjected to lipofection with sgRNA, ssODN, and treatment with small molecules.

[0080] Illumina library preparation and sequencing Three days after lipofection, cells were dissociated using Accutase (Sigma, A6964), pelleted, and resuspended in 15 μl of QuickExtract (Epicentre, QE0905T). ssDNA was generated as a PCR template by incubation at 65°C for 10 min, 68°C for 5 min, and finally at 98°C for 5 min. Primers containing Illumina sequencing adapters for the CALD1, KATNA1, and SLITRK1 target loci were ordered from IDT (Coralville, USA). PCR was performed using the KAPA2G Robust PCR Kit (Peqlab, 07-KK5532-03) with the provided buffer B and 3 μl of cell extract in a total volume of 25 μl in a T100 thermal cycler (Bio-Rad). The PCR thermal cycling profile was: 95°C for 3 minutes; 34 cycles of (95°C for 15 seconds, 65°C for 15 seconds, 72°C for 15 seconds); 72°C for 60 seconds. P5 and P7 Illumina adapters with sample-specific indicators were added to the second PCR reaction (Kircher et al. 2012) using Phusion HF MasterMix (Thermo Scientific, F-531L) and 0.3 μl of the first PCR product. The PCR thermal cycling profile was: 98°C for 30 seconds; 25 cycles of (98°C for 10 seconds, 58°C for 10 seconds, 72°C for 20 seconds); 72°C for 5 minutes. Amplification was confirmed by size-separation agarose gel electrophoresis using EX gel (Invitrogen, G4010-11). The indexed amplicons were purified using Solid Phase Reversible Immobilization (SPRI) beads (Meyer and Kircher 2010). The double-indexed library was sequenced on a MiSeq (Illumina) system, which provided 2x150 bp paired-end sequences. After base calling using Bustard (Illumina), adapters were trimmed using leeHom (Renaud et al. 2014).

[0081] Sequence data analysis Sequencing data from CRISPR genome editing experiments were analyzed using CRISPresso (Pinello et al. 2016) for the percentage of wild-type, targeted nucleotide substitutions (TNS), indels, and a mixture of TNS and indels. The parameters used for the analysis were "-w 20," "--min_identity_score 70," and "--ignore_substitutions" (restricting the analysis to 20-bp windows from amplicons and each gRNA with at least 70% similarity to the wild-type sequence; substitutions were ignored because sequence errors would be incorrectly characterized as NHEJ events). Sequencing data from colonies after single-cell plating (Figure 3B and C) were further analyzed by assessing actual read sequences using SAMtool. The proportion of sequence reads was used to distinguish clonal from mixed colonies. A majority of reads were clearly composed of one sequence (homozygous) or two sequences with similar read numbers (heterozygous). Colonies were counted as clones if they were identical. Because our karyotypes were healthy, we assumed that each cell had two chromosomes. We recorded integrated block mutations, ancestral mutations, and indels for each chromosome in the clonal cells.

[0082] statistical analysis The significance of changes in TNS efficiency was determined using a two-way analysis of variance and Tukey's multiple comparisons pooled across the three genes CALD1, KATNA1, and SLITRK1. Genes and treatments were treated as random and fixed effects, respectively. Therefore, we tested the effect of treatments on their interactions with genes (Zar 1999). Three technical replicates were included for each gene in the analysis. We confirmed whether the assumptions of normal distribution and homogeneity of residuals were met by visual inspection of QQ plots (Field 2005) and residuals plotted against fitted values ​​(Quinn & Keough 2002). These showed that residuals were approximately symmetrically distributed, but with long tails (i.e., excessively large positive and negative residuals), and no clear deviation from the assumption of homogeneity. P values ​​are adjusted for multiple comparisons. Statistical analysis was performed using R.

[0083] Resazurin assay As described in "Oligonucleotide Lipofection," 409-B2 iCRISPR-Cas9n hiPSCs were seeded with or without editing reagents (RNAiMax, gRNA, and ssODN donor for KATNA1 editing). Small molecules or combinations of small molecules were added to the medium, and each condition was performed in duplicate. After 24 hours, the medium was aspirated and 100 μl of fresh medium was added along with 10 μl of resazurin solution (Cell Signaling, 11884). Resazurin is converted to fluorescent resorufin by cellular dehydrogenases, and the resulting fluorescence (excitation: 530-570 nm, emission: 590-620 nm) is recognized as a linear marker of cell viability (O'Brien et al. 2000). Cells were incubated with resazurin at 37°C. The redox reaction was measured every hour by absorbance measurement using a Typhoon 9410 imager (Amershamn Biosciences). After 5 hours, absorbance scans showed good contrast without saturation and were used to quantify absorbance using ImageJ and the "ReadPlate" plugin. Duplicate wells containing medium and resazurin but no cells were used as blanks.

[0084] Microscopy and image analysis 409-B2 iCRISPR-Cas9n hiPSCs were nucleofected with gRNA and blue fluorescent protein (BFP) single-stranded oligos (Figure 8A, 330 ng) in two technical replicates for either mock, NU7026, and CRISPY mix treatments. 2 μg / ml doxycycline (Clontech, 631311) was added to the culture medium for 7 days to allow expression of the nuclear-imported BFP in the precisely edited cells. Cells were then fixed with 4% formaldehyde in DPBS (ThermoFisher, A24881) for 15 min and then added to 100 μg / ml The cells were permeabilized with 1% saponin in DPBS (ThermoFisher, A24881) supplemented with RNAse A (ThermoFisher, EN0531) and 40 μg / ml propidium iodide (ThermoFisher, P3566) for 45 minutes at 37°C and washed three times with DPBS. Nuclei were counterstained with propidium iodide, which intercalates nucleic acids. Images were taken from each of three technical replicates for each treatment using a fluorescence microscope, Axio Observer Z (Zeiss): phase contrast, HcRed channel (BP 580-604 nm, BS Duplicate images (50x magnification) were acquired consisting of the BP 615nm, BP 625-725nm, 10,000ms), and DAPI channels (BP 335-383nm, BS 395nm, BP 420-470nm, 20,000ms). Images were blinded and scanned using Adobe BFP-positive nuclei were counted using the Photoshop CS5 counting tool. Propidium iodide-positive nuclei were quantified using ImageJ by dividing the nuclear area (default threshold) by the average single-nucleus area.

[0085] Karyotype analysis Microscopic analysis of the karyotype was performed after trypsin-induced Giemsa staining by the 'Saechsischer Inkubator für klinische Translation' (Leipzig, Germany) in accordance with international quality guidelines (ISCN 2016: International System of Nomenclature for Human Cytogenetics).

[0086] research design We aimed to test the efficiency of several small molecules for precise genome editing in iPSCs. Compounds SCR7, L755507, and RS-1 have been suggested in the literature as potential CRISPR-Cas effectors, while the other test compounds are not yet known for this purpose. The test compounds are listed in Table 1.

[0087] Table 1: Overview of small molecules evaluated in this study. Target proteins, their functions, and their respective pathways and small molecule functions are shown. References with an asterisk indicate small molecules as CRISPR-Cas effectors. Abbreviations: Alternative NHEJ (Alt-NHEJ), Damage-Dependent Signaling (DDS).

[0088] [Table 1]

[0089] As a tool for analyzing the effects of small molecules on HDR efficiency, we generated iCRISPR-Cas9 and iCRISPR-Cas9D10A iPSC lines that enable doxycycline-inducible expression of Cas9 or Cas9D10A and efficient delivery of ssODN and guide RNA (gRNA) (Gonzalez et al. 2014). A flow diagram of genome editing and analysis is shown in Figure 1. The designs of gRNA, ssODN, and primers used in editing three example genes, CALD1, KATNA1, and SLITRK1, are shown in Figure 2 and Table 2.

[0090] Table 2: Oligonucleotides used in this study. The gRNAs (crRNA targets) and single-stranded DNA donors (ssODNs) for CALD1, KATNA1, SLITRK1, HPRT, DMNT1, AAVS1-iCRISPR, and PRKDC editing, as well as primers for analysis of the Cas9 iCRISPR donor plasmid and Q5 site-directed mutagenesis, are shown. Mutations are in bold, and ancestral mutations are underlined. The donors KATNA1 Cas9D10A2 and SLITRK1 Cas9D10A2 have different silent mutations and were used for Figures 10-12.

[0091] [Table 2-1]

[0092] [Table 2-2]

[0093] The small molecules we selected to test for their ability to block NHEJ were STL127685, a 4-fluorophenyl analog of the SCR7 and Ku70 / 80 inhibitor STL127705 (Weterings et al. 2016), and the DNA-PK inhibitor NU7026 (Suzuki et al. 2016). We selected the WRN helicase inhibitor NSC 19630 to block Alt-NHEJ (Aggarwal et al. 2011). MLN4924, a NEDD8-activating enzyme (NAE) inhibitor, has been shown to inhibit neddylation of CtIP, which increases the extent of DNA end extension at strand breaks and thus regulates DNA double-strand break repair pathway selection, preferentially via HDR (Jimeno et al. 2015). DNA chipping leaves ssDNA, which is coated and stabilized by RPA before undergoing homology-seeking and recombination. We hypothesized that increased RPA availability favors HDR. Fumaropimaric acid (NSC15520) may prevent RPA from binding to p53 and RAD9 (Glanzer et al. 2011) (Glanzer et al. 2013), increasing the amount of RPA available for ssDNA. RAD51 is important for homologous recombination with double-stranded DNA (dsDNA), whereas RAD52 is required for annealing of ssDNA (Grimme et al. 2010). Therefore, we decided to test the effects of the RAD52 inhibitor AICAR (Sullivan et al. 2016), the RAD51 inhibitor B02 (Huang et al. 2011), and the RAD51 promoter RS-1 (Song et al. 2016) on the efficiency of precise genome editing. We also included small molecules described to enhance the repair protein kinase ATM. Resveratrol has been described to have a direct stimulatory effect on the catalytic efficiency of purified ATM in vitro (Lee et al. 2014). The histone deacetylase inhibitor trichostatin A has been described to activate the ATM-dependent DNA damage signaling pathway and increase HR (Lee 2007) (Jimeno et al. 2015).Finally, we included the β3 adrenergic receptor agonist L755507 in the screen.

[0094] result The effect of small molecules on precise genome editing We first tested different concentrations of small molecules to determine whether iCRISPR Cas9D10A We used Cas9D10A and Cas9D10B to verify their effects on precise genome editing of CALD1, KATNA1, and SLITRK1 (Figure 3). As a result, we selected the respective concentrations that resulted in the highest frequency of precise genome editing. When different concentrations resulted in similar effects on editing, we selected the lower concentration. The effects of selected small molecule concentrations on the efficiency of precise genome editing in CALD1, KATNA1, and SLITRK1 by Cas9D10A and Cas9D10B from repeated independent experiments are shown in Figure 4.

[0095] NU7026 treatment increased PGE induced by Cas9D10A (Figure 4A) and Cas9 (Figure 4B) at all three loci. The average changes were 1.5-fold for CALD1, 2.6-fold for KATNA1, and 2.5-fold for SLITRK1 with Cas9D10A, and 1.5-fold for CALD1, 1.6-fold for KATNA1, and 1.2-fold for SLITRK1 with Cas9. We found that in addition to NU7026, TSA and MLN4924 had an enhancing effect on the frequency of PGE induced by Cas9D10A. TSA increased PGE induced by Cas9D10A by 1.5-fold for CALD1, 2.2-fold for KATNA1, and 1.8-fold for SLITRK1; interestingly, no enhancing effect was observed with Cas9. MLN4924 increased PGE1 by 1.2-fold for CALD1, 1.1-fold for KATNA1, and 1.3-fold for SLITRK1 in Cas9D10A. MLN4924 had a slight reducing effect on PGE1 in Cas9. Treatment with NSC 15520 increased PGE1 by 1.4-fold and 1.3-fold for CALD1 in Cas9D10A and Cas9, respectively, but had no effect on PGE1 in KATNA1 and SLITRK1. NSC 19630, AICAR, RS-1, resveratrol, SCR7, L755507, and STL127685 had no clear effect on PGE1 frequencies for the three genes CALD1, KATNA1, and SLITRK1 compared with their respective controls in Cas9D10A, whereas Cas9 showed no effect or a reduced effect. B02 halved both Cas9D10A- and Cas9-induced PGE at all three loci.

[0096] We next tested whether combinations of small molecules had an enhancing effect on PGE2 induced by Cas9D10A or Cas9. We selected small molecules that showed an increase in PGE2 induced by Cas9D10A in at least one gene and never decreased PGE2; these molecules were: NU7026, TSA, MLN4924, NSC 19630, NSC 15520, AICAR and RS-1.

[0097] The effect of small molecule combinations on editing of three different loci (trees) with either Cas9D10A or Cas9 is shown in Figure 5. For editing with Cas9D10A, treatment with NU7026 for PGE resulted in a 2.3- or 1.8-fold higher PGE frequency than without the molecule (Tukey's pairwise comparisons, post-hoc comparisons: p<0.001) (Figure 5A). The combination of NU7026 and TSA resulted in a 1.3- or 1.6-fold higher PGE frequency than either NU7026 or TSA alone (p<0.001). Addition of MLN4924 to the NU7026 and TSA mixture resulted in an additional 1.3-fold increase in PGE (p<0.01). When using Cas9D10A for editing, the additional addition of NSC 15520 slightly increased the average PGE for all loci, but this did not reach statistical significance. The addition of NSC 19630, AICAR, and RS-1 had no measurable effect on PGE.

[0098] We conclude that the small molecule mix that most effectively increases the frequency of PGE induced by Cas9D10A is the combination of NU7026 (20 μM), TSA (0.01 μM), MLN4924 (0.5 μM), and NSC 15520 (5 μM). We refer to this as "CRIS" We named this "PY" nickase mix, which resulted in a 2.8-fold increase in PGE for CALD1 (from 11 to 31%), a 3.6-fold increase in KATNA1 (from 12.8 to 45.8%), and a 6.7-fold increase in SLITRK1 (from 4.7 to 31.6%) in the iCRISPR 409-B2 iPSC line.

[0099] When we used Cas9, which introduces blunt-end DSBs, the addition of other small molecules in addition to NU7026 had no significant effect (Figure 5B). In contrast, CRISPY mix with Cpf1 ribonucleoprotein, which generates staggered DNA breaks and was introduced into 409-B2 hiPSCs by electroporation, increased PGE by 2.9-fold for HPRT and 4.0-fold for DMNT1 (Figure 5D). Addition of NU7026 alone increased PGE by 2.1-fold for HPRT and 2.4-fold for DMNT1.

[0100] To test whether CRISPY Mix increases PGE in other pluripotent stem cell lines, we edited the gene KATNA1 in SC102A1 hiPSCs and H9 hESCs using Cas9n plasmid electroporation, and HPRT in chimpanzee iPSCs using Cpf1 ribonucleoprotein. PGE increased 2.6-fold, 2.8-fold, and 2.3-fold, respectively, a greater increase than NU7026 alone (Figure 5C).

[0101] In further experiments, the effect of the CRISPY mix and its components on the efficiency of targeted nucleotide substitution was examined in the immortalized human embryonic kidney cell line HEK293, the immortalized leukemia cell line K562, and the hematopoietic CD4 + T cells and CD34 + Electroporation of Cpf1 was tested in multiple non-pluripotent cell types, including hematopoietic progenitor cells and primary human keratinocytes (HEKα). Results are shown in Figure 6. NU7026, a component of the CRISPY mix, was found to be effective in all cell lines tested, except HEKα cells. In HEK293 and K562 cells, NU7026 significantly increased PGE efficiency, TSA and NSC15520 moderately increased efficiency, and MLN4924 had a distinct disruptive effect on cell lines with cancer characteristics. MLN4924 also had a disruptive effect on PGE efficiency in primary cells. CRISPY mix without MLN4924 significantly increased PGE efficiency in hematopoietic CD4 + T cells and CD34 +In progenitor cells, NU7026 and NSC15520 have a greater effect on PGE efficiency than NU7026 alone. In primary human keratinocytes (HEKα), NU7026 and NSC15520 also have a disruptive effect on PGE efficiency.

[0102] In further experiments, we tested the toxicity of the CRISPY mix and its components. After editing of KATNA1 with Cas9n double-nicking and 24-hour CRISPY mix treatment, cells showed 75% viability compared to no small molecule treatment, with no additive toxic effects of the components (Figure 7). Importantly, when we simulated five rounds of editing, each consisting of passaging cells with lipofection reagent and CRISPY mix and harvesting them after 3 days, the cells had a healthy karyotype without numerical or large-scale chromosomal aberrations, as shown by trypsin-induced Giemsa staining (Figure 13).

[0103] In yet further experiments, we tested the effect of the CRISPY mix and its component NU7026 on BFP insertion (ssODN) efficiency in human induced pluripotent stem cells. The results are shown in Figure 8. We inserted an 871-nt (including a 50-nt homology arm) sequence encoding a 2A self-cleaving peptide directly opposite the enhanced blue fluorescent protein (BFP) (Subach et al. 2011) in the AAVS1 iCRISPR locus. When the sequence is inserted, doxycycline leads to expression of nuclear-translocated BFP. Nuclei positive for BFP increased 7.1-fold (26.6%) compared to the no-CRISPY control (3.7%), whereas NU7026 alone produced only a 1.6-fold (6%) increase (Figure 8B and 8C). and C) CRISPY mix increases the efficiency of gene fragment insertion in iPSCs.

[0104] Effect of prepared DNA protein kinase catalytic subunit on precise genome editing The K3753R mutation near the ATP-binding site abolishes the kinase activity of DNA-PKcs. In CHOV3 cells, DSB-induced HDR in DNA-PKcs KR cells has been shown to be 2- to 3-fold greater than the elevated HDR levels in DNA-PKcs null cells and 4- to 7-fold greater than cells expressing wild-type DNA-PKcs (Shrivastav et al. 2008 and 2009).

[0105] We generated induced pluripotent stem cell (iPSC) lines containing a doxycycline-inducible Cas9 nickase (iCRISPR-D10A) as described by Gonzalez et al. (2014) and achieved highly efficient targeted DSBs with reduced off-target events (Shen et al. 2014). Using the integrated Cas9D10A, we replaced a lysine near the kinase active site of DNA-PKcs with an arginine (K3752R), inactivating its kinase activity while maintaining its overall structural integrity (Shrivastav et al. 2008, 2009). To compare the efficiency of precise genome editing using wild-type DNA-PKcs with DNA-PKcs KR, we precisely edited the neurite outgrowth genes CALD1, KATNA1, and SLITRK1 back to their ancestral state in the last common ancestor of humans and Neanderthals (Prufer et al. 2014). We also investigated the efficiency of reversing the inactivation of PRKDC (DNA-PKcs) by exchanging R3753 to lysine. Figure 10A shows the PGE efficiency of CALD1, KATNA1, SLITRK1, and PRKDC by iCRISPR-Cas9D10A, expressing either wild-type DNA-PKcs or DNA-PKcs KR. When DNA-PKcs KR was used, the PGE efficiency increased by 2.5-fold (36%), 4.1-fold (78.5%), and 7.8-fold (51.1%) for CALD1, KATNA1, and SLITRK1. This corresponds to a shift in the PGE / NHEJ ratio from 0.23, 0.29, and 0.08 to 3.49, 25.45, and 2.60, respectively. We achieved a PRKDC back mutation in 71.4% of chromosomes. Recombinant C When as9 or Cpf1 were electroporated, PGE efficiency for CALD1 or HPRT increased 4.8-fold (72.2%) or 8.3-fold (43.7%), and the PGE / NHEJ ratio shifted from 0.45 to 12.8 or from 0.08 to 7.1, respectively. Thus, PGE is increased by the KR strain regardless of the type of DSB.

[0106] Given these high PGE efficiencies achieved by DNA-PKcs KR, we next attempted multiplex editing of three genes. When we used multiplex lipofection of gRNA and ssDNA donors, the PGE efficiencies achieved for some genes were only 3–10% (data not shown). When we electroporated gRNA and ssODN for three genes, we achieved PGE efficiencies of 21.3% for CALD1, 32.0% for KATNA1, and 34.0% for SLITRK1 (Figure 11A). Unlike lipofection, cells are prepared as single-cell suspensions for electroporation. The majority of cells were plated for subsequent bulk DNA isolation, while 10 percent were plated as single-cell dilutions to generate colony progeny from a single cell. Analysis of 33 clones showed that the ancestral mutation and silent block mutation did not always integrate together into the chromosome where the targeted nucleotide substitution (TNS) occurred (Figure 11B). Figure 11C shows a heatmap of chromosomes with indel integration and any TNS integration (left panel) or at least ancestral mutation integration (right panel), regardless of whether it is a block mutation or an ancestral mutation. Most clones either remain wild-type or are correctly edited for all three genes. Surprisingly, one-third of the clones have no additional indel integration in both chromosomes for all three genes. The CALD1 TNS gene has TNS without any additional indels. 12.1% of clones have at least ancestral TNS without any additional indels on both chromosomes for all three genes. The drop from 33.3% to 12.1% is primarily due to the design of the CALD1 donor; the right block mutation was far removed from the other two mutations, and it was the only substitution integrated into 30% of the CALD1 TNS-positive chromosomes (9 of 30) (Figure 3A). The incorporation of TNS on both chromosomes for the three genes is more than fourfold higher than expected by chance based on the efficiency of single-gene TNS (7.1% expected for any TNS and 2.9% expected for at least ancestral TNS). The MPGE method presented here is not only efficient but also fast. The use of single-cell dilution plating immediately after electroporation allows for the generation of clones with the desired MPGE in less than two weeks (Figure 11D). Importantly, DNA-PKcs KR hiPSCs maintained a healthy karyotype without numerical or gross chromosomal abnormalities after 26 passages (corresponding to 3 months), as indicated by trypsin-induced Giemsa band formation (Figure 13).

[0107] As we show in further experiments, PGE efficiency in the presence of mutant DNA-PKcs can be further enhanced by CRISPY mix containing Cas9D10A. The DNA-PKcs mutant increased PGE for CALD1, KATNA1, and SLITRK1 by 2.8-fold, 4-fold, 3-fold, and 12.4-fold, respectively (see Figure 12). Furthermore, CRISPY mix further enhanced PGE efficiency compared to that of wild-type DNA-PKcs without small molecule treatment. The PGE efficiencies were 48% (3.7-fold increase), 82% (4.7-fold increase), and 57.5% (19.2-fold increase) for CALD1, KATNA1, and SLITRK1, respectively.

[0108] Consideration The CRISPY mix increased PGE more than any of its individual components in all four pluripotent stem cell lines tested (three human and one chimpanzee) (Figure 5A, C, and D), but this was not the case in other cell lines tested (Figure 6). Indeed, our results indicate that small molecules and their combinations can have opposite effects on PGE in different cell lines. This may be due to cell line reliance on different repair proteins or pathways, suggesting the need for further research into cell-type-specific mechanisms of DNA repair. Studies showing that human ESCs and iPSCs have a high DNA repair capacity (which decreases after differentiation) (Blanpain et al. 2011, Rocha et al. 2013) are consistent with this interpretation. It may also explain discrepancies between some studies (e.g., the DNA ligase IV inhibitor SCR7 and the RAD51 promoter RS-1 promote precise genome editing in some cell types but not others). Therefore, it may be necessary to screen small molecules for their effect on CRISPR editing in each cell type of interest.

[0109] The small molecules that increased PGE induced by Cas9D10A and ssODN donors in pluripotent stem cells, both alone (Figure 4A) and in combination (Figure 5A), were NU7026, TSA, MLN4924, and NSC 15520. The combination also showed a further enhancing effect on PGE induced by Cpf1 compared to single treatment with NU7026, but not Cas9. NU7026 inhibits DNA-PK, a key complex in the NHEJ pathway (Shrivastav et al. 2008), and has previously been shown to increase PGE efficiency in hiPSCs (Suzuki et al. 2016). TSA can activate the ATM-dependent DNA damage signaling pathway (Lee 2007). The Nedd8-activating enzyme (NAE) inhibitor MLN4924 has been shown to inhibit the neddylation of CtIP, which increases the extent of DNA end trimming at strand breaks, thereby promoting HDR (Jimeno et al. 2015). DNA trimming leaves ssDNA, which is then coated and stabilized by RPA before undergoing homology detection and recombination. NSC15520 prevents RPA from binding to p53 and RAD9 (Glanzer et al. 2011) (Glanzer et al. 2013), potentially increasing the amount of RPA available for ssDNA, which may favor HDR. RS-1, SCR7, and L755507, which have conflicting reports of their ability to increase PGE, did not show any clear effect on PGE mediated by either Cas9 or Cas9D10A in our study.

[0110] The enhancing effect of TSA and MLN4924 on PGE, when used with Cas9D10A double nicking or Cpf1, suggests that blunt and staggered DNA cuts (5' overhangs) are repaired by different repair mechanisms, in contrast to the lack of effect of Cas9. The mechanism of HDR by ssODNs is unclear, but a model has been proposed (Bothmer et al. 2017). Bothmer et al. suggested that 5' overhang structures result in higher levels of HDR than 3' overhangs, suggesting that different overhang polarities are involved in different repair pathways.

[0111] However, the negative effect of MLN4924 on Cas9 editing may be due to the shorter ssODN used (compared to the longer ssODN of Cas9D10A). When significant DNA cutting caused by MLN4924 occurs, there is no remaining homology for ssODN annealing if a short donor is used. The presence of a long 5' overhang in Cas9D10A editing provides homologous DNA even after significant cutting. Therefore, the use of a longer donor may make MLN4924 effective in enhancing precise genome editing, even when using Cas9.

[0112] Although RAD51 is clearly important for classical homologous recombination with dsDNA (Shrivastav et al. 2008), RAD52 is required for annealing of ssDNA (Grimme et al. 2010). Therefore, we sought to test whether RAD52, rather than RAD51, could be the driving force behind HDR of ssODNs. Our results on the effects of the RAD52 inhibitor AICAR, the RAD51 inhibitor B02, and the RAD51 promoter RS-1 on PGE efficiency suggest that RAD51, but not RAD52, is important for accurate ssODN editing, as B02-mediated inhibition of RAD51 was halved and RAD52 inhibition had no effect on PGE efficiency. Interestingly, stimulation of RAD51 by RS-1 had no beneficial effect on PGE.

[0113] To increase the efficiency of PGE, we generated an inducible Cas9D10A iPSC line and optimized ssODN delivery. Occasionally, indels were observed in over 90% of cells (Figure 3A). Using the CRISPY small molecule mix, we achieved nearly 50% PGE in hiPSCs, which, to our knowledge, is the highest PGE efficiency for human pluripotent stem cells described to date (Figure 3A). We also demonstrated efficient PGE (20%) for the first time using Cpf1 with the CRISPY mix (Figure 5C). The CRISPY mix provides a simple tool for increasing PGE frequency and may therefore be useful for many researchers and medical professionals to perform precise genome editing using the CRISPR system.

[0114] By utilizing efficient DSB induction using iCRISPR and kinase-dead DNA-PKcs KR as a switch between NHEJ and HDR, highly efficient and precise genome editing is possible (Figure 10). High PGE efficiency is closely related to a high PGE to indel ratio. We found that when multiplexing several genes, all We also show that TNS incorporation into target chromosomes is much higher than would be expected by chance based on the TNS efficiency of a single gene. The HDR inhibitory properties of DNA-PKcs have been shown to be absolutely dependent on its kinase activity (Neal et al. 2011). Similar to our data, HDR in CHOV3 KR cells has been shown to be 4-7 times greater than in cells expressing wild-type DNA-PKcs (Shrivastav et al. 2008 and 2009). Surprisingly, the increase in HDR in CHOV3 KR cells was greater than in CHOV3 DNA-PKcs knockout cells, suggesting that protein structure is required for further enhanced HDR. Phosphorylation of the ABCDE or JK cluster has been shown to promote end processing or inhibit NHEJ, respectively (see Figure 9B). DNA-PKcs deficiency results in reduced levels of the key repair kinase ATM in cultured cell lines and mice (10). Shrivastav et al. showed that restoration of ATM-dependent phosphorylation of DNA-PKcs KR, and possibly ATM-promoted RAD51 turnover, contributes to the hyperrecombination phenotype (Neal et al., 2016). The use of catalytically inactive but structurally intact DNA-PKcs prevents phosphorylation of downstream c-NHEJ proteins and phosphorylation-induced inhibition of ATM kinase activity (Zhou et al., 2016). In this case (e.g., 2017), ATM levels are maintained in a kinase-independent manner, thus resulting in enhanced HDR. Given the central role of DNA-PKcs in c-NHEJ and similar findings of enhanced HDR in Chinese hamster ovary cells by Shrivastav et al., we believe that KR mutations have consistent effects across cell types and species. Because DNA-PKcs and its sequence around K3753 are conserved in vertebrates (Figure 9C), KR mutations may be a valuable genetic modification tool in several animal models. DNA-PKcs orthologs have also been found in mosquitoes, honeybees, and amoeba slime molds, suggesting an ancient ancestor of this enzyme (Dore et al. 2004, Douglas et al. 2007).

[0115] We did not detect any numerical or large-scale chromosomal aberrations in DNA-PKcs KR hiPSCs after 3 months of culture (Figure 13). In addition to targeted genomic breaks, endogenous DSBs periodically arise, as during V(D)J recombination, due to reactive oxygen species and physical stress leading to replication fork collapse. Impairing error-prone c-NHEJ by inactivating the kinase activity of DNA-PKcs leaves cells free to repair DSBs via slow, error-prone a-NHEJ, with enhanced, high-fidelity HDR using sister chromatids, or die. Therefore, speculatively, genome stability may be better in KR cells compared to wild-type cells due to the enhanced, high-fidelity repair via HDR at the expense of loss of heterozygosity.

[0116] We also demonstrated efficient reverse inactivation of DNA-PKcs by mutating R3753 back to lysine (Fig. 10A). Because V(D)J recombination depends on c-NHEJ, it is desirable to reactivate DNA-PKcs after targeted mutation before differentiation into T cells. It may also be beneficial to revert the DNA-PKcs KR mutation before differentiation into non-proliferating cells such as neurons. HDR is limited to dividing cells, and therefore NHEJ is central to DNA repair in post-mitotic cells. However, the c-NHEJ factors Ku70 / 80, XRCC4, and LIG4, along with XLF, are sufficient to repair low-level damage (Gu et al., 2000), a-NHEJ is in any case DNA-PKcs independent.

[0117] This finding supports the CRISPR-facilitated approach, as the CRISPY small molecule mix can be used with catalytically inactive DNA-PKcs to edit multiple loci at once. This has the potential to revolutionize research and tailor genomes. Not only is the editing efficiency very high (up to 82%), but the limited proportion of indels makes fast (multiplexed) and precise genome editing possible (Figures 10 and 11).

[0118] In conclusion, the hyperrecombination effect of DNA-PKcs KR combined with CRISPR technology leads to unexpected efficiencies in precise genome editing. This can be further amplified by the CRISPY small molecule mix in human pluripotent stem cells. This could significantly reduce time and effort in various areas of genomic research. These include exciting applications such as high-throughput genome-wide association screen (GWAS) validation of relevant medically important traits, disease modeling and drug screening, species comparative analysis, ex vivo gene therapy, simplified production of custom animal models, species deextinction, and, one day, the possibility of introducing large de novo synthesized genomic fragments.

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Claims

1. for genome editing in eukaryotic target cells, including mammalian target cells; (i) a catalytically inactive but structurally intact DNA protein kinase catalytic subunit comprising a sequence containing only the K3753R mutation compared to the wild-type sequence set forth in SEQ ID NO: 50 in humans, wherein the position of the mutation in DNA-PKcs or its orthologue in a species other than human corresponds to the position of said mutation in humans; (ii) a nucleic acid molecule encoding the DNA protein kinase catalytic subunit of (i), and / or (iii) a eukaryotic cell that contains or is capable of expressing the DNA protein kinase catalytic subunit of (i). In vitro use of.

2. 2. The use of claim 1, wherein the target cell is a eukaryotic target cell, including a vertebrate target cell.

3. 3. The use of claim 1 or 2, wherein the target cell is a mammalian target cell, including a rodent target cell or a human target cell.

4. The use of claim 3, wherein the target cells are human target cells.

5. 5. The use of claim 4, wherein the target cells are stem cells, including induced pluripotent stem cells or embryonic pluripotent stem cells, of a eukaryotic target organism, including human induced pluripotent stem cells or embryonic pluripotent stem cells.

6. (a) Compound (I), which is an HDAC inhibitor; (b) Compound (II), which is an NAE inhibitor; (c) Compound (III), which is a DNA-PK inhibitor; or (d) Compound (IV), an RPA inhibitor The use of any one of claims 1 to 5, further comprising introducing into a target cell a combination of at least two, at least three, or at least four of said compounds.

7. Compound (I) is trichostatin A, and / or Compound (II) is MLN4924, and / or Compound (III) is NU7026, and / or Compound (IV) is NSC15520; Use according to claim 6.

8. 8. The use of any one of claims 1 to 7, wherein genome editing comprises introducing a staggered cut, including a staggered cut with a 5' overhang, or a blunt-ended cut into the double-stranded genome of the target cell.

9. 9. The use of any one of claims 1 to 8, wherein the genome editing comprises the presence of a DNA cleaving enzyme in the target cell, comprising the presence of a CRISPR / Cas9D10A enzyme, or (ii) the presence of a CRISPR / Cpf1 enzyme, or (iii) the presence of a Cas9 nuclease.

10. 10. The use of any one of claims 1 to 9, wherein genome editing comprises introducing into a target cell a donor DNA molecule having a desired mutation, the donor DNA molecule being a single-stranded or double-stranded DNA molecule, the donor DNA molecule comprising a single-stranded DNA molecule.

11. The use of any one of claims 1 to 10 in combination with knockdown of endogenous DNA-PKcs in target cells.

12. 1. A method for editing the genome of a eukaryotic target cell or organism (excluding humans), comprising administering to the target cell or organism (i) a catalytically inactive but structurally intact DNA-protein kinase catalytic subunit comprising a sequence that contains only the K3753R mutation compared to the wild-type sequence set forth in human SEQ ID NO: 50, wherein the position of the mutation in DNA-PKcs or an orthologue thereof corresponds to the position of said mutation in humans; or (ii) a nucleic acid molecule combination encoding the DNA-protein kinase catalytic subunit of (i); 20. A method comprising:

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