Use of inhibitors to increase the efficiency of CRISPR / Cas insertion

Abstract

This disclosure provides a method for inserting a target polynucleotide into the genome of a eukaryotic cell, the method comprising improving the efficiency of CRISPR / Cas-mediated polynucleotide insertion by adding a microhomology-mediated end-joining (MMEJ) pathway inhibitor to the eukaryotic cell. This disclosure further provides a composition for inserting a target polynucleotide into the genome of a eukaryotic cell, and a kit for inserting a target gene into the genome of a eukaryotic cell.

Description

[Technical Field]

[0001] (Related applications) This application claims priority to U.S. Provisional Application No. 63 / 492,847, filed on 29 March 2023, which is incorporated herein by reference in its entirety for all purposes. [Background technology]

[0002] Developing cost-effective and reliable methods for precise, targeted modifications to the genome of living cells has been a long-standing goal. Genome editing offers the potential to eliminate genes causing specific disorders (i.e., gene "knock-out"), correct genetic defects via gene "knock-in," or provide means for genetic manipulation or insertion to enhance biological processes. Genome editing can be applied to the treatment of numerous disorders, including hereditary disorders, hematological disorders, and cancer, as well as in immunotherapeutic methods.

[0003] Clustered, regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are prokaryotic immune systems first discovered by Ishino in E. coli (Ishino et al., Journal of Bacteriology 169(12):5429-5433 (1987)). Prokaryotic immune systems provide immunity against viruses and plasmids by targeting their nucleic acids in a sequence-specific manner. See also Soret et al., Nature Reviews Microbiology 6(3):181-186 (2008).

[0004] Since its initial discovery, several groups have conducted extensive research focusing on the potential applications of the CRISPR system in genetic engineering, including gene editing (Jinek et al., Science 337(6096):816-821(2012); Cong et al., Science 339(6121):819-823(2013); and Mali et al., Science 339(6121):823-826(2013)). The CRISPR-Cas9 gene editing system has been successfully used in a wide range of organisms and cell lines. In addition to genome editing, the CRISPR system has numerous other applications, particularly in gene expression regulation, gene circuit construction, and functional genomics (as outlined in Sander et al., Nature Biotechnology 32:347-355(2014)).

[0005] Cas9 endonuclease generates double-strand DNA breaks at target sequences upstream of protospacer adjacent motifs (PAMs). The target sequence can then be removed, or the desired sequence can be inserted into the target sequence using the cell's endogenous repair pathways. These endogenous DNA repair pathways include non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), and homology-directed repair (HDR). While NHEJ, MMEJ, and HDR pathways repair double-strand DNA breaks, such repairs can result in insertions or deletions at the break site. NHEJ does not require a homologous template to repair the DNA break. NHEJ repair can be error-prone, but the error rate decreases if the DNA break contains a compatible overhang. NHEJ and MMEJ are mechanistically distinct DNA repair pathways, each involving different subsets of DNA repair enzymes. Unlike NHEJ, which can be accurate in some cases and error-prone in others, MMEJ is always error-prone, resulting in both deletions and insertions at the repair site. Deletions associated with MMEJ are due to microhomology (2-10 base pairs) on both sides of a double-strand break. In contrast, HDR requires a homologous template to direct repair, but HDR repair is typically high-fidelity and error-resistant. Therefore, HDR-mediated repair of double-strand DNA breaks is preferred over NHEJ-mediated or MMEJ-mediated repair. However, in many cell types, HDR activity is limited by NHEJ activity at all cell cycle stages, and HDR is primarily utilized during the S phase of cell growth (Mao et al., Cell Cycle, 7:2902-2906 (2008)). [Overview of the Initiative]

[0006] In some embodiments, the present disclosure relates to a method of increasing the efficiency of CRISPR / Cas-mediated gene insertion. In some embodiments, the method includes inserting a polynucleotide of interest into the genome of a eukaryotic cell, and the method comprises: (a) adding an inhibitor of the MMEJ pathway to a composition comprising the eukaryotic cell; (b) adding a Cas effector protein to the composition; and (c) adding a polynucleotide of interest to the composition, wherein the polynucleotide of interest is inserted into the genome of the eukaryotic cell by homologous recombination repair (HDR) or single-stranded template repair (SSTR).

[0007] In some embodiments, step (a) of the method further comprises adding an inhibitor of the non-homologous end joining (NHEJ) pathway.

[0008] In some embodiments, the method further comprises: (d) adding a polynucleotide comprising an RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof to the composition.

[0009] In some embodiments, the Cas effector protein and the polynucleotide of (d) are added in the form of a ribonucleoprotein (RNP).

[0010] In some embodiments, the Cas effector protein is added in (b) by adding a Cas polynucleotide encoding the Cas effector protein.

[0011] In some embodiments, the target polynucleotide, the polynucleotide from step (d), and the Cas polynucleotide are encoded on a single vector. In some embodiments, the target polynucleotide is added as DNA. In some embodiments, the polynucleotide from step (d) is added as DNA. In some embodiments, the polynucleotide from step (d) is added as RNA. In some embodiments, the Cas effector polynucleotide is added as DNA. In some embodiments, the Cas polynucleotide is added as RNA. In some embodiments, the Cas polynucleotide is added as mRNA.

[0012] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retrovirus, lentivirus, adenovirus, or adeno-associated virus (AAV).

[0013] In some embodiments, the Cas effector protein, the polynucleotide of interest, and the polynucleotide of (d) are added to eukaryotic cells by microinjection, electroporation, or via lipid nanoparticles, liposomes, exosomes, gold nanoparticles, or DNA nanoclew.

[0014] In some embodiments, the vector is added to a composition containing eukaryotes by transfecting eukaryotic cells.

[0015] In some embodiments, the Cas effector protein is Cas9 nuclease, Cas12a nuclease, or Cas12f nuclease. In some embodiments, the Cas effector protein is Cas9 nuclease. In some embodiments, the Cas9 nuclease is Cas9 nuclease fused to reverse transcriptase, Cas9 nuclease fused to DNA polymerase, Cas9 nuclease fused to DN1S, Cas9 nickasase, Cas9 fused to gemin deglon domain, or Cas9 nuclease fused to CTIP.

[0016] In some embodiments, the polynucleotide of interest is added via a vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retrovirus, lentivirus, adenovirus, or adeno-associated virus (AAV).

[0017] In some embodiments, the target polynucleotide contains the target gene. In some embodiments, the target polynucleotide is 1 to 50 base pairs long. In some embodiments, the target polynucleotide is 1 to 10 base pairs long. In some embodiments, the target polynucleotide is 50 to 5000 base pairs long.

[0018] In some embodiments, the polynucleotide of interest is single-stranded. In some embodiments, the polynucleotide of interest is double-stranded. In some embodiments, the polynucleotide of interest is a hybrid polynucleotide comprising a single-stranded region and a double-stranded region. In some embodiments, the hybrid polynucleotide comprises a double-stranded sequence at the 5' and 3' termini and an internal single-stranded sequence. In some embodiments, the polynucleotide of interest is a double-stranded polynucleotide with blunt ends. In some embodiments, the polynucleotide of interest is a double-stranded polynucleotide with a 3' overhang. In some embodiments, the polynucleotide of interest is a double-stranded polynucleotide with a 5' overhang. In some embodiments, the polynucleotide of interest is a circular polynucleotide.

[0019] In some embodiments, the polynucleotide of interest comprises a chemical modification that enhances the activity, distribution, or uptake of the polynucleotide.

[0020] In some embodiments, an inhibitor of the MMEJ pathway is an inhibitor of POL Q / DNA polymerase q. In some embodiments, an inhibitor of POL Q is a compound of formula (I),

[0021]

Chemical formula

[0022]

Chemical formula

[0023] In some embodiments, the inhibitor of POL Q is a compound disclosed herein, or a combination thereof.

[0024] In some embodiments, the inhibitor of POL Q is a compound listed in Table I, Table II, Table III, its pharmaceutically acceptable salt, or a combination thereof.

[0025] In some embodiments, the inhibitor of POL Q is 9-benzyl-8-(2-chloro-4-(2-(4-methylpiperazine-1-yl)ethoxy)phenyl)-6-(1-methylcyclopropoxy)-9H-purine (compound 1), or a salt thereof.

[0026] [ka]

[0027] In some embodiments, the MMEJ pathway inhibitor in the composition containing eukaryotic cells is in concentrations of about 0.01 mM to about 1 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 0.5 mM, about 0.1 mM to about 100 mM, or about 1 mM to about 50 mM.

[0028] In some embodiments, the NHEJ pathway inhibitor is a DNA-dependent protein kinase (DNA-PK) inhibitor. In some embodiments, the DNA-PK inhibitor is M3814, M9831 / VX984, Nu7441, KU0060648, AZD7648, or a combination thereof. In some embodiments, the DNA-PK inhibitor is AZD7648. In some embodiments, the DNA-PK inhibitor is a peptide.

[0029] In some embodiments, the NHEJ pathway inhibitor in the composition containing eukaryotic cells is present in concentrations of approximately 0.01 mM to approximately 1 mM, approximately 0.1 mM to approximately 1 mM, approximately 0.1 mM to approximately 0.5 mM, approximately 0.1 mM to approximately 100 mM, or approximately 1 mM to approximately 50 mM.

[0030] In some embodiments, the MMEJ pathway inhibitor is added to the composition containing eukaryotic cells at 0 minutes to about 48 hours, 0 minutes to about 24 hours, 0 minutes to about 12 hours, 0 minutes to about 6 hours, or 0 minutes to about 1 hour before the Cas effector protein is added to the composition. In some embodiments, the MMEJ pathway inhibitor is added to the composition containing eukaryotic cells at 0 minutes to about 1 hour after the Cas effector protein is added to the composition containing eukaryotic cells.

[0031] In some embodiments, the NHEJ pathway inhibitor is added to the composition containing eukaryotic cells at 0 minutes to about 48 hours, 0 minutes to about 24 hours, 0 minutes to about 12 hours, 0 minutes to about 6 hours, or 0 minutes to about 1 hour before the Cas effector protein is added to the composition. In some embodiments, the NHEJ pathway inhibitor is added to the composition containing eukaryotic cells at 0 minutes to about 1 hour after the Cas effector protein is added to the composition containing eukaryotic cells.

[0032] In some embodiments, the MMEJ pathway inhibitor and the NHEJ pathway inhibitor are added simultaneously to the composition containing eukaryotic cells. In some embodiments, the MMEJ pathway inhibitor and the NHEJ pathway inhibitor are added to the composition containing eukaryotic cells at different time points.

[0033] In some embodiments, an MMEJ pathway inhibitor, an NHEJ pathway inhibitor, and a Cas effector protein are added simultaneously to a composition containing eukaryotic cells.

[0034] In some embodiments, the MMEJ pathway inhibitor is present in the composition containing eukaryotic cells for about 1 to about 300 hours, about 10 to about 100 hours, or about 20 to about 80 hours.

[0035] In some embodiments, the MMEJ pathway inhibitor is added to the composition containing eukaryotic cells at least once, at least twice, or at least three times.

[0036] In some embodiments, the NHEJ pathway inhibitor is present in the composition containing eukaryotic cells for about 1 to about 300 hours, about 10 to about 100 hours, or about 20 to about 80 hours.

[0037] In some embodiments, the NHEJ pathway inhibitor is added to the composition containing eukaryotic cells at least once, at least twice, or at least three times.

[0038] In some embodiments, the composition containing eukaryotic cells is a cell culture. In some embodiments, the cell culture is an in vitro cell culture or an ex vivo cell culture. In some embodiments, the eukaryotic cells are in vivo.

[0039] In some embodiments, the cell culture includes a cell extract.

[0040] In some embodiments, the eukaryotic cell is a lymphocyte. In some embodiments, the lymphocyte includes a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

[0041] In some embodiments, eukaryotic cells are pluripotent stem cells. In some embodiments, pluripotent stem cells are induced pluripotent stem cells (iPSCs).

[0042] In some embodiments, the cell culture is a mammalian cell culture.

[0043] In some embodiments, the Disclosure relates to a method for increasing the efficiency of CRISPR / Cas-mediated gene insertion, comprising inserting a polynucleotide of interest into the genome of a eukaryotic cell containing a genomically integrated Cas polynucleotide. In some embodiments, the Disclosure provides a method for inserting a polynucleotide of interest into the genome of a eukaryotic cell, the method comprising (a) adding a microhomology-mediated end-joining (MMEJ) pathway inhibitor to a composition containing a eukaryotic cell, and (b) adding the polynucleotide of interest to the composition, wherein the genome contains a genomically integrated Cas polynucleotide, and the polynucleotide of interest is inserted into the genome by homologous recombination repair (HDR) or single-strand template repair (SSTR). In some embodiments, the genomically integrated Cas polynucleotide is inducible.

[0044] In some embodiments, the method further comprises adding an inhibitor of the non-homologous end-joining (NHEJ) pathway to the composition.

[0045] In some embodiments, the method further comprises (c) adding a polynucleotide comprising an RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof to the composition.

[0046] In some embodiments, (i) the polynucleotide of interest and (ii) the polynucleotide of (c) are encoded on the vector. In some embodiments, the polynucleotide of interest is added as DNA. In some embodiments, the polynucleotide of (c) is added as DNA. In some embodiments, the polynucleotide of (c) is added as RNA.

[0047] In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retrovirus, lentivirus, adenovirus, or adeno-associated virus (AAV). In some embodiments, the vector is added to a composition containing a eukaryote by transfecting a eukaryotic cell.

[0048] In some embodiments, the Cas effector protein is Cas9 nuclease, Cas12a nuclease, or Cas12f nuclease. In some embodiments, the Cas effector protein is Cas9 nuclease. In some embodiments, the Cas9 nuclease is Cas9 nuclease fused to reverse transcriptase, Cas9 nuclease fused to DNA polymerase, Cas9 nuclease fused to DN1S, Cas9 nickasase, Cas9 fused to gemin deglon domain, or Cas9 nuclease fused to CTIP.

[0049] In some embodiments, the polynucleotide of interest is added via a vector. In some embodiments, the vector is a viral vector. In some embodiments, the viral vector is a retrovirus, lentivirus, adenovirus, or adeno-associated virus (AAV).

[0050] In some embodiments, the polynucleotide of interest contains the gene of interest. In some embodiments, the polynucleotide of interest is 1 to 50 base pairs long, 1 to 10 base pairs long, or 50 to 5000 base pairs long.

[0051] In some embodiments, the target polynucleotide is single-stranded. In some embodiments, the target polynucleotide is double-stranded. In some embodiments, the target polynucleotide is a hybrid polynucleotide comprising single-stranded and double-stranded regions. In some embodiments, the hybrid polynucleotide includes double-stranded sequences at the 5' and 3' ends and an internal single-stranded sequence. In some embodiments, the target polynucleotide is a double-stranded polynucleotide with blunt ends. In some embodiments, the target polynucleotide is a double-stranded polynucleotide with a 3' overhang. In some embodiments, the target polynucleotide is a double-stranded polynucleotide with a 5' overhang. In some embodiments, the target polynucleotide is a cyclic polynucleotide.

[0052] In some embodiments, the polynucleotide includes chemical modifications that enhance the activity, distribution, or uptake of the polynucleotide.

[0053] In some embodiments, the MMEJ pathway inhibitor is a POL Q / DNA polymerase q inhibitor. In some embodiments, the POL Q inhibitor is a compound of formula I, or a combination thereof. In some embodiments, the POL Q inhibitor is a compound disclosed herein, or a combination thereof. In some embodiments, the POL Q inhibitor is a compound disclosed in Tables I, II, and III, a pharmaceutically acceptable salt thereof, or a combination thereof. In some embodiments, the POL Q inhibitor is compound 1, or a pharmaceutically acceptable salt thereof. In some embodiments, the POL Q inhibitor is compound 1.

[0054] In some embodiments, the MMEJ pathway inhibitor in the composition containing eukaryotic cells is in concentrations of about 0.01 mM to about 1 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 0.5 mM, about 0.1 mM to about 100 mM, or about 1 mM to about 50 mM.

[0055] In some embodiments, the NHEJ pathway inhibitor is a DNA-dependent protein kinase (DNA-PK) inhibitor. In some embodiments, the DNA-PK inhibitor is M3814, M9831 / VX984, Nu7441, KU0060648, AZD7648, or a combination thereof. In some embodiments, the DNA-PK inhibitor is AZD7648. In some embodiments, the DNA-PK inhibitor is a peptide.

[0056] In some embodiments, the NHEJ pathway inhibitor in the composition containing eukaryotic cells is present in concentrations of approximately 0.01 mM to approximately 1 mM, approximately 0.1 mM to approximately 1 mM, approximately 0.1 mM to approximately 0.5 mM, approximately 0.1 mM to approximately 100 mM, or approximately 1 mM to approximately 50 mM.

[0057] In some embodiments, an MMEJ pathway inhibitor is added to a composition containing eukaryotic cells containing genomically integrated Cas polynucleotides at 0 minutes to approximately 48 hours, 0 minutes to approximately 24 hours, 0 minutes to approximately 12 hours, 0 minutes to approximately 6 hours, or 0 minutes to approximately 1 hour prior to the induction of genomically integrated Cas polynucleotides.

[0058] In some embodiments, an NHEJ pathway inhibitor is added to a composition containing eukaryotic cells containing genomically integrated Cas polynucleotides at 0 minutes to approximately 48 hours, 0 minutes to approximately 24 hours, 0 minutes to approximately 12 hours, 0 minutes to approximately 6 hours, or 0 minutes to approximately 1 hour prior to the induction of genomically integrated Cas polynucleotides.

[0059] In some embodiments, the MMEJ pathway inhibitor and the NHEJ pathway inhibitor are added simultaneously to a composition containing eukaryotic cells containing genomically integrated Cas polynucleotides. In some embodiments, the MMEJ pathway inhibitor and the NHEJ pathway inhibitor are added at different time points to a composition containing eukaryotic cells containing genomically integrated Cas polynucleotides.

[0060] In some embodiments, an MMEJ pathway inhibitor is added to a composition containing eukaryotic cells containing genomically integrated Cas polynucleotides, simultaneously with the induction of genomically integrated Cas polynucleotides.

[0061] In some embodiments, an NHEJ pathway inhibitor is added to a composition containing eukaryotic cells containing genomically integrated Cas polynucleotides, simultaneously with the induction of genomically integrated Cas polynucleotides.

[0062] In some embodiments, MMEJ pathway inhibitors and NHEJ pathway inhibitors are added to a composition containing eukaryotic cells containing genomically integrated Cas polynucleotides, simultaneously with the induction of genomically integrated Cas polynucleotides.

[0063] In some embodiments, the MMEJ pathway inhibitor is present in a composition containing eukaryotic cells containing genomically integrated Cas polynucleotides for about 1 to about 300 hours, about 10 to about 100 hours, or about 20 to about 80 hours.

[0064] In some embodiments, the MMEJ pathway inhibitor is added at least once, at least twice, or at least three times to a composition containing eukaryotic cells containing genomically integrated Cas polynucleotides.

[0065] In some embodiments, the NHEJ pathway inhibitor is present in a composition containing eukaryotic cells containing genomically integrated Cas polynucleotides for a period of about 1 to about 300 hours, about 10 to about 100 hours, or about 20 to about 80 hours.

[0066] In some embodiments, an NHEJ pathway inhibitor is added at least once, at least twice, or at least three times to a composition containing eukaryotic cells containing genomically integrated Cas polynucleotides.

[0067] In some embodiments, the composition comprising eukaryotic cells containing genomically integrated Cas polynucleotides is a cell culture. In some embodiments, the cell culture is an in vitro cell culture or an ex vivo cell culture.

[0068] In some embodiments, eukaryotic cells containing genomically integrated Cas polynucleotides are in vivo.

[0069] In some embodiments, the cell culture includes cell extracts. In some embodiments, the cell culture is a mammalian cell culture.

[0070] In some embodiments, eukaryotic cells containing genomically integrated Cas polynucleotides are lymphocytes. In some embodiments, the lymphocytes contain chimeric antigen receptors (CARs) or T cell receptors (TCRs).

[0071] In some embodiments, eukaryotic cells containing genomically integrated Cas polynucleotides are pluripotent stem cells. In some embodiments, the pluripotent stem cells are induced pluripotent stem cells (iPSCs).

[0072] In some embodiments, the present disclosure relates to a method for inserting a polynucleotide of interest into the genome of a eukaryotic cell, wherein the method comprises (a) adding a microhomology-mediated end-joining (MMEJ) pathway inhibitor to a composition comprising a eukaryotic cell, and (b) adding (i) a Cas effector protein, (ii) the polynucleotide of interest, and (iii) a polynucleotide comprising an RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof to the composition comprising a polynucleotide of interest, wherein the polynucleotide of interest is inserted into the genome by homologous recombination repair (HDR) or single-strand template repair (SSTR).

[0073] In some embodiments, the method involves adding an inhibitor of the non-homologous end-joining (NHEJ) pathway to a composition containing eukaryotic cells.

[0074] In some embodiments, the Cas effector protein and the polynucleotide containing an RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof are added in the form of a ribonucleoprotein (RNP).

[0075] In some embodiments, the Cas effector protein is encoded by a Cas polynucleotide. In some embodiments, the Cas effector protein and the polynucleotide of interest are encoded on a vector. In some embodiments, the Cas effector protein and the polynucleotide of (iii) are encoded on a vector. In some embodiments, the Cas effector protein, the polynucleotide of interest, and the polynucleotide of (iii) are encoded on a vector. In some embodiments, the polynucleotide is on a vector.

[0076] In some embodiments, the present disclosure relates to a method for increasing the efficiency of homologous recombination repair (HDR) and single-strand template repair (SSTR) gene insertion in eukaryotic cells, the method comprising adding a microhomology-mediated end-joining (MMEJ) pathway inhibitor when performing CRISPR / Cas-mediated gene insertion in eukaryotic cells.

[0077] In some embodiments, the method further includes adding an inhibitor of the non-homologous end-joining (NHEJ) pathway.

[0078] In some embodiments, CRISPR / Cas-mediated gene insertion is CRISPR / Cas9-mediated gene insertion.

[0079] In some embodiments, the present disclosure relates to a method for reducing recombination of the microhomology-mediated end-joining (MMEJ) pathway during CRISPR / Cas-mediated gene insertion in cells, wherein the method includes adding an MMEJ pathway inhibitor to cells when performing Cas-mediated gene insertion.

[0080] In some embodiments, the method further includes reducing non-homologous end joining (NHEJ) recombination during CRISPR / Cas-mediated gene insertion in cells, which includes adding an NHEJ pathway inhibitor to the cells.

[0081] In some embodiments, CRISPR / Cas-mediated gene insertion is CRISPR / Cas9-mediated gene insertion.

[0082] In some embodiments, the disclosure relates to a composition comprising a Cas effector protein, or a vector encoding a Cas effector protein, and an inhibitor of the microhomology-mediated end-joining (MMEJ) pathway. In some embodiments, the composition further comprises an inhibitor of the non-homologous end-joining (NHEJ) pathway.

[0083] In some embodiments, the composition further comprises a polynucleotide comprising at least one RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof.

[0084] In some embodiments, the Cas effector protein is Cas9 nuclease, Cas12a nuclease, or Cas12f nuclease. In some embodiments, the Cas effector protein is Cas9 nuclease. In some embodiments, the Cas9 nuclease is Cas9 nuclease fused to reverse transcriptase, Cas9 nuclease fused to DNA polymerase, Cas9 fused to DN1S, Cas9 nickasase, Cas9 fused to gemin deglon domain, or Cas9 nuclease fused to CTIP.

[0085] In some embodiments, the vector encoding the Cas effector protein is a viral vector.

[0086] In some embodiments, a polynucleotide comprising at least one RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof is encoded on the vector. In some embodiments, the vector is a viral vector.

[0087] In some embodiments, the Cas effector protein and the polynucleotide comprising at least one RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof are in the form of a ribonucleoprotein (RNP).

[0088] In some embodiments, the composition further comprises a pharmaceutically acceptable carrier, diluent, or excipient.

[0089] In some embodiments, the present disclosure relates to a kit comprising a Cas effector protein, or a vector encoding a Cas effector protein, and an inhibitor of the microhomology-mediated end-joining (MMEJ) pathway.

[0090] In some embodiments, the kit further includes an inhibitor of the non-homologous end-joining (NHEJ) pathway.

[0091] In some embodiments, the kit further comprises a polynucleotide containing at least one RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof.

[0092] In some embodiments, the Cas effector protein is Cas9 nuclease, Cas12a nuclease, or Cas12f nuclease. In some embodiments, the Cas effector protein is Cas9 nuclease. In some embodiments, the Cas9 nuclease is Cas9 nuclease fused to reverse transcriptase, Cas9 fused to DNA polymerase, Cas9 fused to DN1S, Cas9 nickasase, Cas9 fused to gemin deglon domain, or Cas9 nuclease fused to CTIP.

[0093] In some embodiments, a polynucleotide comprising at least one RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof is encoded on the vector. In some embodiments, the vector is a viral vector.

[0094] In some embodiments, the Cas effector protein and the polynucleotide comprising at least one RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof are in the form of a ribonucleoprotein (RNP). [Brief explanation of the drawing]

[0095] [Figure 1] This is a schematic diagram illustrating the operation of DNA repair using small molecule inhibitors. In this schematic, components of the CRISPR / Cas genome editing system provide a double-stranded break (DSB) at a specific sequence. DSBs can be repaired by the inaccurate and error-prone microhomology-mediated end joining (MMEJ) or non-homologous end joining (NHEJ) pathways, or by the more accurate homologous recombination repair (HDR) pathway. [Figure 2A] An exemplary method described in the embodiments of this specification is shown. Figure 2A shows an example of cells pretreated for 3 hours with a pharmacological inhibitor of POL Q / DNA polymerase q (PolQ) and / or DNA-dependent protein kinase (DNA-PK). The CRISPR / Cas gene editing system is then added to the cells. After 60 hours, genomic DNA is isolated from the cells and deep-targeted sequencing is performed. The sequencing results are then analyzed by Rational InDel Meta-Analysis (RIMA) to determine the frequency of MMEJ and NHEJ repairs. Figure 2B shows a graphical representation of the RIMA results, where deletions related to microhomology are visualized by the squares shown in the figure. [Figure 2B]An exemplary method described in the embodiments of this specification is shown. Figure 2A shows an example of cells pretreated for 3 hours with a pharmacological inhibitor of POL Q / DNA polymerase q (PolQ) and / or DNA-dependent protein kinase (DNA-PK). The CRISPR / Cas gene editing system is then added to the cells. After 60 hours, genomic DNA is isolated from the cells and deep-targeted sequencing is performed. The sequencing results are then analyzed by Rational InDel Meta-Analysis (RIMA) to determine the frequency of MMEJ and NHEJ repairs. Figure 2B shows a graphical representation of the RIMA results, where deletions related to microhomology are visualized by the squares shown in the figure. [Figure 3] This example demonstrates the effects of inhibiting the MMEJ and NHEJ pathways on DNA repair and accurate integration of DSBs, as described in Example CRISPR-1. [Figure 4] This example demonstrates the effects of inhibiting the MMEJ and NHEJ pathways on CRISPR / Cas editing efficiency, as described in Example CRISPR-1. [Figure 5] This example demonstrates the effects of inhibiting the MMEJ and NHEJ pathways on the CRISPR / Cas-mediated gene knock-in efficiency in mutant sequencing reads, as described in Example CRISPR-2. [Figure 6] This example demonstrates the effects of MMEJ and NHEJ pathway inhibition on CRISPR / Cas-mediated gene knock-in efficiency in mapped sequencing reads, as described in Example CRISPR-2. [Figure 7] The effects of Pol Q and DNA-PK inhibition on MMEJ in mutant reads, as described in Example CRISPR-3, are demonstrated. [Figure 8] This demonstrates the effect of Pol Q inhibition on MMEJ in mapped reads, as described in Example CRISPR-3. [Figure 9]The effects of inhibiting the MMEJ and NHEJ pathways on cellular confluence are demonstrated, as described in Example CRISPR-4. [Figure 10] The effects of inhibiting the MMEJ and NHEJ pathways on transfection efficiency are shown, as described in Example CRISPR-4. [Figure 11] This study demonstrates the effects of inhibiting the MMEJ and NHEJ pathways on DNA repair and accurate integration of DSBs in induced pluripotent stem cells (iPSCs). [Figure 12] This study demonstrates the effects of inhibiting the MMEJ and NHEJ pathways on DNA repair and accurate integration of DSBs in induced pluripotent stem cells (iPSCs). [Figure 13] This study demonstrates the effects of Pol Q and DNA-PK inhibition on DNA repair in CRISPR / Cas-induced DSBs in Cas9-induced iPSCs. [Modes for carrying out the invention]

[0096] This disclosure relates to methods for improving CRISPR / Cas-mediated gene insertion (i.e., gene "knock-in") in eukaryotic cells, compositions for improved CRISPR / Cas-mediated insertion, and kits for improved CRISPR / Cas-mediated gene insertion. Generally, a CRISPR system, such as a CRISPR / Cas system, includes elements (e.g., guide polynucleotides and Cas proteins) that facilitate the formation of a CRISPR complex at a site of a target polynucleotide (e.g., a target DNA sequence). In naturally occurring CRISPR systems (e.g., bacterial immune CRISPR / Cas9 systems), foreign DNA is incorporated into a CRISPR array, which then produces CRISPR-RNA (crRNA). The crRNA contains an RNA guide sequence region complementary to the foreign DNA site and hybridizes with trans-activating CRISPR-RNA (tracrRNA), which is also encoded by the CRISPR system. The tracrRNA can form a secondary structure, such as a stem-loop, and bind to the Cas9 protein. The crRNA / tracrRNA hybrid associates with Cas9, and the crRNA / tracrRNA / Cas9 complex recognizes and cleaves foreign DNA containing a protospacer sequence, thereby conferring immunity against the invading virus or plasmid. The CRISPR / Cas system is further described, for example, in Jinek et al., Science 337(6096):816-821(2012); Cong et al., Science 339(6121):819-823(2013); Mali et al., Science 339(6121):823-826(2013); and Sander et al., Nat Biotechnol 32:347-355(2014).

[0097] The CRISPR / Cas system is engineered to introduce insertions, also known as targeted insertions, into target polynucleotides. Typically, a guide polynucleotide is designed so that the Cas protein generates a double-strand break at the target polynucleotide, and a separate donor template containing the desired sequence is inserted into the cleaved target polynucleotide by a cellular DNA repair mechanism, such as non-homologous end joining (NHEJ) or homologous recombination repair (HDR). The efficiency of the insertion depends on several factors, including the transfection ratio of the donor template, Cas protein, and guide polynucleotide, the sequence and size of the donor template, and the type of DNA repair mechanism induced. For example, HDR provides high-fidelity DNA repair but has a low insertion frequency, while NHEJ has a high insertion frequency but may also introduce mutations into the target DNA.

[0098] In some embodiments, the Disclosure provides compositions, polynucleotides, and / or fusion proteins for improved targeted insertion methods. In some embodiments, the compositions, polynucleotides, and / or fusion proteins of the Disclosure provide more precise insertion of a sequence of interest. In some embodiments, the compositions, polynucleotides, and fusion proteins of the Disclosure provide more efficient insertion of a sequence of interest.

[0099] Unless otherwise defined herein, scientific and technical terms used in this disclosure shall have meanings generally understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include plural forms, and plural terms shall include singular forms. Where used herein, “a” or “an” may mean one or more. Where used herein, in conjunction with the word “comprising,” the words “a” or “an” may mean one or more. Where used herein, “another” or “further” may mean at least a second or more.

[0100] Throughout this application, the term “approximately” is used to indicate that a value includes inherent variations in the error of the method / apparatus used to determine the value, or variations present among the subjects of study. Typically, the term “approximately” means, depending on the context, to include variations of less than approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%.

[0101] The use of the term “or” in the claims is used to mean “and / or” unless it is expressly indicated that it refers only to substitutes, or that the substitutes are not mutually exclusive; however, this disclosure supports the definitions that refer only to substitutes and “and / or.”

[0102] As used herein, “comprising” (and any variations or forms of “comprising,” such as “comprise” and “comprises”), “having” (and any variations or forms of “having,” such as “have” and “has”), “including” (and any variations or forms of “including,” such as “includes” and “include”), or “containing” (and any variations or forms of “containing,” such as “contains” and “contain”) do not exclude additional unlisted elements or steps of methods. Any embodiment considered herein is intended to be carried out with respect to any protein, composition, polynucleotide, vector, cell, method, and / or kit of the Disclosure. Furthermore, the methods and proteins of the Disclosure can be achieved using the compositions, polynucleotides, vectors, cells, and / or kits of the Disclosure.

[0103] The use of the term "for example" and its corresponding abbreviation "eg" (whether italicized or not) means that the specific terms described are representative examples and embodiments of the disclosure, and are not intended to be limited to the specific examples referenced or cited unless otherwise expressly stated.

[0104] As used herein, “between” is a range that includes both ends of the range. For example, the numbers between x and y explicitly include the numbers x and y, as well as any numbers that fall within x and y.

[0105] "Nucleic acid," "nucleic acid molecule," "nucleotide," "nucleotide sequence," "oligonucleotide," or "polynucleotide" all refer to polymer compounds containing covalently linked nucleotides. The term "nucleic acid" includes ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), both of which may be single-stranded or double-stranded. Polynucleotides may include naturally occurring nucleic acid bases (e.g., guanine, adenine, cytosine, thymine, and uracil), modified nucleic acid bases (e.g., hypoxanthine, xanthine, 7-methylguanine, dihydrouracil, 5-methylcytosine, 5-hydroxymethylcytosine), and / or synthetic nucleic acid bases (e.g., isoguanine or isocytosine). Nucleic acids are transcribed from the 5' end to the 3' end. In some embodiments, this disclosure provides polynucleotides containing both RNA and DNA nucleotides. Methods for producing polynucleotides containing both RNA and DNA nucleotides are known in the art and include, for example, ligation or oligonucleotide synthesis methods. In some embodiments, the Disclosure provides polynucleotides that can form complexes with Cas nucleases or Cas niccas described herein. In some embodiments, the Disclosure provides polynucleotides that encode any one of the proteins disclosed herein, for example, Cas nucleases or Cas niccas.

[0106] "Genes" refers to an assembly of nucleotides that encode a polypeptide, and includes cDNA and genomic DNA nucleic acid molecules. In some embodiments, "genes" also refers to non-coding nucleic acid fragments that can act as regulatory sequences before (i.e., 5') and after (i.e., 3') a coding sequence.

[0107] A nucleic acid molecule is "hybridizable" or "can be hybridized" to another nucleic acid molecule (e.g., cDNA, genomic DNA, or RNA) if its single-stranded form can anneal to other nucleic acid molecules under appropriate temperature and solution ionic strength conditions. Hybridization and washing conditions are known and exemplified in Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly in Chapter 11 and Table 11.1. Temperature and ionic strength conditions determine the stringency of hybridization. The stringency of hybridization conditions can be selected to provide the selective formation or maintenance of a desired hybridization product of two complementary polynucleotides in the presence of other potentially cross-reacting or interfering polynucleotides. Stringency conditions are sequence-dependent. Typically, longer complementary sequences hybridize specifically at higher temperatures than shorter complementary sequences. Generally, stringent hybridization conditions involve specifying the ionic strength, concentration of chemical denaturant, pH, and the thermal melting point (T) of a particular polynucleotide at the concentration of the hybridization partner. mThe hybridization temperature is approximately 5°C to 10°C lower than the temperature at which 50% of the sequence hybridizes to a substantially complementary sequence. Generally, nucleotide sequences with a high percentage of G and C bases hybridize under more stringent conditions than nucleotide sequences with a low percentage of G and C bases. Generally, stringency can be increased by raising the temperature, raising the pH, lowering the ionic strength, and / or increasing the concentration of chemical nucleic acid denaturants (such as formamide, dimethylformamide, dimethyl sulfoxide, ethylene glycol, propylene glycol, and ethylene carbonate). Stringent hybridization conditions typically involve salt concentrations or ionic strengths of approximately 1 M, 500 mM, 200 mM, 100 mM, or less than 50 mM. Hybridization temperatures are higher than approximately 20°C, 30°C, 40°C, 60°C, or 80°C. The concentration of chemical modifiers is higher than approximately 10%, 20%, 30%, 40%, or 50%. Since many factors can affect the stringency of hybridization, the combination of parameters may be more significant than the absolute value of any single parameter.

[0108] The term "complementary" is used to describe the relationships between nucleotide bases that can hybridize with each other. For example, with respect to DNA, adenosine is complementary to thymine, and cytosine is complementary to guanine. When two nucleic acids are "complementary," it means that one or more regions of the first nucleic acid can form hydrogen bonds with one or more regions of the second nucleic acid. Complementary nucleic acids do not need to have complementarity at every nucleotide and may include one or more nucleotide mismatches, i.e., points where no hydrogen bonds occur. For example, complementary oligonucleotides may have at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% nucleotide hydrogen bonds. In contrast, with respect to oligonucleotides, "fully complementary" or "100% complementary" means that each nucleotide forms hydrogen bonds without any nucleotide mismatches.

[0109] The term "homologous recombination" refers to the insertion of an exogenous polynucleotide (e.g., DNA) into another nucleic acid (e.g., DNA) molecule, such as the insertion of a vector, polynucleotide fragment, or gene into a chromosome. In some cases, the exogenous polynucleotide targets a specific chromosomal site for homologous recombination. For specific homologous recombination, the exogenous polynucleotide typically contains a homologous region long enough with respect to the chromosomal sequence, enabling complementary binding and integration of the exogenous polynucleotide into the chromosome. Longer homologous regions and greater sequence similarity can increase the efficiency of homologous recombination. In some embodiments, the polynucleotides or compositions described herein facilitate homologous recombination by generating cleavage, such as double-strand breaks, within the nucleic acid sequence.

[0110] The term "homologous recombination repair" or "HDR" refers to a mechanism that repairs double-strand breaks in DNA using a template nucleic acid sequence. The most common form of HDR is homologous recombination. In HDR, double-strand breaks are repaired by a process involving the excision of the 5' end DNA strand at the break, creating a 3' overhang, which serves both as a substrate for proteins required for strand entry and as a primer for DNA repair synthesis. The entry strand then replaces one strand of a double-strand DNA template sequence containing a homologous sequence and pairing with the other strand, resulting in the formation of a hybrid DNA known as a displacement loop. These recombination intermediates are then separated to complete the DNA repair process.

[0111] The term "single-strand template repair" or "SSTR" refers to an alternative mechanism that uses a template nucleic acid sequence to repair double-strand breaks in DNA. In contrast to HDR, SSTR utilizes a single-strand template nucleic acid sequence for double-strand DNA break repair.

[0112] The term "non-homologous end joining pathway" or "NHEJ pathway" refers to an alternative mechanism for repairing double-strand breaks in DNA. In NHEJ, a Ku80 / 70 heterodimer recognizes and binds to the blunt end formed by a double-strand break, and the resulting complex activates DNA-PK activity. This activation of DNA-PK recruits Artemis nuclease, DNA polymerase, and DNA ligase to ultimately repair the double-strand break. NHEJ differs from HDR and homologous recombination in that it does not require a homologous template sequence for repair.

[0113] The term "microhomology-mediated end joining pathway" or "MMEJ pathway" refers to an alternative mechanism for repairing double-strand breaks in DNA. MMEJ is similar to NHEJ in that homologous template sequences are not used for double-strand break repair. However, MMEJ is distinguished from other repair mechanisms by its use of micro-homology sequences to align the damaged DNA strands. MMEJ is independent of Ku protein or DNA-PK, although DNA polymerase q (Pol Q) has been shown to be required for MMEJ. MMEJ is also known as "alternative end joining," "alternative non-homologous end joining," or "Alt-NHEJ."

[0114] As used herein, the term “operably linked” means that the polynucleotide of interest, for example, a polynucleotide encoding a nuclease, is linked to a regulatory element in a manner that enables the expression of the polynucleotide. The regulatory element may be a cis-regulatory element or a trans-regulatory element. Examples of regulatory elements include promoters, enhancers, terminators, 5' and 3' UTRs, insulators, silencers, and operators. In some embodiments, the regulatory element is a promoter. In some embodiments, the polynucleotide expressing the protein of interest is operably linked to a promoter on an expression vector.

[0115] As used herein, “promoter,” “promoter sequence,” or “promoter region” refers to a DNA regulatory region or polynucleotide that can bind to RNA polymerase and is involved in initiating the transcription of a downstream coding or non-coding sequence. In some embodiments, the promoter sequence includes a transcription start site and extends upstream to include a minimum number of bases or elements used to initiate transcription at a level detectable beyond the background. In some embodiments, the promoter sequence includes a transcription start site and a protein-binding domain involved in RNA polymerase binding. Eukaryotic promoters typically include “TATA” boxes and “CAT” boxes. Various promoters, including inducible promoters, can be used to drive the expression of various vectors of this disclosure.

[0116] A “vector” is any means for cloning and / or transferring a nucleic acid into a host cell. A vector may be a replicon to which another DNA segment can bind, resulting in the replication of the bound segment. A “replicon” is any genetic element (e.g., plasmid, phage, cosmid, chromosome, virus) that functions as an autonomous unit of DNA replication in vivo, i.e., can replicate under its own control. In some embodiments, a vector is an episomal vector that is removed / disappeared from a population of cells after a number of cell generations, for example, by asymmetric distribution. The term “vector” includes both viral and nonviral means for introducing nucleic acids into cells in vitro, ex vivo, or in vivo. Nucleic acids may be manipulated using a number of vectors known in the art to incorporate response elements and promoters into genes, etc. A vector may include one or more regulatory regions and / or selectable markers useful for selecting, measuring, and monitoring the results of nucleic acid transfer (e.g., transfer to which tissue, duration of expression).

[0117] Possible vectors include, for example, plasmids or modified viruses, bacteriophages such as lambda derivatives, plasmids such as PBR322 or pUC plasmid derivatives, or Bluescript vectors. For example, insertion of DNA fragments corresponding to response elements and promoters into a suitable vector can be achieved by ligating the appropriate DNA fragments into a selected vector having complementary adherent ends. Alternatively, the ends of the DNA molecule may be enzymatically modified, or any site may be generated by ligating polynucleotides (linkers) to the DNA ends. Such vectors may be engineered to include a selectable marker gene that provides selection of cells into which the marker has been incorporated into the cellular genome. Such markers enable the identification and / or selection of host cells into which the protein encoded by the marker is incorporated and expressed.

[0118] Viral vectors, particularly retroviral vectors, have been used for a wide variety of gene delivery applications in cells and living animals. Viral vectors that can be used include, but are not limited to, retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, pox, baculoviruses, vaccinia, herpes simplex, Epstein-Barr, adenoviruses, geminiviruses, and kalimovirus vectors. In some embodiments, viral vectors are used to provide polynucleotides described herein. In some embodiments, viral vectors are used to provide polynucleotides encoding proteins described herein.

[0119] Vectors can be introduced into desired host cells by known methods including, but not limited to, transfection, transduction, cell fusion, and lipofection. Vectors may contain various regulatory elements, including promoters. In some embodiments, vector design can be based on constructs designed by Mali et al., Nat Methods 10:957-63 (2013).

[0120] Polynucleotides and / or vectors provided herein can be grown using methods known in the art. Once suitable host systems and growth conditions are established, recombinant expression vectors can be grown and prepared in large quantities. Expression vectors that can be used as described herein include, but are not limited to, the following vectors or their derivatives: human or animal viruses such as vaccinia viruses or adenoviruses; insect viruses such as baculoviruses; yeast vectors; bacteriophage vectors (e.g., lambda); and plasmids and cosmid DNA vectors.

[0121] The term "plasmid" refers to an extrachromosomal element that often contains genes that are not part of the cell's central metabolism and is usually in the form of a circular double-stranded DNA molecule. Such elements may be autonomously replicating sequences of single-stranded or double-stranded DNA or RNA, genomic integration sequences, phages or nucleotide sequences, derived from any source, and may be linear, circular or supercoiled, where a number of polynucleotides are conjugated to or recombinant into a unique construct that can introduce a promoter fragment and DNA sequence of a selected gene product, along with a suitable 3' untranslated sequence, into a cell. In some embodiments, plasmids are used to provide the polynucleotides described herein. In some embodiments, plasmids are used to provide polynucleotides encoding the proteins described herein.

[0122] As used herein, the term “transfection” means the introduction of an exogenous nucleic acid molecule, including a vector, into a cell. For example, transfection methods for components of the CRISPR / Cas compositions described herein are known to those skilled in the art. A “transfected” cell is one which contains an exogenous nucleic acid molecule within the cell, and a “transformed” cell is one in which the exogenous nucleic acid molecule within the cell induces a change in the cell’s phenotype. A transfected nucleic acid molecule can be incorporated into the genomic DNA of a host cell and / or maintained outside the chromosome by the cell, either temporarily or for a long period of time. A host cell or organism expressing an exogenous nucleic acid molecule or fragment is referred herein to as a “recombinant” organism, a “transformed” organism, or a “transgenic” organism. In some embodiments, this disclosure provides a host cell containing one of the vectors described herein, for example, a vector containing a Cas polynucleotide, a vector containing a polynucleotide of interest, or a vector containing a polynucleotide containing an RNA guide sequence, a CAS binding region, a DNA template sequence, or a combination thereof.

[0123] The term "host cell" refers to a cell into which a recombinant expression vector has been introduced, or it may also refer to the offspring of such a cell. For example, offspring may not be identical to the parent cell because modifications may occur in later generations due to mutation or environmental influences, but they are still included within the scope of the term "host cell."

[0124] The terms “peptide,” “polypeptide,” and “protein” are used interchangeably herein and refer to polymeric forms of amino acids of any length, and may include encoded and unencoded amino acids, amino acids not found in nature, chemically or biochemically modified or derivatized amino acids, peptides and polypeptides having a modified peptide backbone, and circular / cyclic peptides and polypeptides.

[0125] The start of a protein or polypeptide is known as the "N-terminus" (also called the amino-terminus, NH2-terminus, N-terminus, or amine-terminus), and refers to the free amine group (-NH2) of the first amino acid residue of the protein or polypeptide. The end of a protein or polypeptide is known as the "C-terminus" (also called the carboxyl-terminus, carboxyl-terminus, C-terminus, or COOH-terminus), and refers to the free carboxyl group (-COOH) of the last amino acid residue of the protein or polypeptide.

[0126] As used herein, “amino acid” refers to a compound containing both a carboxyl (-COOH) group and an amino (-NH2) group. “Amino acid” refers to both natural and unnatural (i.e., synthetic) amino acids. Natural amino acids are abbreviated using three-letter and one-letter abbreviations and include alanine (Ala, A); arginine (Arg, R); asparagine (Asn, N); aspartic acid (Asp, D); cysteine ​​(Cys, C); glutamine (Gln, Q); glutamic acid (Glu, E); glycine (Gly, G); histidine (His, H); isoleucine (Ile, I); leucine (Leu, L); lysine (Lys, K); methionine (Met, M); phenylalanine (Phe, F); proline (Pro, P); serine (Ser, S); threonine (Thr, T); tryptophan (Trp, W); tyrosine (Tyr, Y); and valine (Val, V). Non-natural or synthetic amino acids may contain side chains different from those of the natural amino acids provided above, and may include, for example, fluorophores, post-translational modifications, metal ion chelators, photocage and photocrosslinking moieties, independently reactive functional groups, and NMR, IR, and X-ray crystallographic probes. Exemplary non-natural or synthetic amino acids are provided, for example, in Mitra et al., Mater Methods 3:204 (2013) and Wals et al., Front Chem 2:15 (2014). Non-natural amino acids may also include naturally occurring compounds that are not typically incorporated into proteins or polypeptides, such as citrulline (Cit), selenocysteine ​​(Sec), and pyrrolidine (Pyl).

[0127] "Amino acid substitution" refers to a polypeptide or protein that includes one or more substitutions of a wild-type or naturally occurring amino acid at an amino acid residue, with an amino acid different from the wild-type or naturally occurring amino acid. The substituted amino acid may be synthetic or naturally occurring amino acid. In some embodiments, the substituted amino acid is a naturally occurring amino acid selected from the group consisting of A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, and V. In some embodiments, the substituted amino acid is a non-natural or synthetic amino acid. Substitution variants may be described using an abbreviated system. For example, a substitution variant in which the fifth (5th) amino acid residue is substituted may be abbreviated as "X5Y", where "X" is the wild-type or naturally occurring amino acid to be substituted, "5" is the amino acid residue position in the amino acid sequence of the protein or polypeptide, and "Y" is the substituted amino acid, or a non-wild-type or naturally occurring amino acid.

[0128] An “isolated” polypeptide, protein, peptide, or nucleic acid is a molecule taken out of its natural environment. It is also understood that an “isolated” polypeptide, protein, peptide, or nucleic acid may be formulated with excipients, such as diluents or adjuvants, and may still be considered isolated. As used herein, “isolated” does not necessarily imply any particular level of purity of the polypeptide, protein, peptide, or nucleic acid.

[0129] When used in reference to nucleic acid molecules, peptides, polypeptides, or proteins, the term “recombinant” means a new combination of genetic material not known to exist in nature, or arising therefrom. Recombinant molecules can be produced by any of the techniques available in the field of recombinant technology, including, but not limited to, polymerase chain reaction (PCR), gene splicing (e.g., using restriction endonucleases), and solid-phase synthesis of nucleic acid molecules, peptides, or proteins.

[0130] The term "exogenous" means that the referenced molecule or activity is introduced into the host cell. The molecule can be introduced, for example, by integration into the host chromosome, or by introduction of coding nucleic acid into the host genetic material, such as non-chromosomal genetic material, e.g., as a plasmid. "Exogenous" proteins can be introduced into host cells via "exogenous" nucleic acids that code for the protein. The term "endogenous" refers to the referenced molecule or activity that is naturally present in the host cell. "Endogenous" proteins are expressed by nucleic acids contained within the host cell. The term "heterogeneous" refers to a molecule or activity that originates from a source other than the referenced organism / species, while "homogeneous" refers to a molecule or activity that originates from the host organism / species. Therefore, exogenous expression of coding nucleic acids can utilize either heterogeneous or homogeneous coding nucleic acids, or both.

[0131] When used in relation to polypeptides or proteins, the term “domain” means a distinct functional and / or structural unit within a protein. Domains may be responsible for specific functions or interactions and contribute to the overall role of the protein. Domains can exist in a variety of biological contexts. Similar domains may be found in proteins with different functions. Alternatively, domains with low sequence identity (i.e., less than approximately 50%, 40%, 30%, 20%, 10%, 5%, or less than 1%) may have the same function.

[0132] When used in relation to polypeptides or proteins, the term “motif” generally refers to a conserved set of amino acid residues, typically shorter than 20 amino acids, that may be important for protein function. Certain sequence motifs can mediate common functions in various proteins, such as protein binding or targeting to specific intracellular locations. Examples of motifs include, but are not limited to, nuclear localization signals, microbody targeting motifs, motifs that prevent or promote secretion, and motifs that promote protein recognition and binding. Motif databases and / or motif search tools are well-known in the art, such as PROSITE, PFAM, PRINTS, and MiniMotif Miner.

[0133] As used herein, “engineered” protein means a protein that has one or more modifications in it to achieve a desired property. Exemplary modifications include, but are not limited to, insertions, deletions, substitutions, and / or fusions with another domain or protein. A “fusion protein” (also called a “chimeric protein”) is a protein that typically contains at least two domains encoded by two distinct genes, which are transcribed and translated as a single unit, thereby conjugating to produce a single polypeptide having the functional properties of each of the domains. Engineered proteins of the present disclosure include Cas nucleases, Cas niccas, and fusions of Cas proteins with DNA polymerases, DNA ligases, and / or DNA polymerase-binding proteins.

[0134] In some embodiments, the engineered protein is derived from a wild-type protein. As used herein, “wild-type” protein or nucleic acid is a naturally occurring unmodified protein or nucleic acid. For example, wild-type Cas9 protein can be isolated from the organism Streptococcus pyogenes. Wild-type can be contrasted with “mutants” that have one or more modifications in the amino acid and / or nucleotide sequence of the protein or nucleic acid. In some embodiments, the engineered protein may have substantially the same activity as the wild-type protein (e.g., more than about 80%, more than about 85%, more than about 90%, more than about 95%, or more than about 99% of the activity of the wild-type protein). In some embodiments, the Cas nuclease of the fusion protein described herein has substantially the same activity as the wild-type Cas nuclease.

[0135] In some embodiments, the manipulated protein, for example, the Cas9 protein, may have substantially the same amino acid sequence as the wild-type protein (e.g., identity greater than about 80%, greater than about 85%, greater than about 90%, greater than about 95%, or greater than about 99% with the wild-type protein). As used herein, the terms “sequence similarity” or “similarity%” refer to the degree of identity or correspondence between nucleic acid sequences or amino acid sequences. In the context of polynucleotides, “sequence similarity” may refer to nucleic acid sequences in which a change in one or more nucleotide bases results in the substitution of one or more amino acids, but does not affect the functional properties of the protein encoded by the polynucleotide. “Sequence similarity” may also refer to polynucleotide modifications, such as the deletion or insertion of one or more nucleotide bases, which does not substantially affect the functional properties of the resulting transcript. It is understood that this disclosure encompasses more than specific exemplary sequences. Methods for making nucleotide base substitutions are known as methods for determining the retention of the biological activity of the encoded polypeptide.

[0136] Furthermore, those skilled in the art will recognize that the similar polynucleotides included in this disclosure are also defined by their ability to hybridize with the sequences exemplified herein under stringent conditions. The similar polynucleotides of this disclosure are about 70%, at least about 70%, about 75%, at least about 75%, about 80%, at least about 80%, about 85%, at least about 85%, about 90%, at least about 90%, about 95%, at least about 95%, about 99%, at least about 99%, or about 100% identical to the polynucleotides disclosed herein.

[0137] In the context of polypeptides, "sequence similarity" refers to two or more polypeptides in which more than approximately 40% of the amino acids are identical, or more than approximately 60% of the amino acids are functionally identical. Functionally identical or functionally similar amino acids have chemically similar side chains. For example, amino acids can be grouped according to their functional similarity in the following ways: (i) positively charged side chains: Arg, His, Lys; (ii) negatively charged side chains: Asp, Glu; (iii) polar uncharged side chains: Ser, Thr, Asn, Gln; (iv) hydrophobic side chains: Ala, Val, Ile, Leu, Met, Phe, Tyr, Trp; and (v) others: Cys, Gly, Pro.

[0138] In some embodiments, the similar polypeptides of this disclosure have about 40%, at least about 40%, about 45%, at least about 45%, about 50%, at least about 50%, about 55%, at least about 55%, about 60%, at least about 60%, about 65%, at least about 65%, about 70%, at least about 70%, about 75%, at least about 75%, about 80%, at least about 80%, about 85%, at least about 85%, about 90%, at least about 90%, about 95%, at least about 95%, about 97%, at least about 97%, about 98%, at least about 98%, about 99%, at least about 99%, or about 100% identical amino acids. In some embodiments, the similar polypeptides of the present disclosure have about 60%, at least about 60%, about 65%, at least about 65%, about 70%, at least about 70%, about 75%, at least about 75%, about 80%, at least about 80%, about 85%, at least about 85%, about 90%, at least about 90%, about 95%, at least about 95%, about 97%, at least about 97%, about 98%, at least about 98%, about 99%, at least about 99%, or about 100% functionally identical amino acids.

[0139] Sequence similarity can be determined by sequence alignment using methods known in the art, such as BLAST, MUSCLE, Clustal (including ClustalW and ClustalX), and T-Coffee (including variants such as M-Coffee, R-Coffee, and Expresso).

[0140] The percentage of identity of a polynucleotide or polypeptide can be determined by aligning the polynucleotide or polypeptide sequence over a specific comparison window. In some embodiments, sequence identity is determined by aligning only specific portions of two or more sequences. In some embodiments, sequence similarity is determined by aligning only specific domains of two or more sequences. The comparison window may be a segment of at least 20 to about 1000 residues, or at least 50 to 500 residues, from at least 10 to more than 1000 residues, over which sequences can be aligned and compared. Alignment methods for determining sequence identity are well known and can be performed using publicly available databases such as BLAST. For example, in some embodiments, the "percentage of identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul, Proc Nat Acad Sci USA 87:2264-2268 (1990), modified as in Karlin and Altschul, Proc Nat Acad Sci USA 90:5873-5877 (1993). Such algorithms are incorporated into BLAST programs, such as the BLAST+ or NBLAST and XBLAST programs described in Altschul et al., J Mol Biol, 215:403-410 (1990). BLAST protein searches can be performed using a program such as the XBLAST program with a score of 50 and a word length of 3 to obtain amino acid sequences homologous to the protein molecules disclosed herein. If a gap exists between two sequences, Gapped BLAST can be used, as described in Altschul et al., Nucleic Acids Res 25(17):3389-3402 (1997). When using the BLAST and Gapped BLAST programs, the default parameters for each program (e.g., XBLAST and NBLAST) can be used.

[0141] In some embodiments, the polypeptide or polynucleotide has 70%, at least 70%, 75%, at least 75%, 80%, at least 80%, 85%, at least 85%, 90%, at least 90%, 95%, at least 95%, 97%, at least 97%, 98%, at least 98%, 99%, or at least 99%, or 100% sequence identity with a reference polypeptide or polynucleotide (or fragment of a reference polypeptide or polynucleotide) provided herein. In some embodiments, the polypeptide or polynucleotide has about 70%, at least about 70%, about 75%, at least about 75%, about 80%, at least about 80%, about 85%, at least about 85%, about 90%, at least about 90%, about 95%, at least about 95%, about 97%, at least about 97%, about 98%, at least about 98%, about 99%, at least about 99%, or about 100% sequence identity with the reference polypeptide or polynucleotide (or fragment of the reference polypeptide or nucleic acid molecule) provided herein.

[0142] As used herein, “complex” refers to a group of two or more associated polynucleotides and / or polypeptides. In the context of complex formation, the term “associate” or “associate” refers to molecules that are bound to one another via electrostatic interactions, hydrophobic / hydrophilic interactions, and / or hydrogen bonding interactions, without covalent bonding. Molecules containing different parts that are covalently bonded to each other are known. In some embodiments, a complex is formed when all components of the complex are present together (i.e., a self-assembling complex). In some embodiments, a complex is formed via chemical interactions between different components of the complex, such as hydrogen bonding. In some embodiments, the polynucleotides provided herein complex with the proteins provided herein via secondary structure recognition of the polynucleotides by the proteins. In some embodiments, the Cas-binding region of the polynucleotides provided herein includes a secondary structure recognized by the Cas nuclease, Cas nickase, or fusion protein provided herein.

[0143] The term "alkoxy" refers to an alkyl group that is bonded to the rest of a molecule via an oxygen atom. Typical alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy, and tert-butoxy.

[0144] The term "alkoxyalkyl" refers to an alkyl group bonded to an alkoxy group, where the group is bonded to the rest of the molecule via a carbon atom on the alkyl group, i.e., an alkyl group having the structure -RO-R' (where R and R' are the same or different alkyl groups).

[0145] The terms "alkyl" or "alkane" refer to fully saturated linear or branched non-aromatic hydrocarbons. Examples of linear and branched alkyl groups include methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, hexyl, pentyl, and octyl.

[0146] The term "alkylamino" refers to an amino group substituted with at least one alkyl group, i.e., -NRR', NHR, NRR'H + , or NH2R + This refers to a group having the structure shown in the formula, where R and R' are the same or different alkyl groups.

[0147] The terms "alkyne" or "alkynyl" refer to non-aromatic hydrocarbons containing at least one carbon-carbon triple bond. Examples of alkyne groups include acetylene, propyne, and butyne.

[0148] The term "amide" is derived from the general formula RC(=O)NR1R2, or

[0149] [ka] This refers to a group having R, in the formula 1 , R 2 , and R 3is either hydrogen or the same or different alkyl groups, provided that at least one of them is an alkyl group.

[0150] The term "carbamate" is derived from the general formula R1OC(O)NR2R3 or

[0151] [ka] This refers to a group having R, in the formula 1 , R 2 , and R 3 The atoms are either hydrogen or the same or different alkyl groups, with at least one being an alkyl group. Carbamates are connected to the rest of the molecule via the carbon of one of the alkyl groups.

[0152] The term "carbocyclic" refers to partially or fully saturated non-aromatic hydrocarbon ring systems, including cycloalkyls, cycloalkenyls, and cycloalkynyls. Examples of cycloalkyls include cyclopropane, cyclobutane, cyclopentane, cyclohexane, cyclopropene, cyclobutene, cyclopentene, and cyclohexene.

[0153] The term "ester" refers to structure R 1 -C(O)-OR 2 , or

[0154] [ka] This refers to a group having R, in the formula 1 and R 2 These are the same or different alkyl groups. The ester is connected to the rest of the molecule via the carbon of one of the alkyl groups.

[0155] The term "halo" refers to fluoro, chloro, bromo, and iodine. In some embodiments, halo is fluoro or chloro. In another embodiment, halo is fluoro. In yet another embodiment, halo is chloro.

[0156] The term "haloalkyl" refers to an alkyl group in which one or more hydrogen atoms are substituted with a halo.

[0157] The term "hydroxyalkyl" refers to an alkyl group in which one or more hydrogen atoms are replaced by hydroxyls.

[0158] The terms “heterocyclic,” “heterocyclic,” or “heterocyclic” refer to a partially or fully saturated hydrocarbon ring system in which at least one of the ring carbon atoms is replaced by a heteroatom independently selected from nitrogen, oxygen, and sulfur. The heterocyclic group can be bonded to the rest of the molecule via carbon or nitrogen ring member atoms. Heterocyclics include monocyclic heterocyclics as well as spiro, fused, and / or bridging polycyclic heterocyclics, such as bicyclic heterocyclics. Examples of monocyclic heterocyclics include, but are not limited to, tetrahydropyran, tetrahydrofuran, morpholine, azetidine, pyrrolidine, piperidine, piperazine, azepane, diazepane, oxetane, and isoxazolidine. Examples of polycyclic heterocycles include 2-azaspiro[3.3]heptane, 2,6-diazaspiro[3.3]heptane, 1,6-diazaspiro[3.3]heptane, 2-thia-6-azaspiro[3.3]heptane, 3,6-diazabicyclo[3.1.1]heptane, 2,6-diazaspiro[3.4]octane, 3,8-diazabicyclo[3.2.1]octane, and 4,7-diazaspiro[2.5]octane.

[0159] The term "sulfonyl" is derived from the general formula R 1 S(O)2R 2 or

[0160] [ka] This refers to a group having R, in the formula 1 and R 2The atoms are either hydrogen or the same or different alkyl groups, with at least one being an alkyl group. The sulfonyl group is connected to the rest of the molecule via the carbon on one of the alkyl groups.

[0161] In this specification, "C" is defined as a case where x and y are integers. x~y The prefix C is used in terms such as "alkyl". x~y This indicates the numerical range of carbon atoms present in the group. Suitable C 1~3 Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, and i-propyl. 1~4 Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, and i-propyl, n-butyl, i-butyl, s-butyl, and t-butyl. In some cases, the group has two sections containing carbon, in which case the prefix indicates a numerical range of total carbon in the group, for example, C 2~6 An alkoxyalkyl group refers to an alkoxyalkyl group that has 2 to 6 carbon atoms, formed by the combination of an alkyl group and an alkoxy group.

[0162] Cas protein As used herein, “Cas effector protein” is also referred to herein as “Cas protein” and encompasses both Cas nuclease and Cas nickase. Cas effector protein is part of the CRISPR / Cas system described herein. The CRISPR / Cas system comprises a Cas effector protein and a polynucleotide (also referred to herein as “guide polynucleotide”) which can be used for site-directed genome modification. In some embodiments, the CRISPR / Cas system comprises a Cas effector protein and a guide polynucleotide comprising a Cas-binding region (which binds to and / or activates the Cas protein) and a guide sequence (which hybridizes to a target sequence), wherein the Cas effector protein and the guide polynucleotide form a complex described herein. In some embodiments, the CRISPR / Cas system comprises a Cas effector protein, a first polynucleotide containing a guide sequence, and a second polynucleotide containing a Cas binding region, wherein the first and second polynucleotides hybridize with each other to form a complex with the Cas effector protein.

[0163] The CRISPR / Cas system can be classified into types I to VI based on the Cas effector protein in the system. For example, Cas9 is found in type II systems, and Cas12 is found in type V systems. Each type can be further divided into subtypes. For example, type II may include subtypes II-A, II-B, and II-C, and type V may include subtypes VA and VB. The classification of the CRISPR / Cas system and Cas nucleases is further discussed, for example, in Makarova et al., Methods Mol Biol 1311:47-75 (2015); Makarova et al., The CRISPR Journal Oct 2018;325-336; and Koonin et al., Phil Trans R Soc B 374:20180087 (2018). Unless otherwise specified, the Cas nucleases described herein may include any type or variant.

[0164] In some embodiments, the Cas effector protein is a Cas nuclease. Generally, a Cas effector nuclease can produce double-strand polynucleotide breaks, such as double-strand DNA breaks. Generally, a Cas nuclease can contain one or more nuclease domains, such as RuvC and HNH, which can cleave double-strand DNA. In some embodiments, a Cas nuclease contains a RuvC domain and an HNH domain, each of which cleaves one strand of double-strand DNA. In some embodiments, a Cas nuclease produces blunt ends. In some embodiments, the RuvC and HNH of the Cas nuclease cleave each DNA strand at the same position, thereby producing blunt ends. In some embodiments, a Cas nuclease produces adherent ends. In some embodiments, the RuvC and HNH of the Cas nuclease cleave each DNA strand at different positions (i.e., at an "offset"), thereby producing adherent ends. As used herein, the terms “cohesive end,” “staggered end,” or “sticky end” refer to nucleic acid fragments having multiple strands of unequal length. In contrast to “blunt ends,” sticky ends are produced by alternating cuts on double-stranded nucleic acids (e.g., DNA). Sticky or cohesive ends have unpaired nucleotides or protruding single strands with “overhangs,” e.g., 3' or 5' overhangs.

[0165] In some embodiments, the Cas nuclease is a Cas9 nuclease. Exemplary Cas9 nucleases include, but are not limited to, those derived from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus mutans, Listeria innocua, Neisseria meningitidis, Staphylococcus aureus, Klebisella pneumoniae, and numerous other bacteria. Further exemplary Cas9 nucleases are described, for example, in U.S. Patents 8,771,945, 9,023,649, 10,000,772, 10,407,697, and 2014 / 0068797. In some embodiments, the Cas9 nuclease is derived from S. pyogenes (SpCas9).

[0166] In some embodiments, the Cas9 nuclease comprises a sequence disclosed in UniProt ID G3ECR1 (SEQ ID NO: 1), UniProt ID Q99ZW2 (SEQ ID NO: 2), or UniProt ID J7RUA5 (SEQ ID NO: 3). In some embodiments, Cas9 comprises a polypeptide sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to any of SEQ ID NOs: 1-3. In some embodiments, the disclosure provides a polynucleotide encoding a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to any of SEQ ID NOs: 1-3. In some embodiments, Cas9 is encoded by a polynucleotide that is codon-optimized for expression in host cells.

[0167] In some embodiments, the Cas9 nuclease is a type IIB Cas9 nuclease. Generally, type IIB Cas9 proteins can produce adherent ends as described herein. Exemplary type IIB Cas9 proteins include, but are not limited to, those derived from Legionella pneumophila, Francisella novicida, Parasutterella excrementihominis, Sutterella wadsworthensis, Wolinella succinogenes, and numerous other bacteria. Further type IIB Cas9 proteins are described, for example, in WO2019 / 099943.

[0168] In some embodiments, the Cas effector protein is a Cas12 nuclease. In some embodiments, the Cas nuclease is a Cas12a nuclease (formerly known as "Cpf1" or "C2c1"). In some embodiments, the Cas nuclease is a Cas12f nuclease. The Cas12f nuclease is also known in the art as Cas14 (Makarova et al, Nature Rev. Microbiol., 2019, 18:67-83). In some embodiments, the Cas nuclease is a Cas14 nuclease. Cas12 nucleases are generally smaller than Cas9 nucleases and typically capable of producing sticky ends. Exemplary Cas12 proteins include, but are not limited to, those derived from Francisella novicida, Acidaminococcus sp., Lachnospiraceae sp., Prevotella sp., and numerous other bacteria. Further Cas12 nucleases are described, for example, in U.S. Patent No. 9,580,701, U.S. Patent No. 2016 / 0208243, Zetsche et al., Cell 163(3):759-771 (2015), and Chen et al., Science 360:436-439 (2018).

[0169] In some embodiments, the Cas12 nuclease comprises a sequence disclosed in UniProt ID A0Q7Q2 (SEQ ID NO: 4), UniProt ID U2UMQ6 (SEQ ID NO: 5), or UniProt ID T0D7A2 (SEQ ID NO: 6). In some embodiments, Cas12 has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to any of SEQ ID NOs: 4-6. In some embodiments, the disclosure provides a polynucleotide encoding a polypeptide having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity to any of the polypeptides of SEQ ID NOs: 4-6. In some embodiments, Cas12 is encoded by a codon-optimized polynucleotide for expression in host cells.

[0170] In some embodiments, the Cas effector protein is a Cas nickase. A nickase that produces a single-strand break on a double-stranded polynucleotide (e.g., DNA) is distinguished from a nuclease that cleaves both strands of a double-stranded polynucleotide (e.g., DNA). As discussed herein, wild-type Cas nucleases typically contain two catalytic nuclease domains (RuvC and HNH), each responsible for cleaving one strand of double-stranded DNA. Thus, in some embodiments, Cas nickases contain amino acid mutations in the catalytic domain compared to Cas nucleases. Cas nickases are further described, for example, in Cho et al., Genome Res 24:132-141 (2013); Ran et al., Cell 154:1380-1389 (2013); and Mali et al., Nat Biotechnol 31:833-838 (2013).

[0171] In some embodiments, Cas nickase is Cas9 nickase. In some embodiments, Cas nickase is Cas12a nickase. In some embodiments, Cas nickase is type II-B Cas nickase. In some embodiments, Cas nickase is produced by providing mutations in Cas nuclease. For example, SpCas9 nickase contains the D10A mutation or the H840A mutation compared to wild-type SpCas9 nuclease. It will be understood by those skilled in the art that Cas nickase can be produced by determining the corresponding amino acid residues in other Cas nucleases (e.g., Cas12a or type II-B Cas nucleases) using alignment methods such as those described herein.

[0172] In some embodiments, the Cas nuclease or Cas nickase of the composition is not fused to a heterologous protein domain. In some embodiments, the Cas nuclease or Cas nickase is not fused to a DNA polymerase, DNA ligase, or reverse transcriptase.

[0173] In some embodiments, the recombinant Cas effector protein of this disclosure is part of a fusion protein comprising one or more heterologous protein domains (e.g., the recombinant Cas effector protein plus about or at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more domains). The Cas fusion protein may include any additional protein sequences and, optionally, linker sequences between any two domains. Examples of protein domains that can be fused to a recombinant Cas9 protein include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcriptional activation activity, transcriptional repression activity, transcription termination factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-exclusive examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, autofluorescent proteins including green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and blue fluorescent protein (BFP), as well as mCherry.In some embodiments, the recombinant Cas effector protein is fused to a protein or protein fragment that binds to a DNA molecule or other cellular molecule, including but not limited to a maltose-binding protein (MBP), an S-tag, a Lex A DNA-binding domain (DBD), a GAL4 DNA-binding domain, and the herpes simplex virus (HSV) BP16 protein. Additional domains that may form part of the fusion protein containing the Cas effector protein are described in U.S. Patent Publication 2011 / 0059502. In some embodiments, the tagged recombinant Cas effector protein is used to locate the target sequence.

[0174] In some embodiments, the Cas effector protein is fused to a heterologous protein or protein domain. In some embodiments, the Cas effector protein is fused to a reverse transcriptase. In some embodiments, the Cas effector protein is a Cas9 nuclease fused to a reverse transcriptase. Examples of such Cas9-reverse transcriptase fusions are described in Anzalone et al., Nature, 576:149-157 (2019).

[0175] In some embodiments, the Cas effector protein is fused to DNA polymerase. In some embodiments, the Cas effector protein is a Cas9 nuclease fused to DNA polymerase.

[0176] In some embodiments, the Cas effector protein is fused to dominant-negative 53BP1 (TP53BP1, also known as tumor suppressor p53 binding protein 1). In some embodiments, the Cas effector protein is a Cas9 nuclease fused to a dominant-negative 53BP1 protein. In some embodiments, the dominant-negative 53BP1 protein is DN1S. In some embodiments, the Cas effector protein is a Cas9 nuclease fused to DN1S.

[0177] In some embodiments, the Cas effector protein is fused to the geminin degron domain. In some embodiments, the Cas effector protein is a Cas9 nuclease fused to the geminin degron domain. Examples of such proteins are described in Gutschner et al, Cell Reports, 14:1555-1566 (2016).

[0178] In some embodiments, the Cas effector protein is fused to the CtIP (C-terminal binding protein 1) protein. In some embodiments, the Cas effector protein is a Cas9 nuclease fused to the CtIP protein.

[0179] In some embodiments, recombinant Cas effector proteins can form components of an inducible system. The inducible nature of this system allows for spatiotemporal control of gene editing or gene expression using a form of energy. The form of energy may include, but is not limited to, electromagnetic radiation, acoustic energy, chemical energy, and thermal energy. Non-limiting examples of inducible systems include tetracycline-inducible promoters (Tet-On or Tet-Off), small molecule 2-hybrid transcriptional activation systems (FKBP, ABA, etc.), or photoinducible systems (phytochrome, LOV domain, or cryptochrome). In some embodiments, the Cas effector protein is part of a light-inducible transcriptional effector (LITE) for inducing changes in transcriptional activity in a sequence-specific manner. The photoinducible components may include the Cas effector protein, a photoresponsive cytochrome heterodimer (e.g., from Arabidopsis thaliana), and a transcriptional activation / repression domain. Further examples of inducible DNA-binding proteins and methods for their use are provided in International Patent Publications WO2014 / 018423 and WO2014 / 093635, U.S. Patents 8,889,418 and 8,895,308, and U.S. Patent Publications 2014 / 0186919, 2014 / 0242700, 2014 / 0273234, and 2014 / 0335620.

[0180] [Table 1-1]

[0181] [Table 1-2]

[0182] [Table 1-3]

[0183] [Table 1-4]

[0184] nucleotide i. Target array In some embodiments, the polynucleotides of this disclosure are exogenous polynucleotides containing a sequence of interest (SOI) to be inserted into the genome of a eukaryotic cell. In some embodiments, the sequence of interest encodes a gene of interest.

[0185] In some embodiments, a polynucleotide containing an exogenous polynucleotide (SOI) is an exogenous polynucleotide template inserted into the genome of a eukaryotic cell via CRISPR / Cas-mediated homologous recombination. In some embodiments, the SOI contains at least one target mutation to be inserted into the genome of a eukaryotic cell. In some embodiments, the SOI contains a target gene to be inserted into the genome of a eukaryotic cell. In some embodiments, the SOI can be introduced as an exogenous polynucleotide template. In some embodiments, the SOI is a hybrid polynucleotide containing single-stranded and double-stranded regions. In some embodiments, the hybrid polynucleotide contains double-stranded sequences at the 5' and 3' ends and an inner single-stranded sequence (Shy et al, bioRxiv, 2021, preprint published 9 / 2 / 2021). In some embodiments, the exogenous polynucleotide contains blunt ends. In some embodiments, the exogenous polynucleotide template contains adherent ends. In some embodiments, the exogenous polynucleotide template contains adherent ends complementary to the adherent ends in the target sequence.

[0186] The exogenous polynucleotide template may be of any preferred length, for example, about or at least about 10, 15, 20, 25, 50, 75, 100, 150, 200, 250, 500, 1000, 5000, or 10,000 or more nucleotide lengths. In some embodiments, the exogenous polynucleotide template is complementary to a portion of the polynucleotide containing the target sequence. In some embodiments, when optimally aligned, the exogenous polynucleotide template overlaps with one or more nucleotides of the target sequence (for example, about or at least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, or 100 or more nucleotides). In some embodiments, when the exogenous polynucleotide template and the polynucleotide containing the target sequence are optimally aligned, the nearest nucleotide of the exogenous polynucleotide template is within approximately 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 100, 1500, 2000, 2500, 5000, and 10,000 or more nucleotides from the target sequence.

[0187] In some embodiments, the exogenous polynucleotide is a nucleic acid complexed with DNA, such as a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear fragment of single-stranded or double-stranded DNA, an oligonucleotide, a PCR fragment, a naked nucleic acid, or a delivery vehicle such as a liposome. In some embodiments, the exogenous polynucleotide is RNA. In some embodiments, the RNA is messenger RNA (mRNA).

[0188] In some embodiments, an exogenous polynucleotide is inserted into a target sequence using the cell's endogenous DNA repair pathway. In some embodiments, the endogenous DNA repair pathway is HDR. During the repair process, an exogenous polynucleotide template containing an SOI can be introduced into the target sequence. In some embodiments, an exogenous polynucleotide template containing an SOI adjacent to an upstream sequence and a downstream sequence is introduced into the cell, where the upstream and downstream sequences share sequence similarity with either side of the integration site in the target sequence. In some embodiments, the exogenous polynucleotide containing the SOI includes, for example, a mutant gene. In some embodiments, the exogenous polynucleotide includes an endogenous or exogenous sequence to the cell. In some embodiments, the SOI includes a polynucleotide encoding a protein, or a non-coding sequence such as, for example, a microRNA. In some embodiments, the SOI is operably linked to a regulatory element. In some embodiments, the SOI is the regulatory element. In some embodiments, the SOI includes a resistance cassette, for example, a gene conferring resistance to antibiotics. In some embodiments, the SOI includes a mutation in the wild-type target sequence. In some embodiments, the SOI disrupts or modifies the target sequence by creating frameshift mutations or nucleotide substitutions. In some embodiments, the SOI includes a marker. The introduction of a marker into the target sequence can facilitate screening for targeted integration. In some embodiments, the marker is a restriction site, a fluorescent protein, or a selectable marker. In some embodiments, the SOI is introduced as a vector containing the SOI.

[0189] The upstream and downstream sequences in the exogenous polynucleotide template are selected to facilitate homologous recombination between the target sequence and the exogenous polynucleotide. The upstream sequence is a nucleic acid sequence that shares sequence similarity with the sequence upstream of the targeting site for integration (i.e., the target sequence). Similarly, the downstream sequence is a nucleic acid sequence that shares sequence similarity with the sequence downstream of the targeting site for integration. Thus, in some embodiments, the exogenous polynucleotide template containing the SOI is inserted into the target sequence by homologous recombination in the upstream and downstream sequences. In some embodiments, the upstream and downstream sequences in the exogenous polynucleotide template have at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or about 100% sequence identity with the upstream and downstream sequences of the targeted genomic sequence, respectively. In some embodiments, the upstream or downstream sequence has at least about 20, 50, 100, 150, 200, 250, 300, 350, 400, or 500 base pairs, and at most about 600, 750, 1000, 1250, 1500, 1750, or 2000 base pairs. In some embodiments, the upstream or downstream sequence has about 20 to 2000 base pairs, or about 50 to 1750 base pairs, or about 100 to 1500 base pairs, or about 200 to 1250 base pairs, or about 300 to 1000 base pairs, or about 400 to about 750 base pairs, or about 500 to 600 base pairs. In some embodiments, the upstream or downstream sequence has about 50, about 100, about 250, about 500, about 100, about 1250, about 1500, about 1750, about 2000, about 2250, or about 2500 base pairs.

[0190] In some embodiments, the SOI comprises the gene of interest. As used herein, the term “gene of interest” refers to a gene that codes for the biomolecule of interest (e.g., a protein or RNA molecule). In some embodiments, the gene of interest codes for the protein of interest. In some embodiments, the protein of interest includes intracellular proteins, membrane proteins, extracellular proteins, or combinations thereof. In some embodiments, the protein of interest includes nuclear proteins, transcription factors, nuclear membrane transporters, organelle-associated proteins, membrane receptors, catalytic proteins, enzymes, therapeutic proteins, membrane proteins, membrane transport proteins, signaling proteins, immunological proteins, or combinations thereof. In some embodiments, the immunological protein includes antibodies, e.g., IgG, IgA, IgM, IgD, IgE, or combinations thereof. In some embodiments, the immunological protein is a T cell receptor (TCR). In some embodiments, the immunological protein is a chimeric antigen receptor (CAR). In some embodiments, the SOI codes for a copy of a native gene in the host cell. In some embodiments, the SOI codes for a copy of a native gene that is deficient in the host cell. In some embodiments, the host cell contains a mutation in its gene, and the SOI encodes a wild-type copy of the gene. In some embodiments, the host cell contains a wild-type gene, and the SOI encodes a copy of the gene containing the desired mutation. In some embodiments, the SOI encodes a heterologous gene that is not naturally present in the host cell.

[0191] In some embodiments, the gene of interest encodes the RNA of interest. In some embodiments, the RNA of interest includes therapeutic RNA. In some embodiments, the RNA of interest includes messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNA (snRNA), antisense RNA, microRNA (miRNA), small interfering RNA (siRNA), cell-free RNA (cfRNA), or a combination thereof. In some embodiments, the sequence of interest includes the regulatory element of interest. In some embodiments, the SOI is inserted into a target polynucleotide in a host cell so that the regulatory element on the sequence of interest can regulate the intrinsic gene in the host cell. Regulatory elements are described herein and include, for example, promoters, enhancers, silencers, operators, response elements, 5'UTR, 3'UTR, insulators, etc.

[0192] In some embodiments, the SOI-containing polynucleotide is about 1 to about 5000 nucleotides long. In some embodiments, the SOI-containing polynucleotide is about 5 to about 5000 nucleotides long. In some embodiments, the SOI-containing polynucleotide is about 6 to about 1000 nucleotides long. In some embodiments, the SOI-containing polynucleotide is about 7 to about 750 nucleotides long. In some embodiments, the SOI-containing polynucleotide is about 8 to about 500 nucleotides long. In some embodiments, the SOI-containing polynucleotide is about 9 to about 250 nucleotides long. In some embodiments, the SOI-containing polynucleotide is about 10 to about 100 nucleotides long. In some embodiments, the SOI-containing polynucleotide is about 15 to about 90 nucleotides long. In some embodiments, the SOI-containing polynucleotide is about 20 to about 80 nucleotides long. In some embodiments, the SOI-containing polynucleotide is about 25 to about 70 nucleotides long. In some embodiments, the SOI-containing polynucleotide is about 30 to about 50 nucleotides long. In some embodiments, the SOI-containing polynucleotide is about 1 to about 10 nucleotides long. In some embodiments, the SOI-containing polynucleotide is about 1 to about 20 nucleotides long. In some embodiments, the SOI-containing polynucleotide is about 1 to about 30 nucleotides long. In some embodiments, the SOI-containing polynucleotide is about 10 to about 40 nucleotides long. In some embodiments, the SOI-containing polynucleotide is about 1 to about 50 nucleotides long. In some embodiments, the polynucleotides containing SOI are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long.In some embodiments, the polynucleotide containing the SOI is longer than about 10 nucleotides, longer than about 15 nucleotides, longer than about 20 nucleotides, longer than about 25 nucleotides, longer than about 30 nucleotides, longer than about 35 nucleotides, longer than about 40 nucleotides, longer than about 45 nucleotides, or longer than about 50 nucleotides.

[0193] In some embodiments, the SOI is about 3 to about 5000 nucleotides long. In some embodiments, the SOI is about 4 to about 1000 nucleotides long. In some embodiments, the SOI is about 5 to about 900 nucleotides long. In some embodiments, the SOI is about 6 to about 800 nucleotides long. In some embodiments, the SOI is about 7 to about 700 nucleotides long. In some embodiments, the SOI is about 8 to about 600 nucleotides long. In some embodiments, the SOI is about 9 to about 500 nucleotides long. In some embodiments, the SOI is about 50 to about 5000 nucleotides long. In some embodiments, the SOI is about 60 to about 1000 nucleotides long. In some embodiments, the SOI is about 70 to about 900 nucleotides long. In some embodiments, the SOI is about 8 to about 800 nucleotides long. In some embodiments, the SOI is about 90 to about 700 nucleotides long. In some embodiments, the SOI is about 100 to about 500 nucleotides long. In some embodiments, the SOI is about 100 to about 250 nucleotides long. In some embodiments, the SOI is about 10 to about 90 nucleotides long. In some embodiments, the SOI is about 11 to about 80 nucleotides long. In some embodiments, the SOI is about 12 to about 70 nucleotides long. In some embodiments, the SOI is about 15 to about 60 nucleotides long. In some embodiments, the SOI is about 10 to about 50 nucleotides long. In some embodiments, the SOI is about 1 to about 10 nucleotides long. In some embodiments, the SOI is about 1 to about 25 nucleotides long. In some embodiments, the SOI is about 1 to about 50 nucleotides long. In some embodiments, the SOI is approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 nucleotides long.In some embodiments, the SOI is longer than about 10 nucleotides, longer than about 15 nucleotides, longer than about 20 nucleotides, longer than about 25 nucleotides, longer than about 30 nucleotides, longer than about 35 nucleotides, longer than about 40 nucleotides, longer than about 45 nucleotides, or longer than about 50 nucleotides.

[0194] ii. Cas and Cas-related polynucleotides In some embodiments, the Disclosure includes Cas effector proteins of the Disclosure, i.e., nucleotides or polynucleotide sequences that encode Cas polynucleotides.

[0195] In some embodiments, the polynucleotides of this disclosure can form complexes with Cas effector proteins. In some embodiments, the polynucleotides that can form complexes with Cas effector proteins include a guide sequence. In some embodiments, the polynucleotides that can form complexes with Cas effector proteins include a Cas binding region. In some embodiments, the polynucleotides that can form complexes with Cas effector proteins include a DNA template sequence. In some embodiments, the polynucleotides that can form complexes with Cas effector proteins include a guide sequence, a Cas binding region, and a DNA template sequence, or any combination thereof. In some embodiments, the polynucleotide includes a guide sequence, a Cas binding region, and a DNA template sequence in 5' to 3' order.

[0196] In some embodiments, the guide sequence can hybridize with a target polynucleotide, for example, a target polynucleotide in the genome of a host cell. In several embodiments, the guide sequence is complementary to the target polynucleotide. In some embodiments, the target polynucleotide is target DNA intended to be cleaved by a Cas nuclease or Cas nickase. In some embodiments, the guide sequence includes RNA, i.e., an RNA guide sequence. In some embodiments, the guide sequence includes a combination of RNA and DNA. Hybrid RNA-DNA guide sequences are further described, for example, in Rueda et al., Nat Comm 8:1610 (2017).

[0197] In some embodiments, the guide sequence is about 10 to about 40 nucleotides long. In some embodiments, the guide sequence is about 12 to about 30 nucleotides long. In some embodiments, the guide sequence is about 15 to about 20 nucleotides long. In some embodiments, the guide sequence is about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31, about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, or about 40 nucleotides long. In some embodiments, the guide sequence is long enough to hybridize to the target polynucleotide.

[0198] In some embodiments, the Cas-binding domain can bind to a Cas effector protein (e.g., Cas nuclease or Cas nickase), thereby forming a complex with the Cas protein. In some embodiments, the Cas-binding domain includes RNA. In some embodiments, the Cas-binding domain includes a combination of RNA and DNA. Hybrid RNA-DNA sequences that can bind to and / or activate Cas proteins are further described in Rueda et al., Nat Comm 8:1610 (2017).

[0199] In some embodiments, multiple guide RNAs described herein in the methods, kits, and compositions may be used in the same method, kit, or composition. For example, in some embodiments, two, three, four, five, six, seven, eight, nine, or more different guide RNAs may be used simultaneously.

[0200] In some embodiments, the Cas-binding region comprises tracrRNA that binds to and activates the Cas protein. In some embodiments, the Cas-binding region can hybridize with tracrRNA, and the composition further comprises tracrRNA. In some embodiments, tracrRNA can bind to a Cas nuclease or Cas nickase. In some embodiments, tracrRNA can activate a Cas nuclease or Cas nickase. In some embodiments, activation includes initiating or increasing the cleavage activity of the Cas nuclease or Cas nickase. In some embodiments, activation includes promoting the binding of the Cas nuclease or Cas nickase to a target polynucleotide (e.g., guided by a guide sequence). In some embodiments, activation includes a combination of promoting the binding of the Cas nuclease or Cas nickase to a target polynucleotide and initiating or increasing the cleavage activity of the Cas nuclease or Cas nickase. TracrRNA sequences of Cas proteins (e.g., Cas9, Cas12a, or type II-B Cas proteins as described herein) are available from suitable databases, including RNAcentral and Rfam, and are further described, for example, in Chylinski et al., RNA Biol 10(5):726-737 (2013) and Gasiunas et al., Nat Comm 11:5512 (2020).

[0201] In some embodiments, the polynucleotide that can form a complex with the Cas effector molecule includes a DNA template sequence at the 3' end of the polynucleotide. In some embodiments, the DNA template sequence includes single-stranded DNA. In some embodiments, the DNA template sequence includes the sequence of interest. In some embodiments, the DNA template sequence includes a primer-binding sequence and the sequence of interest. In some embodiments, the DNA template sequence includes a template for amplification by DNA polymerase. In some embodiments, the sequence of interest includes a template for amplification by DNA polymerase. In some embodiments, the Cas nuclease or Cas nickase of the composition is guided to the target polynucleotide by a guide sequence, cleaves the target polynucleotide, and one strand of the cleaved target polynucleotide hybridizes to the primer-binding sequence and functions as a primer for DNA polymerase. In some embodiments, the DNA polymerase can synthesize a DNA strand complementary to the SOI to form a double-stranded sequence containing the SOI. In some embodiments, the double-stranded sequence containing the SOI is inserted into a cleaved target polynucleotide, for example, via a ligation or DNA repair pathway as described herein.

[0202] In some embodiments, the DNA template sequence is approximately 5 to 5000 nucleotides long. In some embodiments, the DNA template sequence is approximately 6 to 1000 nucleotides long. In some embodiments, the DNA template sequence is approximately 7 to 750 nucleotides long. In some embodiments, the DNA template sequence is approximately 8 to 500 nucleotides long. In some embodiments, the DNA template sequence is approximately 9 to 250 nucleotides long. In some embodiments, the DNA template sequence is approximately 10 to 100 nucleotides long. In some embodiments, the DNA template sequence is approximately 15 to 90 nucleotides long. In some embodiments, the DNA template sequence is approximately 20 to 80 nucleotides long. In some embodiments, the DNA template sequence is approximately 25 to 70 nucleotides long. In some embodiments, the DNA template sequence is approximately 30 to 50 nucleotides long. In some embodiments, the DNA template sequence is approximately 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 nucleotides long. In some embodiments, the DNA template sequence is more than approximately 10 nucleotides long, more than approximately 15 nucleotides long, more than approximately 20 nucleotides long, more than approximately 25 nucleotides long, more than approximately 30 nucleotides long, more than approximately 35 nucleotides long, more than approximately 40 nucleotides long, more than approximately 45 nucleotides long, or more than approximately 50 nucleotides long.

[0203] In some embodiments, the DNA template sequence includes a primer-binding sequence. In some embodiments, the primer-binding sequence is about 3 to about 50 nucleotides long. In some embodiments, the primer-binding sequence is about 4 to about 45 nucleotides long. In some embodiments, the primer-binding sequence is about 5 to about 40 nucleotides long. In some embodiments, the primer-binding sequence is about 6 to about 35 nucleotides long. In some embodiments, the primer-binding sequence is about 7 to about 30 nucleotides long. In some embodiments, the primer-binding sequence is about 8 to about 25 nucleotides long. In some embodiments, the primer-binding sequence is about 10 to about 20 nucleotides long. In some embodiments, the primer-binding sequence is about 4 to about 30 nucleotides long. In some embodiments, the primer-binding sequence is approximately 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides long. In some embodiments, the primer-binding sequence is long enough to hybridize with the region of the cleaved target DNA sequence.

[0204] In some embodiments, the polynucleotide containing the DNA template sequence includes modified nucleotides, non-B DNA structures, DNA polymerase-recruited moieties, DNA ligase-recruited moieties, or combinations thereof.

[0205] In some embodiments, the polynucleotide containing the DNA template sequence includes modified nucleotides. In some embodiments, the modified nucleotides include abasic site, a covalent linker, xeno nucleic acid (XNA), locked nucleic acid (LNA), peptide nucleic acid (PNA), phosphorothioate bond, DNA damage, DNA photoproduct, modified deoxyribonucleoside, methylated nucleotide, or a combination thereof.

[0206] In some embodiments, the modified nucleotide reduces or prevents over-elongation of the target sequence by DNA polymerase. In some embodiments, reducing or preventing over-elongation of the target sequence by DNA polymerase increases the accuracy of insertion of a double-stranded sequence containing the target sequence. In some embodiments, the modified nucleotide includes an abasid site, also known as a purine-free / pyrimidine-free (AP) site.

[0207] In some embodiments, the modified nucleotide includes a covalent linker. In some embodiments, the covalent linker includes a triethylene glycol (TEG) linker. In some embodiments, the covalent linker includes an amino linker. TEG linkers and amino linkers have been shown to block polymerase elongation. See, for example, Strobel et al., bioRxiv doi:10.1101 / 2019.12.26.888743 (23 January 2020).

[0208] In some embodiments, the modified nucleotides reduce or prevent nuclease degradation of the polynucleotides of this disclosure. In some embodiments, the modified nucleotides include xeno nucleic acids (XNAs). XNAs are synthetic nucleotide analogs having a sugar group different from the deoxyribose of DNA or the ribose of RNA. Exemplary sugar groups of XNAs include, but are not limited to, threose, cyclohexene, glycol, or locked ribose. In some embodiments, XNAs include 1,5-anhydrohexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycol nucleic acid (GNA), locked nucleic acid (LNA), and peptide nucleic acid (PNA). In some embodiments, the modified nucleotides include locked nucleic acid (LNA), also known as bridged nucleic acid (BNA). LNA is a modified RNA nucleotide in which the ribose portion is modified with an extra crosslink connecting the 2' oxygen and 4' carbon atoms. In some embodiments, the modified nucleotide includes peptide nucleic acid (PNA). Unlike the deoxyribose or ribose backbone of DNA or RNA, the backbone of a PNA polymer contains N-(2-aminoethyl)-glycine units linked by peptide bonds, and purine and pyrimidine bases are linked to the PNA backbone by methylene crosslinks and carbonyl groups. In some embodiments, the modified nucleotide includes a phosphorothioate bond. A phosphorothioate bond contains a sulfur atom instead of one of the oxygen atoms in the phosphate group linking two nucleotides. In some embodiments, the presence of XNA, e.g., LNA or PNA, or a phosphorothioate bond in a polynucleotide increases the stability of the polynucleotide against nuclease degradation.

[0209] In some embodiments, the presence of modified nucleotides in a polynucleotide (e.g., the polynucleotides of the compositions provided herein) can mobilize DNA polymerase to the polynucleotide. In some embodiments, mobilizing DNA polymerase includes increasing the likelihood that DNA polymerase recognizes the polynucleotide, for example due to the presence of modified nucleotides therein, promoting the binding of DNA polymerase to the polynucleotide, and / or activating DNA polymerase, for example, initiating or increasing the activity of DNA polymerase. In some embodiments, the mobilized DNA polymerase binds to the cleaved target polynucleotide chain and extends the desired sequence on the DNA template sequence, as described herein.

[0210] In some embodiments, the modified nucleotides include DNA damage. As used herein, “DNA damage (DNA lesion)” refers to a region of a DNA polynucleotide that typically contains base changes, base deletions, and / or sugar changes that indicate DNA damage. DNA damage can be caused by hydrolysis, oxidation, alkylation, depurination, depyrimidation, and / or deamination of nucleic acid bases. In some embodiments, DNA damage can recruit DNA polymerase. In some embodiments, DNA damage includes 8-oxoguanine, thymine glycol, N7-(2-hydroxyethyl)guanine (7HEG), 7-(2-oxoethyl)guanine, or a combination thereof. In some embodiments, DNA damage includes 8-oxoguanine, thymine glycol, or a combination thereof.

[0211] In some embodiments, the modified nucleotides include DNA photoproducts. These DNA photoproducts are ultraviolet (UV)-induced DNA damage, further described, for example, in Yokoyama et al., Int J Mol Sci 15(11):20321-20338 (2014). In some embodiments, the DNA photoproducts can recruit DNA polymerase. In some embodiments, the DNA photoproducts include pyrimidine dimers, cyclobutane pyrimidine dimers (CPDs), pyrimidine (6-4)pyrimidone photoproducts (also called "(6-4) photoproducts"), adenine-thymine heterodimers, Dewar pyrimidinones, or combinations thereof. In some embodiments, the DNA photoproducts include CPDs, (6-4) photoproducts, or combinations thereof.

[0212] In some embodiments, the modified nucleotide comprises a modified deoxyribonucleoside. In some embodiments, the modified deoxyribonucleoside can recruit DNA polymerase. In some embodiments, the modified deoxyribonucleoside comprises a base not typically present in DNA, namely adenine, cytosine, guanine, or thymine. In some embodiments, the modified deoxyribonucleoside comprises deoxyuridine, acrolein-deoxyguanine, malondialdehyde-deoxyguanine, deoxyinosine, deoxyxanthosine, or a combination thereof. In some embodiments, the modified deoxyribonucleoside comprises deoxyuridine.

[0213] In some embodiments, the modified nucleotide comprises one or more methylated nucleotides. In some embodiments, the methylated nucleotide, for example, methylated cytosine, can recruit DNA polymerase. In some embodiments, the methylated nucleotide comprises 5-hydroxymethylcytosine, 5-methylcytosine, or a combination thereof.

[0214] In some embodiments, the DNA template sequence includes a non-B DNA structure. As used herein, “non-B DNA structure” refers to a DNA secondary structure conformation that is not a standard right-handed B-DNA helix. Non-limiting examples of non-B DNA structures include G quadruples, triple-stranded DNA (H-DNA), Z-DNA, cruciform, slip DNA strands, A tract bends, and sticky DNA. Non-B DNA structures are further described, for example, in Guiblet et al., Nucleic Acids Res 49(3):1497-1516 (2021). In some embodiments, the non-B DNA structure can recruit DNA polymerase. In some embodiments, the non-B DNA structure includes hairpin, cruciform, Z-DNA, H-DNA (triple-stranded DNA), G quadruple-stranded DNA (quadriform DNA), slip DNA, sticky DNA, or a combination thereof.

[0215] In some embodiments, the DNA template sequence includes a DNA polymerase recruitment moiety. DNA polymerase recruitment is described herein. Non-limiting examples of DNA polymerases that can be recruited by the DNA polymerase recruitment moiety include bacterial DNA polymerases such as Pol I (including its Klenow fragment), Pol II, Pol III, Pol IV, or Pol V; eukaryotic DNA polymerases such as Polα, Polβ, Polλ, Polγ, Polσ, Polμ, Polδ, Polε, Polη, Polι, Polκ, Polζ, Polθ, REV1, or REV3; isothermal DNA polymerases such as Bst, T4, or Φ29 (phi29) DNA polymerase; thermostable DNA polymerases such as Taq, Pfu, KOD, Tth, or Pwo DNA polymerase; or variants or homologs thereof.

[0216] In some embodiments, the polynucleotides of this disclosure may be chemically crosslinked to one or more moieties or conjugates that enhance the activity, cell distribution, or cell uptake of the polynucleotide. These moieties or conjugates may include conjugate groups covalently bonded to functional groups such as primary or secondary hydroxyl groups. Examples of conjugate groups include, but are not limited to, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, polyethers, groups that enhance the pharmacodynamic properties of oligomers, and groups that enhance the pharmacokinetic properties of oligomers. Preferred conjugate groups include, but are not limited to, cholesterol, lipids, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin, and dyes. Examples of groups that enhance pharmacodynamic properties include groups that improve uptake, groups that enhance resistance to degradation, and / or groups that enhance sequence-specific hybridization with target nucleic acids. Groups that enhance pharmacokinetic properties include groups that improve the uptake, distribution, metabolism, or excretion of target nucleic acids.

[0217] The conjugate portion includes lipid portions such as cholesterol (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NYA Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), and thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992,20,533-538), aliphatic chains, e.g., dodecanediol or undecyl residues (Saison-Behmoaras et al.,EMBO J.,1991,10,1111-1118; Kabanov et al.,FEBS Lett.,1990,259,327-330; Svinarchuk et al.,Biochimie,1993,75,49-54), phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium, 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett.,1995,36,3651-3654; Shea et al.,Nucl.Acids Res., 1990, 18, 3777-3783), polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), palmitoyl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther.Examples include, but are not limited to, 1996, 277, and 923-937.

[0218] The conjugate may contain a "protein transduction domain" or PTD (also known as a CPP cell-permeable peptide), which may refer to a polypeptide, polynucleotide, carbohydrate, or organic or inorganic compound that facilitates translocation across lipid bilayers, micelles, cell membranes, organelle membranes, or vesicle membranes. A PTD conjugated to another molecule (which can range from small polar molecules to large macromolecules and / or nanoparticles) facilitates the molecule's translocation across the membrane (e.g., from extracellular space to intracellular space, or from cytosol to organelle). In some embodiments, the PTD is covalently attached to the amino terminus of an exogenous polypeptide (e.g., a site-directed modified polypeptide). In some embodiments, the PTD is covalently attached to the carboxyl terminus of an exogenous polypeptide (e.g., a site-directed modified polypeptide). In some embodiments, the PTD is covalently attached to a nucleic acid (e.g., DNA-targeted RNA, a polynucleotide encoding DNA-targeted RNA, a polynucleotide encoding a site-directed modified polypeptide, etc.).Examples of PTDs include: minimal undecapeptide protein transduction domains (sequence number 7, corresponding to residues 47-57 of HIV-1 TAT containing YGRKKRRQRRR); polyarginine sequences containing a sufficient number of arginines to direct cell entry (e.g., 3, 4, 5, 6, 7, 8, 9, 10, or 10-50 arginines); VP22 domains (Zender et al. (2002) Cancer Gene Ther. 9(6):489-96); Drosophila Antennapedia protein transduction domains (Noguchi et al. (2003) Diabetes 52(7):1732-1737); cleaved human calcitonin peptides (Trehin et al. (2004) Pharm. Research 21:1248-1256); polylysine (Wender et al. al.(2000)Proc.Natl.Acad.Sci.USA97:13003-13008);RRQRRTSKLMKR (SEQ ID NO: 8); transportan GWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO: 9);KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO: 10); and RQIKIWFQNRRMKWKK (SEQ ID NO: 11). Examples of PTDs include YGRKKRRQRRR (SEQ ID NO: 12), RKKRRQRRR (SEQ ID NO: 13); and arginine homopolymers of 3 to 50 arginine residues. Examples of PTD domain amino acid sequences include, but are not limited to, the following. YGRKKRRQRRR (SEQ ID NO: 14); RKKRRQRR (SEQ ID NO: 15); YARAAARQARA (SEQ ID NO: 16); THRLPRRRRRR (SEQ ID NO: 17); and GGRRARRRRRR (SEQ ID NO: 18). In some embodiments, PTD is activatable CPP (ACPP) (Aguilera et al. (2009) Integr Biol (Camb) June; 1(5-6): 371-381).ACPP contains a polycationic CPP (e.g., Arg9 or "R9") linked to a matching polyanion (e.g., Glu9 or "E9") via a cleavable linker, thereby reducing the net charge to near zero and inhibiting adhesion and uptake to cells. Upon cleavage of the linker, the polyanion is released, locally exposing polyarginine and its inherent adhesive properties, thus "activating" the ACPP and allowing it to traverse the membrane.

[0219] In some embodiments, the polynucleotides of this disclosure are codon-optimized for expression in eukaryotic cells. In some embodiments, the polynucleotide sequence encoding stiCas9 is codon-optimized for expression in animal cells. In some embodiments, the polynucleotide sequence encoding recombinant Cas effector protein is codon-optimized for expression in human cells. In some embodiments, the polynucleotide sequence encoding recombinant Cas effector protein is codon-optimized for expression in plant cells. Codon optimization is the adjustment of codons to match the tRNA abundance of the expression host in order to increase the yield and efficiency of recombinant or heterologous protein expression. Codon optimization methods are commonplace in the art and can be performed using software programs such as Integrated DNA Technologies' Codon Optimization tool, Entelechon's Codon Usage Table Analysis tool, GENEMAKER's Blue Heron software, Aptagen's Gene Forge software, DNA Builder Software, General Codon Usage Analysis software, publicly available OPTIMIZER software, and Genscript's OptimumGene algorithm.

[0220] CRISPR-Cas system In some embodiments, the disclosure encompasses a CRISPR-Cas system comprising a naturally occurring or non-naturally occurring Cas effector protein and a polynucleotide encoding a sequence of interest. In some embodiments, the CRISPR-Cas system comprises a naturally occurring or non-naturally occurring Cas effector protein, a polynucleotide encoding a sequence of interest, and a polynucleotide capable of complexing with the Cas effector protein. In some embodiments, the polynucleotide capable of complexing with the Cas effector protein comprises a guide sequence, a Cas-binding region, and a DNA template region.

[0221] In some embodiments, the CRISPR-Cas system comprises a regulatory element operably linked to a polynucleotide sequence encoding a recombinant Cas effector protein provided herein, and a polynucleotide comprising a guide sequence that complexes with the recombinant Cas effector protein.

[0222] In some embodiments, the regulatory element linked to the polynucleotide sequence encoding the recombinant Cas effector protein is a promoter. In some embodiments, the regulatory element is a eukaryotic promoter. In some embodiments, the regulatory element is a viral promoter. In some embodiments, the regulatory element is a eukaryotic regulatory element, i.e., a eukaryotic promoter. In some embodiments, the eukaryotic regulatory element is a mammalian promoter.

[0223] In some embodiments, the polynucleotides that can form complexes with the Cas effector proteins of the CRISPR-Cas system are RNA molecules. RNA molecules that bind to CRISPR-Cas components and target them to specific locations within target DNA are referred herein as “guide RNA,” “gRNA,” or “small molecule guide RNA,” and may also be referred herein as “DNA targeting RNA.” A guide polynucleotide, e.g., guide RNA, comprises at least two nucleotide segments: at least one “DNA-binding segment” and at least one “polypeptide-binding segment.” “Segment” means a portion, section, or region of a molecule, e.g., a continuous stretch of nucleotides in a guide polynucleotide molecule. Unless otherwise defined, the definition of “segment” is not limited to a specific number of total base pairs.

[0224] In some embodiments, the DNA-binding segment of a guide polynucleotide (or "DNA targeting sequence") hybridizes with a target sequence within the cell. In some embodiments, the DNA-binding segment of a guide polynucleotide, for example, a guide RNA, contains a polynucleotide sequence complementary to a specific sequence in the target DNA.

[0225] In some embodiments, the guide polynucleotides of the Disclosure have a guide sequence that hybridizes to a target sequence in a eukaryotic cell. In some embodiments, the eukaryotic cell is an animal or human cell. In some embodiments, the eukaryotic cell is a human, rodent, or bovine cell line or cell strain. Examples of such cells, cell lines, or cell strains include, but are not limited to, mouse myeloma (NSO) cell lines, Chinese hamster ovary (CHO) cell lines, HT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BHK (baby hamster kidney cell), VERO, SP2 / 0, YB2 / 0, Y0, C127, L cells, COS, e.g., COS1 and COS7, QC1-3, HEK-293, VERO, PER.C6, HeLA, EBl, EB2, EB3, oncolytic or hybridoma cell lines. In some embodiments, the eukaryotic cells are CHO cell lines. In some embodiments, the cells are CHO-K1 cells, CHO-K1 SV cells, DG44 CHO cells, DUXB11 CHO cells, CHOS, CHO GS knockout cells, CHO FUT8 GS knockout cells, CHOZN, or CHO-derived cells. CHO GS knockout cells (e.g., GSKO cells) are, for example, CHO-K1 SV GS knockout cells. CHO FUT8 knockout cells are, for example, POTELLIGENT CHOK1 SV (Lonza Biologics, Inc.). Eukaryotic cells may also be, for example, bird cells, cell lines, or cell strains such as EBX cells, EB14, EB24, EB26, EB66, or EBvl3.

[0226] In some embodiments, eukaryotic cells are human cells. In some embodiments, human cells are stem cells. Stem cells may be pluripotent stem cells, including, for example, embryonic stem cells (ESCs), adult stem cells, induced pluripotent stem cells (iPSCs), tissue-specific stem cells (e.g., hematopoietic stem cells), and mesenchymal stem cells (MSCs). In some embodiments, human cells are any differentiated form of the cells described herein. In some embodiments, eukaryotic cells are cells derived from any primary cells in culture.

[0227] In some embodiments, eukaryotic cells are hepatocytes such as human hepatocytes, animal hepatocytes, or non-parenchymal cells. For example, eukaryotic cells may be cultureable metabolically qualified human hepatocytes, cultureable induced human hepatocytes, cultureable human hepatocytes, suspension-qualified human hepatocytes (including 10-donor and 20-donor pooled hepatocytes), human hepatic Kupffer cells, human hepatic stellate cells, canine hepatocytes (including single and pooled beagle hepatocytes), mouse hepatocytes (including CD-1 and C57BI / 6 hepatocytes), rat hepatocytes (including Sprague-Dawley, Wistar Han, and Wistar hepatocytes), monkey hepatocytes (including cynomolgus or rhesus monkey hepatocytes), feline hepatocytes (including Domestic Shorthair hepatocytes), and rabbit hepatocytes (including New Zealand White hepatocytes).

[0228] In some embodiments, eukaryotic cells are plant cells. For example, plant cells may be those of crop plants such as cassava, maize, sorghum, wheat, or rice. Plant cells may be those of algae, trees, or vegetables. Plant cells may be those of monocots or dicots, or of crops or cereal plants, production plants, fruits, or vegetables. For example, plant cells can be those of trees, such as citrus trees like orange, grapefruit, or lemon trees; peach or nectarine trees; apple or pear trees; nut-bearing trees like almond, walnut, or pistachio trees; nightshade plants like potatoes, Brassica plants, and Lactuca plants; spinach plants; Capsicum plants; and plants like cotton, tobacco, asparagus, carrots, cabbage, broccoli, cauliflower, tomatoes, eggplants, peppers, lettuce, spinach, strawberries, blueberries, raspberries, blackberries, grapes, coffee, and cocoa.

[0229] In some embodiments, the guide sequence of the guide polynucleotide is about 5 to about 50 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 6 to about 45 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 7 to about 40 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 8 to about 35 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 9 to about 30 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 10 to about 20 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 12 to about 20 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 14 to about 20 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 16 to about 20 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 18 to about 20 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 5 to about 10 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 6 to about 10 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 7 to about 10 nucleotides. In some embodiments, the guide sequence of the guide polynucleotide is about 8 to about 10 nucleotides. The length of the guide sequence can be determined by those skilled in the art using guide sequence design tools such as, for example, CRISPR Design Tool (Hsu et al., Nat Biotechnol 31(9):827-832 (2013)), ampliCan (Labun et al., bioRxiv 2018, doi:10.1101 / 249474), CasFinder (Alach et al., bioRxiv 2014, doi:10.1101 / 005074), or CHOPCHOP (Labun et al., Nucleic Acids Res 2016, doi:10.1093 / nar / gkw398).

[0230] In some embodiments, the guide polynucleotide of the Disclosure, e.g., guide RNA, includes a polypeptide-binding sequence / segment. The polypeptide-binding segment (or "protein-binding sequence") of the guide polynucleotide, e.g., guide RNA, interacts with the polynucleotide-binding domain of the Cas effector protein of the Disclosure. Such polypeptide-binding segments or sequences are known to those skilled in the art and are disclosed, for example, in U.S. Patent Publications 2014 / 0068797, 2014 / 0273037, 2014 / 0273226, 2014 / 0295556, 2014 / 0295557, 2014 / 0349405, 2015 / 0045546, 2015 / 0071898, 2015 / 0071899, and 2015 / 0071906, the entirety of which is incorporated herein by reference. In some embodiments, the polypeptide-binding segment of a guide polynucleotide binds to Cas9. In some embodiments, the polypeptide-binding segment of a guide polynucleotide binds to the recombinant Cas9 protein provided herein.

[0231] In some embodiments, the guide polynucleotide is at least about 10, 15, 20, 25, or 30 nucleotides, and at most about 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 nucleotides. In some embodiments, the guide polynucleotide is about 10 to about 150 nucleotides. In some embodiments, the guide polynucleotide is about 20 to about 120 nucleotides. In some embodiments, the guide polynucleotide is about 30 to about 100 nucleotides. In some embodiments, the guide polynucleotide is about 40 to about 80 nucleotides. In some embodiments, the guide polynucleotide is about 50 to about 60 nucleotides. In some embodiments, the guide polynucleotide is about 10 to about 35 nucleotides. In some embodiments, the guide polynucleotide is about 15 to about 30 nucleotides. In some embodiments, the guide polynucleotide is about 20 to about 25 nucleotides.

[0232] Guide polynucleotides, such as guide RNA, can be introduced into target cells as isolated molecules, such as RNA molecules, or they can be introduced into cells using an expression vector containing DNA encoding the guide polynucleotide, such as guide RNA.

[0233] In some embodiments, the guide polynucleotide of the CRISPR-Cas system is ligated to a direct repeat sequence. The direct repeat, or DR sequence, is an array of repeat sequences at the CRISPR locus, spaced apart by short stretches (spacers) of non-repetitive sequences. The spacer sequences target protospacer adjacent motifs (PAMs) on the target sequence. When the non-coding portion of the CRISPR locus (i.e., the guide polynucleotide and tracrRNA) is transcribed, the transcript is cleaved at the DR sequence into short crRNAs containing individual spacer sequences that direct the Cas9 nuclease to the PAMs. In some embodiments, the DR sequence is RNA. In some embodiments, the DR sequence is encoded by nucleic acid. In some embodiments, the DR sequence is ligated to the guide polynucleotide. In some embodiments, the DR sequence is ligated to the guide sequence of the guide polynucleotide. In some embodiments, the DR sequence includes a secondary structure. In some embodiments, the DR sequence includes a stem-loop structure. In some embodiments, the DR sequence is 10 to 20 nucleotides long. In some embodiments, the DR sequence is at least 16 nucleotides. In some embodiments, the DR sequence is at least 16 nucleotides and includes a single stem-loop. In some embodiments, the DR sequence includes an RNA aptamer. In some embodiments, the secondary structure or stem-loop in the DR is recognized by a nuclease for cleavage. In some embodiments, the nuclease is a recombinase. In some embodiments, the nuclease is RNase III.

[0234] In some embodiments, the CRISPR-Cas system of the present disclosure further comprises tracrRNA. The "tracrRNA," or transactivated CRISPR-RNA, forms an RNA double helix with pre-crRNA or pre-CRISPR-RNA and is then cleaved by the RNA-specific ribonuclease RNase III to form a crRNA / tracrRNA hybrid. In some embodiments, the guide RNA comprises the crRNA / tracrRNA hybrid. In some embodiments, the tracrRNA component of the guide RNA activates the Cas effector protein. In some embodiments, the guide polynucleotide of the CRISPR-Cas system comprises a tracrRNA sequence. In some embodiments, the CRISPR-Cas system comprises a separate polynucleotide comprising a tracrRNA sequence.

[0235] In some embodiments, the polynucleotide encoding the recombinant Cas effector protein and the guide polynucleotide are on a single vector. In some embodiments, the polynucleotide encoding the recombinant Cas effector protein, the guide polynucleotide (or a nucleotide that can be transcribed into the guide polynucleotide), and the tracrRNA are on a single vector. In some embodiments, the polynucleotide encoding the recombinant Cas effector protein, the guide polynucleotide (or a nucleotide that can be transcribed into the guide polynucleotide), the tracrRNA, and the direct repeat sequence are on a single vector. In some embodiments, the vector is an expression vector. In some embodiments, the vector is a mammalian expression vector. In some embodiments, the vector is a human expression vector. In some embodiments, the vector is a plant expression vector.

[0236] In some embodiments, the polynucleotide encoding the recombinant Cas effector protein and the guide polynucleotide are a single nucleic acid molecule. In some embodiments, the polynucleotide encoding the recombinant Cas effector protein, the guide polynucleotide, and the tracrRNA are a single nucleic acid molecule. In some embodiments, the polynucleotide encoding the recombinant Cas effector protein, the guide polynucleotide, the tracrRNA, and the direct repeat sequence are a single nucleic acid molecule. In some embodiments, the single nucleic acid molecule is an expression vector. In some embodiments, the single nucleic acid molecule is a mammalian expression vector. In some embodiments, the single nucleic acid molecule is a human expression vector. In some embodiments, the single nucleic acid molecule is a plant expression vector.

[0237] In some embodiments, recombinant Cas effector proteins and guide polynucleotides can form a complex. In some embodiments, the complex of recombinant Cas effector proteins and guide polynucleotides does not exist in nature.

[0238] cell In some embodiments of this disclosure, eukaryotic cells are eukaryotic cells. In some embodiments, eukaryotic cells are animal or human cells. In some embodiments, eukaryotic cells are human, rodent, or bovine cell lines (cell lines or cell strains). Examples of such cells, cell lines (cell lines or cell strains) include, but are not limited to, mouse myeloma (NSO) cell lines, Chinese hamster ovary (CHO) cell lines, HT1080, H9, HepG2, MCF7, MDBK Jurkat, NIH3T3, PC12, BHK (baby hamster kidney cells), VERO, SP2 / 0, YB2 / 0, Y0, C127, L cells, COS, e.g., COS1 and COS7, QC1-3, HEK-293, VERO, PER.C6, HeLa, EBl, EB2, EB3, oncolytic or hybridoma cell lines. In some embodiments, eukaryotic cells are CHO cell lines. In some embodiments, the eukaryotic cell is a CHO cell. In some embodiments, the cell is a CHO-K1 cell, a CHO-K1 SV cell, a DG44 CHO cell, a DUXB11 CHO cell, a CHOS cell, a CHO GS knockout cell, a CHO FUT8 GS knockout cell, a CHOZN cell, or a CHO-derived cell. A CHO GS knockout cell (e.g., a GSKO cell) is, for example, a CHO-K1 SV GS knockout cell. A CHO FUT8 knockout cell is, for example, a POTELLIGENT CHOK1 SV (Lonza Biologics, Inc.). The eukaryotic cell may also be a bird cell, cell line, or cell strain, such as EBX cells, EB14, EB24, EB26, EB66, or EBvl3.

[0239] In some embodiments, eukaryotic cells are human cells. In some embodiments, human cells are stem cells. Stem cells can be pluripotent stem cells, including, for example, embryonic stem cells (ESCs), adult stem cells, induced pluripotent stem cells (iPSCs), tissue-specific stem cells (e.g., hematopoietic stem cells), and mesenchymal stem cells (MSCs). In some embodiments, cells are pluripotent stem cells. In some embodiments, cells are induced pluripotent stem cells. In some embodiments, human cells are any differentiated form of the cells described herein. In some embodiments, eukaryotic cells are cells derived from any primary cells in culture.

[0240] In some embodiments, eukaryotic cells are hepatocytes such as human hepatocytes, animal hepatocytes, or non-parenchymal cells. For example, eukaryotic cells may be cultureable metabolically qualified human hepatocytes, cultureable induced human hepatocytes, cultureable human hepatocytes, suspension-qualified human hepatocytes (including 10-donor and 20-donor pooled hepatocytes), human hepatic Kupffer cells, human hepatic stellate cells, canine hepatocytes (including single and pooled beagle hepatocytes), mouse hepatocytes (including CD-1 and C57BI / 6 hepatocytes), rat hepatocytes (including Sprague-Dawley, Wistar Han, and Wistar hepatocytes), monkey hepatocytes (including cynomolgus or rhesus monkey hepatocytes), feline hepatocytes (including Domestic Shorthair hepatocytes), and rabbit hepatocytes (including New Zealand White hepatocytes).

[0241] In some embodiments, the eukaryotic cells are hematopoietic cells. In some embodiments, the hematopoietic cells are myeloid progenitor cells. In some embodiments, the hematopoietic cells are lymphocyte progenitor cells. In some embodiments, the hematopoietic cells are mast cells, megakaryocytes, platelets, basophils, neutrophils, eosinophils, dendritic cells, monocytes, or macrophages. In some embodiments, the hematopoietic cells are natural killer cells (NK cells), T lymphocytes, or B lymphocytes. In some embodiments, the T lymphocytes or B lymphocytes contain chimeric antigen receptors (CARs).

[0242] In some embodiments, eukaryotic cells are plant cells. For example, plant cells may be plant cells of crop plants such as cassava, maize, sorghum, wheat, or rice. Plant cells may be plant cells of algae, trees, or vegetables. Plant cells may be plant cells of monocots or dicots, or of crops or cereal plants, production plants, fruits, or vegetables. For example, plant cells can be those of trees, such as citrus trees like orange, grapefruit, or lemon trees; peach or nectarine trees; apple or pear trees; nut-bearing trees like almond, walnut, or pistachio trees; nightshade plants like potatoes, Brassica plants, and Lactuca plants; spinach plants; Capsicum plants; and plants like cotton, tobacco, asparagus, carrots, cabbage, broccoli, cauliflower, tomatoes, eggplants, peppers, lettuce, spinach, strawberries, blueberries, raspberries, blackberries, grapes, coffee, and cocoa.

[0243] In some embodiments, the eukaryotic cells are tissue cultures of any of the aforementioned cells. In some embodiments, the eukaryotic cells are in the form of tissue extracts of any of the aforementioned cells.

[0244] In some embodiments, eukaryotic cells contain genomically integrated Cas polynucleotides. In some embodiments, eukaryotic cells contain inducible genomically integrated Cas polynucleotides.

[0245] Delivery system Various methods for delivering CRISPR-Cas systems are known in the art. Preferred delivery systems include microinjection, electroporation, transfection, or hydrodynamic delivery of polynucleotides encoding Cas effector proteins, polynucleotides containing the sequence of interest, and / or polynucleotides capable of forming complexes with Cas effector proteins. In some embodiments, the delivery system includes delivery particles. Examples of such delivery systems, including nanoparticles, cell-permeable peptides, and DNA nanoclues, are disclosed in Lino et al., Drug Delivery, 25(1):1234-1257 (2018).

[0246] In some embodiments, the CRISPR-Cas system of this disclosure, comprising a Cas effector protein, a polynucleotide encoding the Cas effector protein, a polynucleotide encoding a sequence of interest, and / or a polynucleotide capable of forming a complex with the Cas effector protein, is delivered by delivery particles. The delivery particles are a biological delivery system or formulation comprising particles. “Particles,” as defined herein, are entities having a maximum diameter of about 100 microns (μm). In some embodiments, particles have a maximum diameter of about 10 μm. In some embodiments, particles have a maximum diameter of about 2000 nanometers (nm). In some embodiments, particles have a maximum diameter of about 1000 nm. In some embodiments, particles have a maximum diameter of about 900 nm, about 800 nm, about 700 nm, about 600 nm, about 500 nm, about 400 nm, about 300 nm, about 200 nm, or about 100 nm. In some embodiments, particles have a diameter of about 25 nm to about 200 nm. In some embodiments, the particles have a diameter of about 50 nm to about 150 nm. In some embodiments, the particles have a diameter of about 75 nm to about 100 nm.

[0247] The delivery particles may be provided in any form, including but not limited to solid, semi-solid, emulsion, or colloidal particles. In some embodiments, the delivery particles are lipid-based systems, liposomes, micelles, microvesicles, exosomes, or gene guns. In some embodiments, the delivery particles comprise a CRISPR-Cas system. In some embodiments, the delivery particles comprise a CRISPR-Cas system comprising a recombinant Cas effector protein and a polynucleotide capable of forming a complex with the Cas effector protein, wherein the polynucleotide comprises a guide polynucleotide. In some embodiments, the delivery particles comprise a Cas effector protein, a polynucleotide containing a sequence of interest, and a polynucleotide capable of forming a complex with the Cas effector protein and comprising a guide polynucleotide. In some embodiments, the delivery particles comprise a CRISPR-Cas system comprising a recombinant Cas effector protein and a polynucleotide capable of forming a complex with the Cas effector protein and comprising a guide polynucleotide, wherein the recombinant Cas effector protein and the polynucleotide are in the complex. In some embodiments, the delivery particle comprises a CRISPR-Cas system comprising a recombinant Cas effector protein, a polynucleotide that forms a complex with the Cas effector protein and includes a guide polynucleotide, and a polynucleotide that includes tracrRNA.

[0248] In some embodiments, the Cas effector protein-polynucleotide complex of the present disclosure is a ribonucleoprotein (RNP), which is delivered via hydrodynamic delivery, nanoparticles, vesicles, cell-permeable peptides, or DNA nanoclues.

[0249] In some embodiments, the delivery particle further comprises lipids, sugars, metals, or proteins. In some embodiments, the delivery particle is a lipid envelope. mRNA delivery using a lipid envelope or a lipid-containing delivery particle is described, for example, in Su et al., Molecular Pharmacology 8(3):774-784 (2011). In some embodiments, the delivery particle is a sugar-based particle, for example, GalNAc. Sugar-based particles are described in WO 2014 / 118272 and Nair et al., J.Am.Chem.Soc.136(49):16958-16961 (2014).

[0250] In some embodiments, the delivery particles are nanoparticles. Nanoparticles encompassed in this disclosure may be provided in different forms, for example, as solid nanoparticles (e.g., metals such as silver, gold, iron, and titanium), nonmetals, lipid-based solids, polymers, suspensions of nanoparticles, or combinations thereof. Metallic, dielectric, and semiconductor nanoparticles, as well as hybrid structures (e.g., core-shell nanoparticles), can be prepared. Nanoparticles fabricated from semiconductor materials may be labeled as quantum dots if they are small enough (typically less than 10 nm) to cause quantization of electronic energy levels. Such nanoscale particles can be used in biomedical applications as drug carriers or contrast agents and may be adapted for similar purposes in this disclosure.

[0251] Preparation of delivery particles is further described in U.S. Patent Publications 2011 / 0293703, 2012 / 0251560, and 2013 / 0302401, and U.S. Patents 5,543,158, 5,855,913, 5,895,309, 6,007,845, and 8,709,843.

[0252] In some embodiments, the vesicle comprises the CRISPR-Cas system of the Disclosure. A “vesicle” is a small intracellular structure having fluid surrounded by a lipid bilayer. In some embodiments, the CRISPR-Cas system of the Disclosure is delivered by the vesicle. In some embodiments, the vesicle comprises a recombinant Cas effector protein and a guide polynucleotide. In some embodiments, the vesicle comprises a Cas effector protein and a guide polynucleotide, wherein the Cas effector protein and the guide polynucleotide are in a complex. In some embodiments, the vesicle comprises a CRISPR-Cas system comprising a Cas effector protein, a polynucleotide that can form a complex with the Cas effector protein and includes a guide polynucleotide, and a polynucleotide that includes tracrRNA. In some embodiments, the vesicle comprises a CRISPR-Cas system comprising a Cas effector protein, a polynucleotide that can form a complex with the Cas effector protein and includes a guide polynucleotide, and tracrRNA.

[0253] In some embodiments, a vesicle comprising a Cas effector protein and a polynucleotide capable of forming a complex with the Cas effector protein and comprising a guide polynucleotide is an exosome or liposome. In some embodiments, the vesicle is an exosome. In some embodiments, the exosome is used to deliver the CRISPR-Cas system of the present disclosure. The exosome is an endogenous nanovesicle (i.e., having a diameter of about 30 to about 100 nm) that transports RNA and proteins and can deliver RNA to the brain and other target organs. Engineered exosomes for delivering exogenous biological material to target organs are described, for example, by Alvarez-Erviti et al., Nature Biotechnology 29:341 (2011), El-Andaloussi et al., Nature Protocols 7:2112-2116 (2012), and Wahlgren et al., Nucleic Acids Research 40(17):e130 (2012).

[0254] In some embodiments, liposomes are used to deliver the CRISPR-Cas system of this disclosure. Liposomes are spherical vesicle structures having at least one lipid bilayer and can be used as vehicles for the administration of nutrients and pharmaceuticals. Liposomes are often composed of phospholipids, particularly phosphatidylcholine, but can also be composed of other lipids such as egg phosphatidylethanolamine. Types of liposomes include, but are not limited to, multimembrane vesicles, small monomembrane vesicles, large monomembrane vesicles, and spiral vesicles. See, for example, Spuch and Navarro, Journal of Drug Delivery, Article ID 469679 (2011). Liposomes for the delivery of biological materials such as CRISPR-Cas components have been described, for example, by Morrissey et al., Nature Biotechnology 23(8):1002-1007 (2005), Zimmerman et al., Nature Letters 441:111-114 (2006), and Li et al., Gene Therapy 19:775-780 (2012).

[0255] In some embodiments, Cas effector proteins can be delivered using cell-permeable peptides fused to the Cas effector proteins.

[0256] In some embodiments, the Cas effector proteins and polynucleotides of this disclosure can be delivered in the form of DNA nanoclues. A DNA nanoclue is a spherical structure containing DNA that can be loaded with a payload such as a Cas effector protein (Sun et al., J.Am.Chem.Soc., 136:14722-14725). DNA nanoclues have been used in vitro for the delivery of Cas9 editing systems (Lino et al., Drug Delivery, 25(1):1234-1257).

[0257] In some embodiments, the viral vector comprises the CRISPR-Cas system of the Disclosure. In some embodiments, the CRISPR-Cas system of the Disclosure is delivered by the viral vector. In some embodiments, the viral vector comprises recombinant Cas9 and a guide polynucleotide. In some embodiments, the viral vector comprises a Cas effector protein and a guide polynucleotide, wherein the Cas effector protein and the guide polynucleotide are in a complex. In some embodiments, the viral vector comprises a CRISPR-Cas system comprising a Cas effector protein, a polynucleotide that can form a complex with the Cas effector protein and includes a guide polynucleotide, and a polynucleotide that includes tracrRNA. In some embodiments, the viral vector comprises a CRISPR-Cas system comprising a Cas effector protein, a polynucleotide that can form a complex with the Cas effector protein and includes a guide polynucleotide, and tracrRNA. In some embodiments, the viral vector is a viral vector of a retrovirus, lentivirus, adenovirus, or adeno-associated virus. Examples of viral vectors are provided herein.

[0258] In some embodiments, retroviruses, lentiviruses, adenoviruses, and / or adeno-associated virus (AAV) vectors can be used as viral vectors comprising elements of the CRISPR-Cas system described herein. In some embodiments of this disclosure, the Cas effector protein is expressed intracellularly by cells transduced by the viral vector.

[0259] In some embodiments, the Cas protein and methods of this disclosure are used for ex vivo gene editing, such as CAR-T therapy. These embodiments may involve modification of cells derived from a human donor. In these cases, viral vectors may also be used. However, there is an additional option of directly transfecting cultured cells with the Cas9 protein (along with in vitro transcribed guide RNA and donor DNA).

[0260] Inhibitors of the microhomology-mediated end-binding (MMEJ) pathway As used herein, an MMEJ pathway inhibitor is any compound, molecule, or entity that inhibits, antagonistizes, blocks, or reduces the activity and / or level of any component of the MMEJ pathway. In some embodiments, the MMEJ pathway inhibitor is a PolQ inhibitor. In some embodiments, the MMEJ pathway inhibitor is a POL Q / DNA polymerase q inhibitor.

[0261] In some embodiments, the inhibitor of POL Q is a compound of formula (I),

[0262] [ka] or any stereoisomer thereof or a pharmaceutically acceptable salt thereof, During the ceremony, R 1 and R 2 However, each is independently H, halo, C1-C3 alkyl, C1-C3 alkoxy, C1-C3 haloalkyl, C1-C3 hydroxyalkyl, -CN, C2-C4 alkyne, or C2-C6 alkoxyalkyl. Q 1 Q 2 , and Q 3 However, independently, N, CLR, or CR x Q 1 Q 2 , and Q 3 One or fewer of these are CLRs, L is a bond, -O-, -C(O)-, -O(CH2) p C(O)-, -C(O)NR y -, -O(CH2) p C(O)NR y -, -O(CH2) p NR y , -NR y , -(CH2) p~ , -(CH2) p NR y , -(CH2) p O-, -(CH2) p [[ID=2?]]C(O)-, -(CH2) p C(O)O-, -O(CH2) p and p is independently 1, 2, or 3 R is H, R a , R b , R c , or R d and R a is an optionally substituted 3- to 10-membered heterocycle with 1 to 4 substituents selected from amino, carboxy, halo, hydroxy, oxo, -CN, -S(O)2OH, C1-C4 alkylamino, C1-C5 alkoxy, C2-C5 alkoxyalkyl, 4- to 6-membered heterocycle, and C1-C7 alkyl, where the C1-C7 alkyl is optionally substituted with 1 to 4 substituents selected from amino, carboxy, halo, hydroxy, oxo, -CN, C2-C8 ester, and C1-C5 alkoxy R b is C1-C7 alkyl, where one or two methylene groups from the C1-C7 alkyl are optionally replaced by NR e or O, and one or two single bonds in the C2-C7 alkyl chain are optionally replaced by a double bond or a triple bond, and the C1-C7 alkyl is optionally substituted with 1 to 4 substituents selected from halo, oxo, hydroxy, carboxyl, amino, -CN, C2-C4 alkynyl, C2-C6 carbamate, C1-C8 amide, C1-C4 sulfonyl, C1-C4 sulfonamide, C1-C4 alkylamino, C1-C5 alkoxy, C3-C6 carbocycle, and 3- to 10-membered heterocycle Note: There seems to be a typo in line where it says "C(O)-, -(CH2) " which might be an error in the original text. I've translated it as is for the purpose of following the instructions. Also, in line it says "R is H, R " which is an unusual notation but again, translated as presented. The C3-C6 carbon ring is optionally substituted with 1-4 substituents selected from hydroxyl, halo, and carboxyl groups. A 3-10 membered heterocycle is optionally substituted with 1-4 substituents selected from amino, carboxy, halo, hydroxy, oxo, -CN, -S(O)2OH, C1-C4 alkylamino, C1-C5 alkoxy, C2-C5 alkoxyalkyl, 4-6 membered heterocycle, and C1-C7 alkyl, and the C1-C7 alkyl is optionally substituted with 1-4 substituents selected from amino, carboxy, halo, hydroxy, oxo, -CN, C2-C8 ester, and C1-C5 alkoxy. R c However, it is a C3-C6 carbon ring optionally substituted with 1-4 substituents selected from hydroxyl, halo, and carboxyl. R d However, it is a C1-C4 sulfonyl or C1-C4 sulfonamide. R y However, it is H, C1-C3 alkyl, or C1-3 haloalkyl, R x However, it is H, halo, hydroxy, -CN, -NH2, C1-C3 alkoxy, C1-C3 alkyl, or C1-3 haloalkyl, R e However, it is H, halo, C1-C8 alkyl, or C1-C8 haloalkyl, X is a C1-C4 alkylene, Y is a phenyl or 5-6 membered heteroaryl, and the phenyl or heteroaryl is optionally substituted with 1-3 substituents selected from halo, C1-C3 alkyl, C1-C3 alkoxy, -CN, C1-C3 haloalkyl, and cyclopropyl. G is either N or CH, G a and G b However, N, CH, or CR 5 G a and G b Only one of them is N or CH, and G a and G b Only one of them is CR5 And, R 5 but,

[0263] [ka] And, Z a and Z b However, independently, they are either C1-C3 alkyl or C1-C3 haloalkyl, or Z a and Z b However, it forms a 3-6 membered carbon ring or heteroring. Z c However, is it H, -CN, C1-C3 alkyl, C1-C3 haloalkyl, or C2-C4 alkyne? or a combination thereof. In some embodiments, the inhibitor of POL Q is one of the compounds disclosed herein or a combination thereof.

[0264] In some embodiments, the inhibitor of POL Q is one of the compounds listed in Table I, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0265] In some embodiments, the inhibitor of POL Q is one of the compounds listed in Tables I, II, and III, their pharmaceutically acceptable salts, or a combination thereof.

[0266] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where G is N.

[0267] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where G is CH.

[0268] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where G a However, it's a CR5.

[0269] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where G b However, it's a CR5.

[0270] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where G b However, it is N.

[0271] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where G b However, it is CH.

[0272] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where G b However, it is N.

[0273] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where G b However, it is CH.

[0274] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Z a and Z b However, they are independently C1-C3 alkyl groups.

[0275] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Z a and Z b However, it is -CH3.

[0276] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where G a Or G b However, it is CR5, R 5 but,

[0277] [ka] In the equation, p is between 1 and 4.

[0278] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where G a Or G b However, it is CR5, R 5 but,

[0279] [ka] And in the equation, p is 2.

[0280] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where G a Or G b However, it is CR5, R 5 but,

[0281] [ka] And in the equation, p is 1.

[0282] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Z c However, it is -CH3.

[0283] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Z c However, it is -CN.

[0284] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Y is phenyl or a 5-6 membered heteroaryl, and the phenyl or heteroaryl is optionally substituted with 1 to 3 substituents independently selected from halo, C1-C3 alkyl, C1-C3 alkoxy, -CN, and C1-C3 haloalkyl.

[0285] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Y is phenyl.

[0286] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Y is an N-heteroaryl compound.

[0287] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Y is pyridine.

[0288] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Y is substituted.

[0289] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein Y is substituted with -Cl.

[0290] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein Y is substituted with -CH3.

[0291] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Y is unsubstituted.

[0292] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where R 1 But it's a halo.

[0293] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where R 1 However, it is -Cl.

[0294] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where R 1 However, it is -F.

[0295] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where R 1 However, it is -CH3.

[0296] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where R 2 However, it is -H.

[0297] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3However, it is CLR.

[0298] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 However, it is CLR.

[0299] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 2 However, it is CLR.

[0300] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 3 However, it is CLR.

[0301] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR is CLR, and L is a bond, -O-, -(CH2) p O- or -O(CH2) p - is 。

[0302] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 However, CLR is CLR, and L is a bond, -O-, -(CH2) p O- or -O(CH2) p - is

[0303] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 2 However, CLR is CLR, and L is a bond, -O-, -(CH2) p O- or -O(CH2)p - is

[0304] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 3 However, CLR is CLR, and L is a bond, -O-, -(CH2) p O- or -O(CH2) p - is

[0305] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR is the binding component, and L is the binding component.

[0306] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 However, CLR is the bond. 。

[0307] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 2 However, CLR is the bond. 。

[0308] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 3 However, CLR is the binding component, and L is the binding component.

[0309] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR is CLR, and L is -O-.

[0310] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 However, CLR is -O- 。

[0311] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 2 However, CLR is -O- 。

[0312] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 3 However, CLR is -O- 。

[0313] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR is, and L is -(CH2) p It is O.

[0314] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 However, CLR is, and L is -(CH2) p It is O.

[0315] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 2 However, CLR is, and L is -(CH2) p It is O.

[0316] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 3 However, CLR is, and L is -(CH2) p It is O.

[0317] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR is CLR, and L is -O(CH2) p - is

[0318] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 However, CLR is CLR, and L is -O(CH2) p - is

[0319] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 2 However, CLR is CLR, and L is -O(CH2) p - is

[0320] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 3 However, CLR is CLR, and L is -O(CH2) p - is

[0321] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR is CLR, and L is -O(CH2)2-.

[0322] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 However, CLR is CLR, and L is -O(CH2)2-.

[0323] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 2 However, CLR is CLR, and L is -O(CH2)2-.

[0324] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 3 However, CLR is CLR, and L is -O(CH2)2-.

[0325] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR is CLR, and L is -C(O)-, -O(CH2) p C(O)-, -C(O)NR y -, -O(CH2) p C(O)NR y -,-(CH2) p C(O)-, or -(CH2) p It is C(O)O-.

[0326] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 However, CLR is CLR, and L is -C(O)-, -O(CH2) p C(O)-, -C(O)NR y -, -O(CH2) p C(O)NR y -,-(CH2) p C(O)-, or -(CH2)p It is C(O)O-.

[0327] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 2 However, CLR is CLR, and L is -C(O)-, -O(CH2) p C(O)-, -C(O)NR y -, -O(CH2) p C(O)NR y -,-(CH2) p C(O)-, or -(CH2) p It is C(O)O-.

[0328] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 3 However, CLR is CLR, and L is -C(O)-, -O(CH2) p C(O)-, -C(O)NR y -, -O(CH2) p C(O)NR y -,-(CH2) p C(O)-, or -(CH2) p It is C(O)O-.

[0329] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR is H, R a or R b That is the case.

[0330] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 However, CLR is H, R a or R b That is the case.

[0331] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 2 However, CLR is H, R a or R b That is the case.

[0332] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 3 However, CLR is H, R a or R b That is the case.

[0333] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR is, R is R a That is the case.

[0334] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And R a However, it is a 3- to 10-membered N-complex ring.

[0335] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And R a However, it is a 4- to 7-membered N-complex ring.

[0336] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And R a However, it is a 6-membered N-complex ring.

[0337] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And R a However, these are piperidine, 1,2-diadinane, 1,3-diadinane, 1,4-diadinane, 1,2-oxazinanane, 1,3-oxazinanane, or 1,4-oxazinanane.

[0338] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And R a However, the molecule is substituted with 1 to 4 substituents selected from amino, carboxy, halo, hydroxy, oxo, -CN, -S(O)2OH, C1-C4 alkylamino, C1-C5 alkoxy, C2-C5 alkoxyalkyl, 4-6 membered heterocycles, and C1-C7 alkyl, and the C1-C7 alkyl molecule is optionally substituted with 1 to 4 substituents selected from amino, carboxy, halo, hydroxy, oxo, -CN, C2-C8 esters, and C1-C5 alkoxy.

[0339] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR aAnd R a However, it is substituted with C1-C7 alkyl groups, and the C1-C7 alkyl groups are optionally substituted with 1 to 4 substituents selected from amino, carboxy, halo, hydroxy, oxo, -CN, C2-C8 esters, and C1-C5 alkoxy groups.

[0340] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And R a However, it is substituted with oxo-substituted C1-C7 alkyl groups.

[0341] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And R a However, it is substituted with C1-C7 alkyl groups that are substituted with C1-C5 alkoxy groups.

[0342] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And R a However, it is substituted with methyl.

[0343] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And L is -O(CH2) p - and R aHowever, it is a 3- to 10-membered N-heterogenetic ring with arbitrary substitutions.

[0344] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And L is -O(CH2) p - and R a However, it is a 3- to 10-membered N-heterogenetic ring with arbitrary substitutions.

[0345] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And L is -O(CH2)2-, and R a However, it is a 3- to 10-membered N-heterogenetic ring with arbitrary substitutions.

[0346] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And L is -O(CH2)2-, and R a However, it is a 4- to 7-membered N-complex ring with arbitrary substitutions.

[0347] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And L is -O(CH2)2-, and R a However, it is an unsubstituted 4- to 7-membered N-heterogenetic ring.

[0348] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and Q 1 Q 2 , or Q 3 However, CLR a And L is -O(CH2)2-, and R a However, it is a 4- to 7-membered N-heterocycle substituted with hydroxyl, methyl, or amino.

[0349] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And L is -O(CH2)3-, and R a However, it is a 3- to 10-membered N-heterogenetic ring with arbitrary substitutions.

[0350] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And L is -O(CH2)3-, and R a However, it is a 4- to 7-membered N-complex ring.

[0351] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And L is -O(CH2)3-, and R a However, it is an unsubstituted 4- to 7-membered N-heterogenetic ring.

[0352] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and Q 1 Q2 , or Q 3 However, CLR a And L is -O(CH2)3-, and R a However, it is a 4- to 7-membered N-heterocycle substituted with hydroxyl, methyl, or amino.

[0353] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And L is a bond, and R a However, it is a 3- to 10-membered N-heterogenetic ring with arbitrary substitutions.

[0354] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And L is a bond, and R a However, it is a 4- to 7-membered N-complex ring.

[0355] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And L is a bond, and R a However, it is an unsubstituted 4- to 7-membered N-heterogenetic ring.

[0356] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and Q 1 Q 2 , or Q 3 However, CLR a And L is a bond, and R aHowever, it is a 4- to 7-membered N-heterocycle substituted with hydroxyl, methyl, or amino.

[0357] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And L is -O-, and R a However, it is a 3- to 10-membered N-heterogenetic ring with arbitrary substitutions.

[0358] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And L is -O-, and R a However, it is a 4- to 7-membered N-complex ring.

[0359] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR a And L is -O-, and R a However, it is an unsubstituted 4- to 7-membered N-heterogenetic ring.

[0360] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, and Q 1 Q 2 , or Q 3 However, CLR a And L is -O-, and R a However, it is a 4- to 7-membered N-heterocycle substituted with hydroxyl, methyl, or amino.

[0361] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR is, R is R b That is the case.

[0362] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And R b However, it is substituted with 1 to 4 substituents selected from halo, oxo, hydroxy, carboxyl, amino, -CN, C2-C4 alkynyl, C2-C6 carbamate, C1-C8 amide, C1-C4 sulfonyl, C1-C4 sulfonamide, C1-C4 alkylamino, C1-C5 alkoxy, C3-C6 carbon ring, and 3-10 membered heterocycles, and the C3-C6 carbon ring is optionally substituted with 1 to 4 substituents selected from hydroxy, halo, and carboxyl, and the 3-10 membered heterocycle The elementary ring is optionally substituted with 1 to 4 substituents selected from amino, carboxy, halo, hydroxy, oxo, -CN, -S(O)2OH, C1-C4 alkylamino, C1-C5 alkoxy, C2-C5 alkoxyalkyl, 4-6 membered heterocycles, and C1-C7 alkyl groups, and the C1-C7 alkyl group is optionally substituted with 1 to 4 substituents selected from amino, carboxy, halo, hydroxy, oxo, -CN, C2-C8 esters, and C1-C5 alkoxy groups.

[0363] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And R bHowever, it is substituted with at least one C3-C6 carbon ring, and the C3-C6 carbon ring is substituted with 1-4 substituents selected from hydroxyl, halo, and carboxyl.

[0364] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And R b However, it is substituted with at least one C3-C6 carbon ring, and the C3-C6 carbon ring is substituted with 1-4 substituents selected from hydroxyl and halo.

[0365] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And R b However, it is substituted with hydroxyl.

[0366] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And R b However, it is a 3- to 10-membered complex ring.

[0367] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And R b However, it is substituted with an N-heterogene.

[0368] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And R b However, it is substituted with 4- to 7-membered N-heterogenetic rings.

[0369] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And R b However, the heterocycle is substituted with a 3-10 membered heterocycle, for example, an N-heterocycle (not limited to), for example, a 4-7 membered N-heterocycle (not limited to), and the heterocycle is substituted with 1-4 substituents selected from amino, carboxy, halo, hydroxy, oxo, -CN, -S(O)2OH, C1-C4 alkylamino, C1-C5 alkoxy, C2-C5 alkoxyalkyl, 4-6 membered heterocycle, and C1-C7 alkyl, and the C1-C7 alkyl is optionally substituted with 1-4 substituents selected from amino, carboxy, halo, hydroxy, oxo, -CN, C2-C8 ester, and C1-C5 alkoxy.

[0370] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And R b However, the heterocycles are substituted with 3- to 10-membered heterocycles, for example, N-heterocycles (not limited to these), or 4- to 7-membered N-heterocycles (not limited to these), and the heterocycles are substituted with C1- to C7 alkyl groups, oxo groups, and / or halos.

[0371] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And R b However, it is substituted with amino, C1-C8 amide, and / or C1-C4 alkylamino compounds.

[0372] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And R b However, it is substituted with oxo, hydroxy, and / or carboxy.

[0373] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is -O-, and R b However, these are C1-C5 alkyl groups that have been optionally substituted.

[0374] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is -O-, and R b However, these are C1-C3 alkyl groups that have been optionally substituted.

[0375] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2, or Q 3 However, CLR b And L is -O-, and R b However, these are C1-C5 alkyl groups substituted with 1-4 substituents selected from amino, carboxy, oxy, and hydroxy.

[0376] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is -O-, and R b However, these are C1-C3 alkyl groups substituted with 1-4 substituents selected from amino, carboxy, oxy, and hydroxy.

[0377] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is -O- and R b However, these are C1-C5 alkyl groups substituted with 1-4 substituents selected from -CN, C2-C4 alkynyl, C2-C6 carbamate, and C1-C8 amide.

[0378] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is -O- and R b However, these are C1-C3 alkyl groups substituted with 1-4 substituents selected from -CN, C2-C4 alkynyl, C2-C6 carbamate, and C1-C8 amide.

[0379] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is -O-, and R b However, these are unsubstituted C1-C5 alkyl groups.

[0380] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is -O-, and R b However, these are unsubstituted C1-C3 alkyl groups.

[0381] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is -O-, and R b However, it is a C1-C7 alkyl group that has been optionally substituted, and one or two methylene groups from the C1-C7 alkyl group are NR e Alternatively, it is replaced with O.

[0382] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is -O-, and R b However, these are C1-C7 alkyl groups that have been optionally substituted, and one methylene group from the C1-C7 alkyl group is replaced by an NH group.

[0383] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is -O-, and R b However, it is a C1-C7 alkyl group that has been optionally substituted, and one methylene group from the C1-C7 alkyl group has been replaced by NCH3.

[0384] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is a bond, and R b However, these are C1-C5 alkyl groups that have been optionally substituted.

[0385] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is a bond, and R b However, these are C1-C3 alkyl groups that have been optionally substituted.

[0386] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is a bond, and R b However, these are C1-C5 alkyl groups substituted with 1-4 substituents selected from amino, carboxy, oxy, and hydroxy.

[0387] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is a bond, and R b However, these are C1-C3 alkyl groups substituted with 1-4 substituents selected from amino, carboxy, oxy, and hydroxy.

[0388] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is a bond, and R b However, these are C1-C5 alkyl groups substituted with 1-4 substituents selected from -CN, C2-C4 alkynyl, C2-C6 carbamate, and C1-C8 amide.

[0389] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is a bond, and R b However, these are C1-C3 alkyl groups substituted with 1-4 substituents selected from -CN, C2-C4 alkynyl, C2-C6 carbamate, and C1-C8 amide.

[0390] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is a bond, and R b However, these are unsubstituted C1-C5 alkyl groups.

[0391] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is a bond, and R b However, these are unsubstituted C1-C3 alkyl groups.

[0392] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is a bond, and R b However, it is a C1-C7 alkyl group that has been optionally substituted, and one or two methylene groups from the C1-C7 alkyl group are NR e Alternatively, it is replaced with O.

[0393] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is a bond, and R b However, these are C1-C7 alkyl groups that have been optionally substituted, and one methylene group from the C1-C7 alkyl group is replaced by an NH group.

[0394] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR b And L is a bond, and R b However, it is a C1-C7 alkyl group that has been optionally substituted, and one methylene group from the C1-C7 alkyl group has been replaced by NCH3.

[0395] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR c That is the case.

[0396] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR c And R c However, it is substituted with 1 to 4 substituents selected from hydroxyl, halo, and carboxyl.

[0397] In some embodiments, the inhibitor of POL Q is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, where Q 1 Q 2 , or Q 3 However, CLR c And R c However, it is substituted with 1 to 4 substituents selected from hydroxyl and halo.

[0398] In some embodiments, the inhibitor of POL Q is one of the compounds listed in Table I, a pharmaceutically acceptable salt thereof, or a combination thereof.

[0399] In some embodiments, the inhibitor of POL Q is one of the compounds listed in Tables I, II, and III, their pharmaceutically acceptable salts, or a combination thereof.

[0400] [Table 2-1]

[0401] [Table 2-2]

[0402] Table 2-3

[0403] Table 2-4

[0404] Table 2-5

[0405] Table 2-6

[0406] Table 2-7

[0407] Table 2-8

[0408] Table 3

[0409] Table 4-1

[0410] Table 4-2

[0411] Table 4-3

[0412] Table 4-4

[0413] Table 4-5

[0414] Table 4-6

[0415] Table 4-7

[0416] Table 4-8

[0417] Table 4-9

[0418] Table 4-10

[0419] Table 4-11

[0420] Table 4-12

[0421] Table 4-13

[0422] Table 4-14

[0423] Table 4-15

[0424] Table 4-16

[0425] Table 4-17

[0426] Table 4-18

[0427] Table 4-19

[0428] Table 4-20

[0429] Table 4-21

[0430] Table 4-22

[0431] Table 4-23

[0432] [Table 4-24]

[0433] [Table 4-25]

[0434] [Table 4-26]

[0435] In some embodiments, the inhibitor of POL Q is 9-benzyl-8-(2-chloro-4-(2-(4-methylpiperazine-1-yl)ethoxy)phenyl)-6-(1-methylcyclopropoxy)-9H-purine (compound 1), or a salt thereof.

[0436] [ka]

[0437] In some embodiments, the inhibitor of POL Q is a compound disclosed herein, a pharmaceutically acceptable salt thereof, or a combination thereof. In some embodiments, the inhibitor of POL Q is a compound disclosed in Tables I, II, and III, a pharmaceutically acceptable salt thereof, or a combination thereof. In some embodiments, the inhibitor of POL Q is compound 1 or a pharmaceutically acceptable salt thereof. In some embodiments, the inhibitor of POL Q is compound 1.

[0438] In some embodiments, the MMEJ pathway inhibitor is added to a composition containing eukaryotic cells at a concentration of about 0.01 mM to about 1 mM. In some embodiments, the concentration of the MMEJ pathway inhibitor is about 0.01 mM to about 0.75 mM, about 0.01 mM to about 0.5 mM, about 0.01 mM to about 0.25 mM, about 0.01 mM to about 0.1 mM, about 0.01 mM to about 75 mM, about 0.01 mM to about 50 mM, about 0.01 mM to about 25 mM, about 0.01 to about 25 mM, about 0.01 to about 20 mM, about 0.01 mM to about 15 mM, about 0.01 mM to about 10 mM, or about 0.01 mM to about 1 mM. In some embodiments, the concentration of the MMEJ pathway inhibitor is approximately 0.1 mM to 1 mM, 1 mM to 1 mM, 10 mM to 1 mM, 15 mM to 1 mM, 20 mM to 1 mM, 25 mM to 1 mM, 50 mM to 1 mM, 75 mM to 1 mM, 0.1 mM to 1 mM, 0.25 mM to 1 mM, 0.5 mM to 1 mM, or 0.75 mM to 1 mM. In some embodiments, the concentration of the MMEJ pathway inhibitor is approximately 0.1 mM to 1 mM, 0.1 mM to 0.75 mM, approximately 0.1 mM to 0.5 mM, approximately 0.1 mM to 0.25 mM, approximately 0.1 mM to 0.1 mM, approximately 0.1 mM to 75 mM, approximately 0.1 mM to 50 mM, approximately 0.1 mM to 25 mM, approximately 0.1 mM to 20 mM, approximately 0.1 mM to 15 mM, approximately 0.1 mM to 10 mM, or approximately 0.1 mM to 1 mM. In some embodiments, the concentration of the MMEJ pathway inhibitor is approximately 1 mM to 10 mM, approximately 1 mM to 15 mM, approximately 1 mM to 20 mM, approximately 1 mM to 25 mM, approximately 1 mM to 50 mM, approximately 1 mM to 0.1 mM, approximately 1 mM to 0.25 mM, approximately 1 mM to 0.5 mM, approximately 1 mM to 0.75 mM, or approximately 1 mM to 1 mM. In some embodiments, the concentration of the MMEJ pathway inhibitor is approximately 0.01 mM to 100 mM, approximately 0.1 mM to 90 mM, approximately 0.2 mM to 80 mM, approximately 0.3 mM to 70 mM, approximately 0.4 mM to 60 mM, approximately 0.5 mM to 50 mM, approximately 1 mM to 50 mM, approximately 2 mM to 45 mM, approximately 3 mM to 40 mM, approximately 4 mM to 35 mM, approximately 5 mM to 30 mM, approximately 6 mM to 25 mM, approximately 7 mM to 20 mM, or approximately 8 mM to 15 mM.In some embodiments, the concentration of the MMEJ pathway inhibitor is approximately 0.01 mM to 0.1 mM, 0.01 to 1 mM, 0.05 mM to 0.1 mM, 0.5 mM to 1 mM, 0.5 mM to 5 mM, 0.5 mM to 10 mM, 0.1 mM to 1 mM, 0.1 mM to 5 mM, 0.1 mM to 10 mM, 0.1 mM to 5 mM, 0.1 mM to 10 mM, 1 mM to 5 mM, 1 mM to 10 mM, 1 mM to 15 mM, 1 mM to 20 mM, 1 mM to 25 mM, 1 mM to 50 mM, 5 mM to 10 mM, 5 mM to 15 mM, 5 mM to 20 mM, or 5 mM to 25 mM. In some embodiments, the concentration of the MMEJ pathway inhibitor is approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mM.

[0439] In some embodiments, the concentration of the MMEJ pathway inhibitor is 0.01 mM to about 1 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 0.5 mM, about 0.1 mM to about 100 mM, or about 1 mM to about 50 mM.

[0440] In some embodiments, the MMEJ pathway inhibitor is administered approximately 0 minutes to 96 hours before Cas effector protein is added, approximately 0 minutes to 72 hours before Cas effector protein is added, approximately 0 minutes to 48 hours before Cas effector protein is added, approximately 0 minutes to 36 hours before Cas effector protein is added, approximately 0 minutes to 24 hours before Cas effector protein is added, and approximately 0 minutes to 1 It is added to the composition containing eukaryotic cells at 8 hours prior, approximately 0 to 12 hours prior to the addition of the Cas effector protein, approximately 0 to 6 hours prior to the addition of the Cas effector protein, approximately 0 to 3 hours prior to the addition of the Cas effector protein, approximately 0 to 2 hours prior to the addition of the Cas effector protein, approximately 0 to 1 hour prior to the addition of the Cas effector protein, or approximately 0 to 30 minutes prior to the addition of the Cas effector protein. In some embodiments, an inhibitor of the MMEJ pathway is added to a composition containing eukaryotic cells approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 hours before the Cas effector protein is added.

[0441] In some embodiments, an MMEJ pathway inhibitor is added to a composition containing eukaryotic cells at the same time as the Cas effector protein is added.

[0442] In some embodiments, the MMEJ pathway inhibitor is added to a composition containing eukaryotic cells at 0 to approximately 30 minutes after the addition of the Cas effector protein, at 0 to approximately 1 hour after the addition of the Cas effector protein, at 0 to approximately 3 hours after the addition of the Cas effector protein, at 0 to approximately 6 hours after the addition of the Cas effector protein, at 0 to approximately 12 hours after the addition of the Cas effector protein, at 0 to approximately 18 hours after the addition of the Cas effector protein, at 0 to approximately 24 hours after the addition of the Cas effector protein, at 0 to approximately 36 hours after the addition of the Cas effector protein, at 0 to approximately 48 hours after the addition of the Cas effector protein, at 0 to approximately 72 hours after the addition of the Cas effector protein, or at 0 to approximately 96 hours after the addition of the Cas effector protein. In some embodiments, an inhibitor of the MMEJ pathway is added to a composition containing eukaryotic cells approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 hours after the addition of the Cas effector protein.

[0443] In some embodiments, the MMEJ pathway inhibitor is present in a composition containing eukaryotic cells for approximately 1 to 300 hours, approximately 10 to 200 hours, approximately 10 to 100 hours, approximately 20 to 80 hours, approximately 30 to 70 hours, or approximately 40 to 100 hours. In some embodiments, the MMEJ pathway inhibitor is present in a composition containing eukaryotic cells for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, or 300 hours.

[0444] In some embodiments, the MMEJ pathway inhibitor is added to the composition containing eukaryotic cells at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times.

[0445] Inhibitors of the non-homologous end-joining (NHEJ) pathway As used herein, an NHEJ pathway inhibitor is any compound, molecule, or entity that inhibits, antagonizes, blocks, or reduces the activity and / or level of any component of the NHEJ pathway. An NHEJ inhibitor may be an antibody or its antigen-binding fragment, peptide, soluble protein, siRNA, antisense oligonucleotide, aptamer, or small molecule compound that inhibits, antagonizes, blocks, or reduces the activity and / or level of any component of the NHEJ pathway. In some embodiments, the NHEJ pathway inhibits, antagonizes, blocks, or reduces the activity and / or level of Ku70, Ku80, DNA ligase IV, XLF (non-homologous end binding factor 1, XRCC4-like factor), or DNA-dependent protein kinase (DNA-PK). In some embodiments, the DNA-PK inhibitor is M3814, M9831 / VX984, Nu7441, KU0060648, AZD7648, Nu5455, vanillin, woltmannin, or a combination thereof. In some embodiments, the DNA-PK inhibitor is AZD7648.

[0446] In some embodiments, an NHEJ pathway inhibitor is added to a composition containing eukaryotic cells at a concentration of about 0.01 mM to about 1 mM. In some embodiments, the concentration of the NHEJ pathway inhibitor is about 0.01 mM to about 0.75 mM, about 0.01 mM to about 0.5 mM, about 0.01 mM to about 0.25 mM, about 0.01 mM to about 0.1 mM, about 0.01 mM to about 75 mM, about 0.01 mM to about 50 mM, about 0.01 mM to about 25 mM, about 0.01 to about 25 mM, about 0.01 to about 20 mM, about 0.01 mM to about 15 mM, about 0.01 mM to about 10 mM, or about 0.01 mM to about 1 mM. In some embodiments, the concentration of the NHEJ pathway inhibitor is approximately 0.1 mM to 1 mM, 1 mM to 1 mM, 10 mM to 1 mM, 15 mM to 1 mM, 20 mM to 1 mM, 25 mM to 1 mM, 50 mM to 1 mM, 75 mM to 1 mM, 0.1 mM to 1 mM, 0.25 mM to 1 mM, 0.5 mM to 1 mM, or 0.75 mM to 1 mM. In some embodiments, the concentration of the NHEJ pathway inhibitor is approximately 0.1 mM to 1 mM, 0.1 mM to 0.75 mM, approximately 0.1 mM to 0.5 mM, approximately 0.1 mM to 0.25 mM, approximately 0.1 mM to 0.1 mM, approximately 0.1 mM to 75 mM, approximately 0.1 mM to 50 mM, approximately 0.1 mM to 25 mM, approximately 0.1 mM to 20 mM, approximately 0.1 mM to 15 mM, approximately 0.1 mM to 10 mM, or approximately 0.1 mM to 1 mM. In some embodiments, the concentration of the NHEJ pathway inhibitor is approximately 1 mM to 10 mM, approximately 1 mM to 15 mM, approximately 1 mM to 20 mM, approximately 1 mM to 25 mM, approximately 1 mM to 50 mM, approximately 1 mM to 0.1 mM, approximately 1 mM to 0.25 mM, approximately 1 mM to 0.5 mM, approximately 1 mM to 0.75 mM, or approximately 1 mM to 1 mM. In some embodiments, the concentration of the NHEJ pathway inhibitor is approximately 0.01 mM to 100 mM, approximately 0.1 mM to 90 mM, approximately 0.2 mM to 80 mM, approximately 0.3 mM to 70 mM, approximately 0.4 mM to 60 mM, approximately 0.5 mM to 50 mM, approximately 1 mM to 50 mM, approximately 2 mM to 45 mM, approximately 3 mM to 40 mM, approximately 4 mM to 35 mM, approximately 5 mM to 30 mM, approximately 6 mM to 25 mM, approximately 7 mM to 20 mM, or approximately 8 mM to 15 mM.In some embodiments, the concentration of the NHEJ pathway inhibitor is approximately 0.01 mM to 0.1 mM, approximately 0.01 to 1 mM, approximately 0.05 mM to 0.1 mM, approximately 0.5 mM to 1 mM, approximately 0.5 mM to 5 mM, approximately 0.5 mM to 10 mM, approximately 0.1 mM to 1 mM, approximately 0.1 mM to 5 mM, approximately 0.1 mM to 10 mM, approximately 1 mM to 5 mM, approximately 1 mM to 10 mM, approximately 1 mM to 15 mM, approximately 1 mM to 20 mM, approximately 1 mM to 25 mM, approximately 1 mM to 50 mM, approximately 5 mM to 10 mM, approximately 5 mM to 15 mM, approximately 5 mM to 20 mM, or approximately 5 mM to 25 mM. In some embodiments, the concentration of the NHEJ pathway inhibitor is approximately 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.7, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mM.

[0447] In some embodiments, the concentration of the NHEJ pathway inhibitor is 0.01 mM to about 1 mM, about 0.1 mM to about 1 mM, about 0.1 mM to about 0.5 mM, about 0.1 mM to about 100 mM, or about 1 mM to about 50 mM.

[0448] In some embodiments, the NHEJ pathway inhibitor is administered approximately 0 minutes to 96 hours before Cas effector protein is added, approximately 0 minutes to 72 hours before Cas effector protein is added, approximately 0 minutes to 48 hours before Cas effector protein is added, approximately 0 minutes to 36 hours before Cas effector protein is added, approximately 0 minutes to 24 hours before Cas effector protein is added, and approximately 0 minutes to 1 It is added to the composition containing eukaryotic cells at 8 hours prior, approximately 0 to 12 hours prior to the addition of the Cas effector protein, approximately 0 to 6 hours prior to the addition of the Cas effector protein, approximately 0 to 3 hours prior to the addition of the Cas effector protein, approximately 0 to 2 hours prior to the addition of the Cas effector protein, approximately 0 to 1 hour prior to the addition of the Cas effector protein, or approximately 0 to 30 minutes prior to the addition of the Cas effector protein. In some embodiments, an NHEJ pathway inhibitor is added to a composition containing eukaryotic cells approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 hours before the Cas effector protein is added.

[0449] In some embodiments, an NHEJ pathway inhibitor is added to a composition containing eukaryotic cells at the same time as the Cas effector protein is added.

[0450] In some embodiments, the NHEJ pathway inhibitor is added to a composition containing eukaryotic cells at 0 to approximately 30 minutes after the addition of the Cas effector protein, at 0 to approximately 1 hour after the addition of the Cas effector protein, at 0 to approximately 3 hours after the addition of the Cas effector protein, at 0 to approximately 6 hours after the addition of the Cas effector protein, at 0 to approximately 12 hours after the addition of the Cas effector protein, at 0 to approximately 18 hours after the addition of the Cas effector protein, at 0 to approximately 24 hours after the addition of the Cas effector protein, at 0 to approximately 36 hours after the addition of the Cas effector protein, at 0 to approximately 48 hours after the addition of the Cas effector protein, at 0 to approximately 72 hours after the addition of the Cas effector protein, or at 0 to approximately 96 hours after the addition of the Cas effector protein. In some embodiments, an NHEJ pathway inhibitor is added to a composition containing eukaryotic cells approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 hours after the addition of the Cas effector protein.

[0451] In some embodiments, the NHEJ pathway inhibitor is present in a composition containing eukaryotic cells for a period of about 1 to about 300 hours, about 10 to about 200 hours, about 10 to about 100 hours, about 20 to about 80 hours, about 30 to about 70 hours, or about 40 to about hours. In some embodiments, an inhibitor of the NHEJ pathway is present in a composition containing eukaryotic cells for approximately 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, or 300 hours.

[0452] In some embodiments, an NHEJ pathway inhibitor is added to a composition containing eukaryotic cells at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more times.

[0453] In some embodiments, the NHEJ pathway inhibitor is added to the eukaryotic cell-containing composition after the MMEJ pathway inhibitor has been added to the composition, and before the MMEJ pathway inhibitor is added to the eukaryotic cell-containing composition. In some embodiments, the NHEJ pathway inhibitor and the MMEJ pathway inhibitor are added simultaneously to the eukaryotic cell-containing composition.

[0454] In some embodiments, the MMEJ pathway inhibitor and the NHEJ pathway inhibitor are added to the composition containing eukaryotic cells before the Cas effector protein is added. In some embodiments, the MMEJ pathway inhibitor and the NHEJ pathway inhibitor are added to the composition containing eukaryotic cells after the Cas effector protein is added. In some embodiments, the MMEJ pathway inhibitor and the NHEJ pathway inhibitor are added to the composition containing eukaryotic cells at the same time as the Cas effector protein is added. In some embodiments, the MMEJ pathway inhibitor is added to the composition containing eukaryotic cells before the Cas effector protein is added, and the NHEJ pathway inhibitor is added after the Cas effector protein is added. In some embodiments, the MMEJ pathway inhibitor is added to the composition containing eukaryotic cells after the Cas effector protein is added, and the NHEJ pathway inhibitor is added before the Cas effector protein is added.

[0455] All references cited herein, including patents, patent applications, papers, textbooks, etc., and references cited within them, are incorporated herein by reference in their entirety, unless they have already been cited. [Examples]

[0456] CRISPR implementation examples Examples: Effects of MMEJ and NHEJ inhibitors on the CRISPR-1 double-strand break repair pathway.

[0457] The effects of MMEJ and NHEJ pathway inhibitors on the CRISPR-Cas-induced DNA double-strand break repair pathway were investigated using the process schematically shown in Figure 2A. Briefly, HEK293T cells were seeded in 96-well plates 20 hours before transfection with plasmids encoding SpCas9 and CD34-targeting guide RNA (sgRNA), both in the presence and absence of single-stranded oligonucleotide donors (ssDNA). Two hours before transfection, the cells were treated with inhibitors under the following conditions: a) 1 μM DNAPK inhibitor AZD7648, b) compound 1, titrated with 1 μM DNAPK inhibitor AZD7648 in a 1:2 dilution series ranging from 10 μM to 0.01 μM, and the corresponding DMSO control. 70 hours after transfection, cell confluence and eGFP-based transfection efficiency were determined using Incucyte SX5. Genomic DNA was extracted, and the editing results were analyzed by deep-targeted amplicon sequencing and RIMA2 analysis (Figure 2A) to determine the knock-in frequency and mutagenic DNA repair events such as NHEJ and MMEJ results (Figure 2B). The results of these experiments are shown in Figure 3.

[0458] To demonstrate the effect of DNA repair inhibitors on CRISPR / Cas editing efficiency, HEK293T cells were treated with the DNA-PK inhibitor AZD7648 (1 mM) alone and in combination with the Pol Q inhibitor, compound 1, at specified concentrations, followed by CRISPR / Cas9-mediated gene targeting. The results are shown in Figure 4. These studies demonstrate that inhibition of the NHEJ and / or MMEJ pathways, combined with CRISPR / Cas gene targeting, leads to more accurate HDR pathway-mediated DNA double-strand break repair, minimizing contributions from the more error-prone MMEJ and NHEJ pathways.

[0459] Examples: Effects of MMEJ and NHEJ inhibitors on CRISPR-2-CRISPR / Cas-mediated knock-in efficiency.

[0460] The effects of NHEJ and MMEJ inhibitors on CRISPR / Cas-mediated knock-in efficiency were determined for both mutant and mapped reads. Briefly, HEK293T cells were cultured, transfected, and then treated with various concentrations of the NHEJ inhibitor (AZD7648) alone and in combination with the MMEJ inhibitor (compound 1) according to the protocol described in Example CRISPR-1. Subsequently, genomic DNA was isolated, and knock-in efficiency was analyzed. The results are shown in Figure 5 (mutant sequencing reads) and Figure 7 (mapped sequencing reads).

[0461] Examples: Effect of MMEJ inhibition on CRISPR-3 mutant sequencing reads and mapped sequencing reads.

[0462] HEK293T cells were cultured, transfected, and treated with the DNA-PK inhibitor AZD7648 (1 mM) alone and in combination with a specified concentration of Pol Q inhibitor compound 1, followed by CRISPR / Cas9-mediated gene knock-in. The effect of MMEJ pathway inhibition was evaluated in mutant sequencing reads and mapped sequencing reads. The treatment of CRISPR / Cas-edited cells with MMEJ inhibitors is shown in Figure 7 (MMEJ mutant reads) and Figure 8 (MMEJ mapped reads).

[0463] Examples: Effects of inhibiting the NHEJ and MMEJ pathways on cellular confluence and transfection efficiency in CRISPR-4-CRISPR / Cas-transfected cells.

[0464] HEK293T cells were cultured, transfected, and treated with NHEJ and MMEJ inhibitors as described in Example CRISPR-1. Cell confluence and transfection efficiency were evaluated in transfected cells treated with NHEJ and MMEJ inhibitors. The results are shown in Figure 9 (cell confluence) and Figure 10 (transfection efficiency).

[0465] Examples: Effects of NHEJ and MMEJ inhibitors on the double-strand break repair pathway in CRISPR-5 induced pluripotent stem cells (iPSCs).

[0466] The effects of inhibiting the NHEJ and / or MMEJ pathways on the CRISPR-Cas-induced DNA double-strand break repair pathway in iPSCs were investigated. Briefly, Cas9 expression in iPSCs was induced with 100 ng / mL doxycycline 24 hours prior to reverse transfection with sgRNA (gMEJ) targeting the CD34 site and single-strand oligonucleotide donors (ssDNA). Immediately after transfection, cells were treated with inhibitors under the following conditions: a) 1 μM DNAPK inhibitor AZD7648, b) compound 1, which was titrated with 1 μM DNAPK inhibitor AZD7648 in a 1:3 dilution series ranging from 10 μM to 0.1 μM, and the corresponding DMSO control. 72 hours after transfection, the percentages of double-strand break repair by the HDR, NHEJ, and MMEJ pathways were determined as discussed in Example CRISPR-1. The results of these experiments are shown in Figure 11 (mapped reads) and Figure 12 (mutant reads).

[0467] Examples: Effects of NHEJ and MMEJ inhibitors on single-strand template repair (SSTR) gene insertion in CRISPR-6-iPSCs.

[0468] The effects of inhibiting the NHEJ and MMEJ pathways on editing efficiency in Cas9-inducible iPSCs were investigated. In short, Cas9-inducible iPSCs were cultured and transfected with sgRNA and ssDNA polynucleotides as described in Example CRISPR-5. The results are shown in Figure 13.

[0469] Synthesis method Suitable processes for preparing the compound of formula (I) or its pharmaceutically acceptable salts are exemplified by variations of the following representative processes, unless otherwise specified, G, G a , G b , Q1, Q2, Q3 and R 1 , R 2 , R a , R b , R c , R d X, Y, Z a / Z aa , Z b / Z bb , Z c / Z cc This has one of the meanings defined above. The required starting materials can be obtained by standard procedures in organic chemistry. The preparation of such starting materials, along with variations of the representative processes below, is described in the attached examples. Alternatively, the required starting materials can be obtained by procedures similar to those exemplified, which are within the scope of the ordinary art of organic chemists.

[0470] [ka]

[0471] The compound of formula (I) can be prepared, for example, by the following: a) 1. Another compound of formula (I) (wherein Q) under conditions known in the art as suitable for the Mitsunobu reaction. 1 Q 2 Or Q 3 However, by reaction of a C-OH group with a primary or secondary alcohol (for example, Synthesis Example 1), Alternatively, by reaction with a primary or secondary halide, with or without protecting groups for other functional groups, under typical conditions for nucleophilic substitution, for example, in the presence of a suitable solvent (e.g., DMA or DMF), a suitable base (e.g., potassium carbonate or cesium carbonate), and at a suitable temperature (0-120°C).

[0472] 2. Another compound of formula (I) (wherein Q) that is optionally catalyzed by a metal complex such as a palladium catalyst suitable for the Buchwald-Hartwig amination reaction under conditions known in the art. 1 Q 2 Or Q 3 By reaction of a C-LG (where LG is a leaving group such as a halogen) with a suitable amine (e.g., Synthesis Example 19).

[0473] 3. Under conditions known in the art (e.g., a reaction in the presence of a strong base such as sodium hydride for alkoxide formation), optionally, another compound of formula (I) (wherein Q) is catalyzed by a metal complex such as a palladium catalyst suitable for ether formation reactions (e.g., RockPhos Pd G3). 1 Q 2 Or Q 3 (where C-LG is a leaving group such as a halogen) reacts with a suitable alcohol (for example, Synthesis Example 20).

[0474] More generally, compounds of formula (I) can be prepared from compounds of formula (I) (e.g., amide coupling in synthesis example N1, reductive amination in synthesis example N6).

[0475] Q 1 Q 2 Or Q 3 However, is it C-OH, or Q 1 Q 2 Or Q 3 However, the compound of formula (I), which is C-LG, can be prepared by the following example. b) G a However, CR 5 And R 5 but,

[0476] [ka] In this case, the reaction is carried out by the reaction of another compound of formula (II) with the compound of formula (III) (wherein LG is a leaving group known in the art, for example, a halide such as F, Cl or Br, or trifluoromethanesulfonic acid (triflate)). The reaction conditions can be carried out with or without protecting other functional groups, using a suitable base (for example, sodium hydride or LHMDS), a suitable solvent (e.g., THF), and a suitable temperature (e.g., 0°C to ambient temperature).

[0477] [ka]

[0478] The compound of formula (II) can be prepared by the reaction of the compound of formula (IV) and the compound of formula (V). The reaction conditions involved a one-step procedure as described in section (d). The reaction can be transformed into a two-step procedure involving the isolation of the intermediate compound of formula (VI). The reaction conditions are described in section (d).

[0479] Alternatively, the compound of formula (II) can be prepared by the reaction of the compound of formula (IVa) with the compound of formula (V). The reaction conditions are described in section (d).

[0480] [ka] Alternatively, the compound of formula (IV) can be prepared by the reaction of formula (IVa) by the reduction of a nitro group to an amino group, as described in section (d).

[0481] [ka]

[0482] When X=CH2, the compound of formula (IVa) can be prepared by a reaction between the compound of formula (VII) and the compound of formula (VIIIa) under conditions known in the art to be suitable for reductive amination.

[0483] Alternatively, the compound of formula (IVa) can be prepared by a reaction between the compound of formula (VIIa) and the compound of formula (VIII). The reaction conditions may include an inert solvent (e.g., DMF) in the presence of a base (e.g., triethylamine) and a suitable temperature (e.g., room temperature).

[0484] [ka]

[0485] Alternatively, compound (II) can be obtained from a reaction between the compound of formula (IIa) and the compound of formula (VIIIb) under conditions known in the art to be suitable for nucleophilic substitution, or from a reaction between the compound of formula (IIa) and the compound of formula (VIIIc) under conditions known in the art to be suitable for the Mitsunobu reaction.

[0486] [ka]

[0487] c)G b However, CR 5 And R 5 but

[0488] [ka] In that case, the reaction of another compound of formula (IX) with a compound of formula (X) under conditions known in the art as suitable for the Mitsunobu reaction,

[0489] Alternatively, the compound may be modified by a nucleophilic substitution reaction between another compound of formula (IX) and the compound of formula (Xa) (wherein LG is a leaving group known in the art, such as a halide of Cl, Br, or I). The conditions for the nucleophilic substitution reaction may be such that a suitable base (e.g., potassium carbonate), a suitable solvent (e.g., acetonitrile, DMF, or DMA), and a suitable temperature (0-120°C), with or without protecting other functional groups.

[0490] [ka]

[0491] The compound of formula (IX) can be prepared by the reaction of the compound of formula (XI) and the compound of formula (V), with or without the use of a protecting group for the hydroxyl group.

[0492] The compound of formula (IX) can also be prepared by the reaction of the compound of formula (XIa) with the compound of formula (V), with or without a protecting group for the hydroxyl group.

[0493] Alternatively, the compound of formula (XI) can be prepared by reducing the nitro group to an amino group from the compound of formula (XIa).

[0494] The conditions for the above reaction are illustrated in section (d).

[0495] [ka]

[0496] The compound of formula (XIa) can be prepared by the reaction of the compound of formula (XII) and the compound of formula (VIII) with or without a protecting group for the hydroxyl group (wherein LG is a leaving group known in the art, such as a halide (F or Cl, etc.) or trifluoromethanesulfonic acid (triflate)).

[0497] [ka]

[0498] d) By reaction of the compound of formula (XIII) with the compound of formula (V). The reaction conditions consist of a one-step procedure and may be carried out using a suitable solvent (e.g., EtOH, isopropanol, dioxane, or DMSO) and a suitable temperature (60-120°C), optionally in the presence of a mild oxidizing agent (e.g., iron(III) chloride and / or atmospheric oxygen) and / or an acid (e.g., p-toluenesulfonic acid, acetic acid) and / or a catalyst (e.g., copper(II) acetate in Synthesis Example 8). The reaction can be converted into a two-step procedure with isolation of the intermediate compound of formula (XIV), for which a mild oxidizing agent (e.g., iron(III) chloride and / or oxygen) is added for the second step.

[0499] Alternatively, by the reaction of the compound of formula (XIIIa) with the compound of formula (V). For this reaction, a suitable solvent (e.g., NMP and water) may be used in the presence of a mild reducing agent such as sodium dithionate (also known as sodium hydrosulfite) at a suitable temperature (e.g., 80-120°C).

[0500] Alternatively, the compound of formula (XIII) can be prepared from the compound of formula (XIIIa) by reducing the nitro group to an amino group (for example, in the presence of iron and using a suitable solvent such as ethanol).

[0501] [ka]

[0502] The compound of formula (XIIIa) can be produced by the reaction of the compound of formula (XV) with another compound of formula (VIII) (wherein LG is a leaving group known in the art, for example, a halide (such as F or Cl) or trifluoromethanesulfonic acid (triflate) or methanesulfonyl).

[0503] [ka]

[0504] G a However, CR 5 And R 5 but,

[0505] [ka] In this case, the compound of formula (XIIIa) can be prepared by the reaction of the compound of formula (IVa) with the compound of formula (III). The reaction conditions can be a suitable solvent (e.g., THF) and a suitable temperature (e.g., near ambient temperature) in the presence of a suitable base (e.g., sodium hydride or LHMDS), with or without protecting other functional groups.

[0506] [ka]

[0507] G a However, CR 5 And R 5 but,

[0508] [ka] In that case, the compound of formula (XIIIa) is also suitable for the Mitsunobu reaction by reaction of another compound of formula (XVI) with the compound of formula (III) under conditions known in the art, Alternatively, the compounds can be prepared by a nucleophilic substitution reaction (wherein LG is a leaving group known in the art, such as a halide of Cl, Br, or I) with another compound of formula (XVI). The conditions for the nucleophilic substitution reaction can be a suitable base (e.g., potassium carbonate), a suitable solvent (e.g., acetonitrile, DMF, or DMA), and a suitable temperature (0-120°C), with or without protecting other functional groups.

[0509] [ka]

[0510] Compounds of formula (XVI) can be prepared by the reaction of another compound of formula (XVII) with another compound of formula (VIII), with or without the use of hydroxyl and other functional group protecting groups.

[0511] [ka]

[0512] (e) By reaction of a compound of formula (XVIII) (where LG is a leaving group known in the art, such as a halide such as Cl, Br, or I) with a compound of formula (XIX) (where FG is a functional group suitable for cross-coupling reactions (e.g., Suzuki reaction), such as a boronic acid ester or boronic acid). Reaction conditions are illustrated in Synthesis Example 5.

[0513] [ka]

[0514] The compound of formula (XVIII) can be prepared from the compound of formula (XX) by bromination (when LG is Br), for example, as described in Synthesis Example 5.

[0515] [ka]

[0516] (f) From the reaction between the compound of formula (XXI) and the compound of formula (VIIIb) under conditions known in the art to be suitable for nucleophilic substitution (e.g., Synthesis Example 23).

[0517] Alternatively, from a reaction between a compound of formula (XXI) and a compound of formula (VIIIc) under conditions known in the art to be suitable for the Mitsunobu reaction.

[0518] [ka]

[0519] It is understood that the compound of formula (XXI), like the compound of formula (I), can be prepared by the reactions already exemplified in sections (a) to (e).

[0520] General experimental conditions and abbreviations The compounds described herein are further illustrated in the following synthesis examples. The compounds were named using Chemdraw version 20.0.2.51. These examples are given for illustrative purposes only and are not limiting. In general: Reagents and solvents (all anhydrous HPLC grade) were obtained from commercial suppliers and used without further purification unless otherwise specified. All reagents were weighed and handled in air unless otherwise specified. Brine refers to a saturated solution of NaCl. Concentration under reduced pressure refers to the use of a rotary evaporator.

[0521] Unless otherwise specified, the operations were performed at ambient temperature, i.e., within the range of 17-25°C, and under an atmosphere of an inert gas such as nitrogen.

[0522] Evaporation was carried out by rotary evaporation under reduced pressure using a hot water bath or a similar bath, or by using a Genevac or Biotage v10 evaporator in a vacuum. After removing residual solids by filtration, the work-up procedure was performed.

[0523] Flash chromatography purification was performed using automated Teledyne Isco CombiFlash® Rf, Teledyne Isco CombiFlash® Companion®, or CHEETAH® MP200 systems incorporating UV detection with pre-packed silica gel columns (40–60 μm) or C18 spherical columns (20–35 μm), using the chromatographic conditions detailed in the corresponding experimental data.

[0524] Preparative reverse-phase HPLC is performed using an Agilent 1290 Infinity II preparative system equipped with an SQ MS detector (multimode ESI / APCI source) and a Waters CSH C18 OBD column (5 micron silica, 30 mm diameter, 100 mm length); a Waters MassLynx system with integrated MS detection and an XBridge or Xselect CSH Prep C18 OBD column (5 μm silica, 30 mm diameter, 150 mm length); or an XBridge (10 μm, 19 mm diameter, 150 mm length) or Sunfire system with integrated UV detection. The analysis was performed using a Gilson GX-281, equipped with one of the following C18 columns (10 μm, 19 mm diameter, 250 mm length): water (containing 0.1-0.3% aqueous ammonium), water (containing 0.05% aqueous ammonia and 10 mmol of NH4HCO3), water (containing 0.1% formic acid), or water (containing 0.05% TFA) and a mixture that reduces the polarity of acetonitrile or methanol.

[0525] Preparative SFC purification was performed using either a Sepiatec P100 SFC system with a QDa MS detector or a Waters Prep 100 SFC system, with the chromatographic conditions detailed in the corresponding experimental data.

[0526] Preparative chiral HPLC was performed using a Gilson GX-281 system equipped with integrated UV detection and one of the following columns: Chiralpak AS, AD, Chiralcel OD, OJ; Chiralpak IA, IB, IC, ID, IE, IF, IG, IH columns (Daicel Chemical Industries, Ltd.); (R,R)-Whelk-O1, (S,S)-Whelk-O1 columns (Regis technologies, Inc.); CHIRAL Cellulose-SB, SC, SA columns (YMC Co., Ltd.). The system was equipped with different column sizes (250×20mm, 250×30mm) and used either a specified percentage of ethanol in hexane (%Et / Hex) or isopropanol in hexane (%IPA / Hex) as the constant solvent system.

[0527] The yield, if any, is not necessarily the maximum achievable value.

[0528] Generally, the structure of the final product of formula I is confirmed by nuclear magnetic resonance (NMR) spectroscopy; 1 ¹H-NMR chemical shift values ​​were measured on a delta scale and cited in ppm units for measurements against TMS or residual solvent peaks as internal standards; proton magnetic resonance spectra were measured using a Bruker Avance 500 spectrometer at a proton frequency of 500 MHz, a Bruker Avance 400, Bruker Avance III HD, or Bruker Avance Neo spectrometer at a proton frequency of 400 MHz, or a Bruker Avance III, Avance III HD, or Avance III NEO spectrometer at a proton frequency of 300 MHz; measurements were performed at ambient temperature unless otherwise specified. The following abbreviations were used: s, single; d, double; t, triple; q, quadruple; m, multiple; dd, double double; ddd, double double double; dt, triple double; br s: broad signal; hept: hept.

[0529] Generally, the final product of formula I is also characterized by mass spectrometry after liquid chromatography (LCMS or ULC); reversed-phase C18 silica was used at a flow rate of 1 mL / min, and detection was performed by electrospray mass spectrometry and UV / vis absorbance recording in the wavelength range of 220–320 nm. Analytical ULC was performed using a Waters Acquity UPLC CSH C18 column (dimensions 2.1 × 50 mm and particle size 1.7 microns). Gradient analysis was used with a mixture of decreasing polarity as the eluent, e.g., water (containing 0.1% v / v formic acid or 0.3% v / v ammonia) as solvent A and a mixture of decreasing polarity of acetonitrile as solvent B. A typical 1.7-minute analytical ULC method uses a solvent gradient over 1.3 minutes at approximately 1 mL / min, from a 97:3 mixture of solvents A and B to a 3:97 mixture of solvents A and B, respectively. Furthermore, LCMS was performed using electrospray ionization in cation detection mode, with a Shimadzu LCMS-2020 equipped with a 20ADXR pump, SIL-20ACXR autosampler, CTO-20AC column oven, M20A PDA detector, and LCMS 2020 MS detector. LC was performed in two settings: 1) a Halo C18 column (2.0 μm, 3.0 × 30 mm) combined with a gradient of water and formic acid-FA (0.1%) (A) and CH3CN and FA (0.1%) (B) (5-100% B in 1.2 min) at a flow rate of 1.5 mL / min; 2) a Poroshell HPH C18 column (2.7 μm, 3.0 × 50 mm) combined with a gradient of 46 mM ammonium carbonate aqueous solution / ammonium buffer (pH 10) (A) and MeCN (B) (5-95% B in 2 min) at a flow rate of 1.2 mL / min; 3) a Halo C18 column (2.0 μm A 3.0 × 30 mm column was combined with a gradient of water and TFA (0.05%) (A) and CH3CN and TFA (0.05%) (B) (5-95% B in 2 minutes) at a flow rate of 1.5 mL / min. The column oven (CTO-20AC) temperature was 40.0°C. The injection volume was 1 μL. PDA (SPD-M20A) detection was in the range of 190-400 nm.MS detector configured with electrospray ionization as the ionization source; acquisition mode: scan; spray gas flow rate: 1.5 L / min; dry gas flow rate: 15 L / min; detector voltage: tuning voltage ±0.2 kV; DL temperature: 250°C; heat block temperature: 250°C; scan range: 90.00~900.00 m / z. Unless otherwise specified, reported molecular ions are understood to correspond to [M+H]+, rounded to the nearest unit. Typically, molecules with multiple isotopic patterns (e.g., ) are considered unless otherwise specified. 35 Cl, 79 Br, 12 For C), only the most common lower isotopes are reported.

[0530] Ion exchange purification was generally performed using SCX-2 (Biotage, propyl sulfonic acid-functionalized silica, manufactured using trifunctional silane, non-end cap) cartridges.

[0531] The purity of the intermediate was evaluated by thin-layer chromatography, mass spectrometry, HPLC (high-performance liquid chromatography), and / or NMR analysis.

[0532] The following abbreviations were used

[0533] [Table 5]

[0534] Synthesis Example 1 9-Benzyl-6-isopropoxy-8-(2-methyl-4-(2-(4-methylpiperazine-1-yl)ethoxy)phenyl)-9H-purine

[0535] [ka]

[0536] DIAD (0.260 mL, 1.34 mmol) was added dropwise to 4-(9-benzyl-6-isopropoxy-9H-purine-8-yl)-3-methylphenol (200 mg, 0.53 mmol), 2-(4-methylpiperazine-1-yl)ethane-1-ol (116 mg, 0.80 mmol), and PPh3 (392 mg, 1.50 mmol) in THF (20 mL) under nitrogen at 0°C. The resulting mixture was stirred at room temperature for 16 hours. The residue was purified by preparative TLC (Â) and then further purified by preparative HPLC (XBridge Shield RP18 OBD column, 5 μm silica, 30 mm diameter, 150 mm length) using water (containing 0.05% NH3 water) and a mixture that reduced the polarity of MeCN as the eluent. The fraction containing the desired compound was evaporated to dryness to obtain 9-benzyl-6-isopropoxy-8-(2-methyl-4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl)-9H-purine (76 mg, 28%) as a yellow oil. 1 H NMR(300MHz,DMSO-d6):1.42(6H,d),1.95(3H,s),2.16(3H,s),2.33(8H,m),2.70(2H,t),4.13(2H,t ),5.26(2H,s),5.62(1H,p),6.84-6.94(4H,m),7.18-7.31(4H,m),8.55(1H,s).m / z:ES+[M+H]+501.

[0537] 4-(9-benzyl-6-isopropoxy-9H-purine-8-yl)-3-methylphenol, used as a starting material, was prepared as follows.

[0538] N4-benzyl-6-chloropyrimidine-4,5-diamine

[0539] [ka]

[0540] A mixture of phenylmethaneamine (47.0 g, 439 mmol) and triethylamine (74.0 g, 732 mmol) was added dropwise to 4,6-dichloropyrimidine-5-amine (60 g, 366 mmol) in DMA (400 mL) under air at 100°C for 20 minutes. The resulting solution was stirred at 100°C for 8 hours. The reaction mixture was then poured into water (1000 mL) while stirring. The resulting precipitate was collected by filtration, washed with water (500 mL), and dried at 60°C to obtain N4-benzyl-6-chloropyrimidine-4,5-diamine (60.0 g, 70%) as a pale yellow solid.

[0541] 1 H NMR(300MHz,DMSO-d6):4.65(2H,d),5.11(2H,s),7.19-7.29(1H,m),7.29-7.45(5H,m),7.76(1H,s).m / z:ES+[M+H]+235.

[0542] 4-(9-benzyl-6-chloro-9H-purine-8-yl)-3-methylphenol

[0543] [ka]

[0544] 4-hydroxy-2-methylbenzaldehyde (580 mg, 4.26 mmol) was added to N4-benzyl-6-chloropyrimidine-4,5-diamine (500 mg, 2.13 mmol) in 1,4-dioxane (40 mL) and iron(III) chloride (6.91 g, 6.39 mmol) supported on silica gel. The resulting mixture was stirred at 100°C for 3 days. The solvent was removed under reduced pressure. The filtrate was collected by filtration, the precipitate was washed with ELISA (100 mL), and dried under vacuum to obtain the crude product. The crude product was purified by flash C18-flash chromatography in water (containing 0.05% NH4HCO3) with an elution gradient of 5-80% MeCN. The pure fraction was evaporated to dryness to obtain 4-(9-benzyl-6-chloro-9H-purine-8-yl)-3-methylphenol (230 mg, 31%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6):1.94(3H,s),5.34(2H,s),6.70-6.75(2H,m),6.87-6 .92(2H,m),7.19-7.26(4H,m),8.81(1H,s),9.94(1H,s).m / z:ES+[M+H]+351.

[0545] 4-(9-benzyl-6-isopropoxy-9H-purine-8-yl)-3-methylphenol

[0546] [ka]

[0547] NaH (50.2 mg, 1.25 mmol) was gradually added to 4-(9-benzyl-6-chloro-9H-purine-8-yl)-3-methylphenol (220 mg, 0.63 mmol) and IPA (0.097 mL, 1.25 mmol) in THF (10 mL). The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was quenched with saturated NaHCO3 (50 mL) and extracted with Âx (3 × 20 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to obtain 4-(9-benzyl-6-isopropoxy-9H-purine-8-yl)-3-methylphenol (210 mg, 89%) as a yellow solid. 1 H NMR(300MHz,DMSO-d6):1.41(6H,d),1.90(3H,s),5.26(2H,s),5.61(1H,p),6.68-6.75(2 H,m),6.84-6.90(2H,m),7.13-7.25(4H,m),8.54(1H,s),9.85(1H,s).m / z:ES+[M+H]+375.

[0548] Synthesis Example 2 2-(2-chloro-4-(2-(4-methylpiperazine-1-yl)ethoxy)phenyl)-1-(3-chlorobenzyl)-5-isopropoxy-1H-benzo[d]imidazole

[0549] [ka]

[0550] 2-(2-chloro-4-(2-(4-methylpiperazine-1-yl)ethoxy)phenyl)-1-(3-chlorobenzyl)-1H-benzo[d]imidazole-5-ol (125 mg, 0.24 mmol), 2-bromopropane (0.046 mL, 0.49 mmol), and potassium carbonate (169 mg, 1.22 mmol) were suspended in acetonitrile (5 mL) and sealed in a microwave tube. The reaction mixture was heated in a microwave reactor at 100 °C for 12 hours and then cooled to room temperature. The solid was removed by filtration, and the filtrate was evaporated to dryness. The crude product was purified by preparative HPLC (Waters CSH C18 OBD column, 5 μm silica, 30 mm diameter, 100 mm length) using water (containing 1% aqueous ammonia) and a mixture that reduced the polarity of MeCN as the eluent. The fraction containing the desired compound was evaporated to dryness to obtain 2-(2-chloro-4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl)-1-(3-chlorobenzyl)-5-isopropoxy-1H-benzo[d]imidazole (14 mg, 10%). 1 H NMR(500MHz,CD3OD):1.32(3H,s),1.33(3H,s),2.27(3H,s),2.43-2.72(6H,m),2.84(2H,t),3.30(2H,p),4.19(2H,d),4.55-4.64 (1H,m),5.26(2H,s),6.86(1H,dt),6.93(2H,dd),7.01(1H,dd),7.15-7.23(4H,m),7.31(1H,d),7.35(1H,d).m / z:ES+[M+H]+553.

[0551] The starting material used, 2-(2-chloro-4-(2-(4-methylpiperazine-1-yl)ethoxy)phenyl)-1-(3-chlorobenzyl)-1H-benzo[d]imidazole-5-ol, was prepared as follows.

[0552] 2-(2-chloro-4-(2-(4-methylpiperazine-1-yl)ethoxy)phenyl)-1-(3-chlorobenzyl)-5-methoxy-1H-benzo[d]imidazole

[0553] [ka]

[0554] A solution of crude 2-chloro-4-(2-(4-methylpiperazin-1-yl)ethoxy)benzaldehyde (578 mg, 1.02 mmol) in NMP (3 mL) was added in one step to a stirred suspension of N-(3-chlorobenzyl)-4-methoxy-2-nitroaniline (299 mg, 1.02 mmol, commercially available from Princeton BiolMolecular Research Inc., ACD Identifier: MFCD12564576) and sodium dithionite (628 mg, 3.07 mmol) in water (1 mL). The resulting solution was stirred under reflux for 18 hours. The reaction mixture was diluted with  (50 mL) and washed sequentially with NaHCO3 (50 mL), water (50 mL), and saturated brine (5 mL). The organic layer was dried over MgSO4, filtered, and evaporated over silica gel (1 g). The obtained powder was purified by flash silica chromatography using ammonia as a modifier with an elution gradient of 0-20% MeOH in DCM. The pure fraction was evaporated to dryness to obtain 2-(2-chloro-4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl)-1-(3-chlorobenzyl)-5-methoxy-1H-benzo[d]imidazole (213 mg, 40%) as a white solid. The impure fractions were combined and concentrated under vacuum, and purified by preparative HPLC (Waters CSH C18 OBD column, 5 μm silica, 30 mm diameter, 100 mm length) using water (containing 1% NH3 water) and a mixture that reduces the polarity of MeCN as the eluent. The fraction containing the desired compound was evaporated to dryness to obtain 2-(2-chloro-4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl)-1-(3-chlorobenzyl)-5-methoxy-1H-benzo[d]imidazole (92 mg, 17%) as a white solid. 1H NMR(500MHz,CDCl3):2.30(3H,s),2.48(4H,s),2.63(4H,s),2.84(2H,t),3.87(3H,s),4.14(2H,t),5.17(2H,s),6.82(1H,d),6 .88(1H,dd),6.91(1H,dd),6.95(1H,s),7.04-7.1(2H,m),7.15(1H,t),7.18-7.22(1H,m),7.35(2H,s).m / z:ES+525[M+H]+525.

[0555] 2-(2-chloro-4-(2-(4-methylpiperazine-1-yl)ethoxy)phenyl)-1-(3-chlorobenzyl)-1H-benzo[d]imidazole-5-ol

[0556] [ka]

[0557] A solution of boron tribromide in dichloromethane (1.52 mL, 1.52 mmol) was added dropwise over 2 minutes at 0°C under nitrogen to a stirred solution of 2-(2-chloro-4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl)-1-(3-chlorobenzyl)-5-methoxy-1H-benzo[d]imidazole (0.20 g, 0.38 mmol) in anhydrous dichloromethane (1 mL). The resulting suspension was stirred at room temperature for 35 minutes. The reaction mixture was quenched with 2 M HCl (5 mL) and evaporated to remove DCM. DMSO (2 mL) was added, and the resulting solution was purified by flash reversed-phase silica chromatography using a 5-95% MeCN elution gradient in water with 0.1% formic acid as a modifier. The pure fraction was evaporated to dryness to obtain 2-(2-chloro-4-(2-(4-methylpiperazin-1-yl)ethoxy)phenyl)-1-(3-chlorobenzyl)-1H-benzo[d]imidazole-5-ol (150 mg, 77%) as a white solid. 1H NMR(500MHz,CDCl3):2.64(3H,s),2.81-2.96(6H,m),3.06(4H,s),4.11(2H,t),5.15(2H,s),6.83(2H,td),6.8 7(1H,dd),6.93(1H,s),7-7.06(2H,m),7.12-7.22(2H,m),7.29-7.35(2H,m),8.38(1H,s).m / z:ES+[M+H]+511.

[0558] The starting material used, 2-chloro-4-(2-(4-methylpiperazin-1-yl)ethoxy)benzaldehyde, is described in Synthesis Example 4.

[0559] Synthesis Example 3 3-(4-(1-benzyl-5-isopropoxy-1H-benzo[d]imidazole-2-yl)-3-chlorophenoxy)propan-1-amine

[0560] [ka]

[0561] TFA (2 mL, 25.96 mmol) was slowly added at 0°C to tert-butyl (3-(4-(1-benzyl-5-isopropoxy-1H-benzo[d]imidazole-2-yl)-3-chlorophenoxy)propyl) carbamate (100 mg, 0.18 mmol) in DCM (5 mL). The resulting mixture was stirred at room temperature for 2 hours. The reaction mixture was evaporated to obtain a crude oil. The crude product was purified by preparative HPLC (XBridge Shield RP18 OBD column, 5 μm silica, 30 mm diameter, 150 mm length) using water (containing 0.05% NH3 water) and a mixture that reduces the polarity of MeCN as the eluent. The fraction containing the desired compound was evaporated to dryness to obtain 3-(4-(1-benzyl-5-isopropoxy-1H-benzo[d]imidazole-2-yl)-3-chlorophenoxy)propan-1-amine (27 mg, 33%) as a yellow oily substance, which solidified upon standing. 1¹H NMR (400MHz, CDCl3): 1.39 (6H,d), 1.97-2.07 (2H,m), 2.99 (2H,t), 4.13 (2H,t), 4.58 (1H,p), 5.23 (2H,s), 6.88 (2H,dt), 6.97-7.03 (2H,m), 7.06-7.13 (2H,m), 7.25 (3H,dd), 7.33-7.41 (2H,m). 2H was not observed. m / z: ES+[M+H]+450.

[0562] The tert-butyl(3-(4-(1-benzyl-5-isopropoxy-1H-benzo[d]imidazole-2-yl)-3-chlorophenoxy)propyl) carbamate used as a starting material was prepared as follows.

[0563] N-benzyl-4-isopropoxy-2-nitroaniline

[0564] [ka]

[0565] Phenylmethaneamine (2.37 g, 22.1 mmol) was slowly added at room temperature to DIEA (7.02 mL, 40.2 mmol) and 1-fluoro-4-isopropoxy-2-nitrobenzene (4 g, 20.1 mmol, commercially available) in DMA (10 mL). The resulting mixture was stirred at 100 °C for 18 hours and then cooled to room temperature. The reaction mixture was poured into water (50 mL) and extracted with pharmaceutically acceptable ethyl acetate (3 × 50 mL). The organic layer was sequentially washed with saturated NH₄Cl (20 mL × 1), saturated NaHCO₃ (20 mL × 1), and saturated brine (20 mL × 1). The organic layer was dried over Na₂SO₄, filtered, and evaporated to obtain the crude product. The residue was purified by preparative TLC (petroleum ether: siRNA = 1:6) to obtain N-benzyl-4-isopropoxy-2-nitroaniline (4.50 g, 78%) as a red oily substance, which solidified upon standing. 1H NMR(400MHz,DMSO-d6):1.22(6H,d),4.47(1H,hept),4.60(2H,d),6.88(1H,d),7.18(1H, dd),7.25(1H,ddd),7.29-7.39(4H,m),7.51(1H,d),8.51(1H,t).m / z(ES+),[M+H]+=287.

[0566] 4-(1-benzyl-5-isopropoxy-1H-benzo[d]imidazole-2-yl)-3-chlorophenol - Synthesis Example A1

[0567] [ka]

[0568] A solution of sodium dithionite (7.30 g, 41.9 mmol) in water (5.00 mL) was added dropwise at room temperature to a stirred mixture of N-benzyl-4-isopropoxy-2-nitroaniline (3 g, 10.5 mmol) and 2-chloro-4-hydroxybenzaldehyde (1.80 g, 11.5 mmol) in NMP (20 mL). The resulting mixture was stirred at 100 °C for 18 hours. The reaction mixture was poured into saturated brine (75 mL) and extracted with ethyl acetate (3 × 100 mL). The organic layer was dried over Na₂SO₄, filtered, and evaporated to obtain a yellow gum-like substance. The crude product was purified by flash silica chromatography with an elution gradient of 0–100% petroleum ether in ethyl acetate. The pure fraction was evaporated to dryness to obtain 4-(1-benzyl-5-isopropoxy-1H-benzo[d]imidazole-2-yl)-3-chlorophenol (2.30 g, 56%) as a yellow oily substance, which solidified upon standing. 1 H NMR(300MHz,DMSO-d6):1.27(6H,d),4.60(1H,p),5.25(2H,s),6.85(2H,td),6.93-7.04 (3H,m),7.15-7.30(4H,m),7.30(1H,t),7.37(1H,d),10.41(1H,s).m / z:ES+[M+H]+393.

[0569] Tert-butyl(3-(4-(1-benzyl-5-isopropoxy-1H-benzo[d]imidazole-2-yl)-3-chlorophenoxy)propyl)carbamate - Synthesis Example A2

[0570] [ka]

[0571] DIAD (1.00 g, 4.96 mmol) was added dropwise to 4-(1-benzyl-5-isopropoxy-1H-benzo[d]imidazole-2-yl)-3-chlorophenol (1.5 g, 3.82 mmol), tert-butyl(3-hydroxypropyl)carbamate (0.803 g, 4.58 mmol), and Ph3P (1.50 g, 5.73 mmol) in THF (20 mL) at 0°C under nitrogen. The resulting mixture was stirred at room temperature for 16 hours. The solvent was removed under reduced pressure. The reaction mixture was diluted with Depositphotos:petroleum ether (200 mL, 1:5). The solid was filtered off, and the organic layer was sequentially washed with saturated NH4Cl (30 mL), saturated NaHCO3 (30 mL), and saturated brine (30 mL x 2). The organic layer was dried over Na2SO4, filtered, and evaporated to obtain the crude product. The crude product was purified by flash silica chromatography with an elution gradient of 0-30% siRNA in petroleum ether. The pure fraction was evaporated to dryness to obtain tert-butyl(3-(4-(1-benzyl-5-isopropoxy-1H-benzo[d]imidazole-2-yl)-3-chlorophenoxy)propyl)carbamate (900 mg, 43%) as a yellow gum-like substance. 1 H NMR(400MHz,CDCl3):1.38(6H,d),1.46(9H,s),2.03(2H,q),3.35(2H,q),4.07(2H,t),4.58(1H,hept),4.74(1H,s),5.22(2H ,s),6.87(2H,ddd),6.95-7.02(2H,m),7.06(1H,d),7.09(1H,d),7.25(3H,dd),7.35(1H,d),7.39(1H,d).m / z:ES+[M+H]+550.

[0572] Synthesis Example 4 9-Benzyl-8-(2-chloro-4-(2-(4-methylpiperazine-1-yl)ethoxy)phenyl)-6-(1-methylcyclopropoxy)-9H-purine

[0573] [ka]

[0574] To a stirred solution of (E)-N-benzyl-5-((2-chloro-4-(2-(4-methylpiperazin-1-yl)ethoxy)benzylidene)amino)-6-(1-methylcyclopropoxy)pyrimidine-4-amine (calculated as 82 g crude product, 129 mmol, 1.00 equivalent) in IPA (820 mL), FeCl3 (32 g, 193 mmol, 1.50 equivalent) was added at room temperature under a nitrogen atmosphere. The reaction mixture was stirred at 80 °C for 1.5 hours. The resulting mixture was concentrated under vacuum. The reaction mixture was diluted with 500 ml of H2O, the aqueous layer was adjusted to pH=10 with NaOH, and extracted with DCM / IPA (6:1). The combined organic layers were washed with saturated NaHCO3 and brine (1000 mL x 5), dried over Na2SO4, and concentrated. The residue was applied to a silica gel column eluted with DCM / ammonia solution (3.5 M in MeOH) (1:0~1:20). The resulting mixture was further purified by SFC (OptiChiral-C9-5 column, 5 μm silica, 30 mm diameter, 250 mm length) eluted with 50% scCO2 and MeOH (containing 0.1% 2 M NH3-MeOH), concentrated under vacuum at less than 40°C to obtain a yellow solid, which was slurryed in Et2O (10V) for 2 hours. The resulting mixture was filtered, and the filtrate cake was dried under vacuum to obtain 9-benzyl-8-(2-chloro-4-(2-(4-methylpiperazine-1-yl)ethoxy)phenyl)-6-(1-methylcyclopropoxy)-9H-purine (32.4 g, 45%) as a pale yellow solid. 1H NMR(400MHz,CDCl3):0.76-0.88(2H,m),1.01-1.26(2H,m),1.80(3H,s),2.31(3H,s),2.37-2.57(4H,m),2.64(4H,br s),2.85(2H,t),4.15(2H,t),5.34(2H,s),6.82(1H,dd),6.93(2H,dd),7.06( 1H,d),7.13-7.21(2H,m),7.14-7.19(2H,m),8.67(1H,s).m / z:ES+[M+H]+533.

[0575] (E)-N-benzyl-5-((2-chloro-4-(2-(4-methylpiperazine-1-yl)ethoxy)benzylidene)amino)-6-(1-methylcyclopropoxy)pyrimidine-4-amine, used as a starting material, was prepared as follows.

[0576] N-benzyl-6-chloro-5-nitropyrimidine-4-amine

[0577] [ka]

[0578] To a stirred solution of 4,6-dichloro-5-nitropyrimidine (400 g, 2.01 mol, 1.00 equivalent) in DCM (4000 mL), TEA (228 g, 2.26 mol, 1.1 equivalent) was added at room temperature under a nitrogen atmosphere. Phenylmethaneamine (243.1 g, 2.26 mol, 1.1 equivalent) was added at 0°C. The reaction mixture was stirred at room temperature for 30 minutes. The resulting mixture was washed with brine (1000 mL x 5), dried over anhydrous Na2SO4, and concentrated. The residue was applied to a silica gel column using petroleum ether / ethyl acetate (2:1~1:1). This yielded N-benzyl-6-chloro-5-nitropyrimidine-4-amine (327 g, 60%) as a yellow solid. 1 H NMR (300MHz, CDCl3):4.82(2H,d),7.28-7.47(5H,m),7.82(1H,s),8.43(1H,s).m / z:ES+[M+H]+265.

[0579] N-benzyl-6-(1-methylcyclopropoxy)-5-nitropyrimidine-4-amine

[0580] [ka]

[0581] To a stirred solution of N-benzyl-6-chloro-5-nitropyrimidine-4-amine (210 g, 0.79 mol, 1.00 equivalent) in THF (2100 mL), 1-methylcyclopropan-1-ol (114.3 g, 1.59 mmol, 2.00 equivalent) was added at room temperature under a nitrogen atmosphere. LHMDS (1980 mL, 1.98 mol, 2.50 equivalent) was added at 0°C. The reaction mixture was stirred at room temperature for 18 hours. The reaction mixture was diluted with 1000 mL of NH4Cl and extracted with ethyl acetate (1500 mL x 3). The combined organic layer was washed with brine (2000 mL x 2), dried over Na2SO4, and concentrated. The residue was applied to a silica gel column using petroleum ether / ethyl acetate (1:50~1:20). This yielded N-benzyl-6-(1-methylcyclopropoxy)-5-nitropyrimidine-4-amine (108 g, 45%) as a yellow oily substance. 1 H NMR(400MHz,DMSO-d6):0.76(2H,t),0.92(2H,t),1.62(3H,s),4.70(2H,d),7.1 7-7.26(1H,m),7.26-7.39(4H,m),8.33(1H,s),8.86(1H,t).m / z:ES+[M+H]+301.

[0582] N4-benzyl-6-(1-methylcyclopropoxy)pyrimidine-4,5-diamine

[0583] [ka]

[0584] To a stirred solution of N-benzyl-6-(1-methylcyclopropoxy)-5-nitropyrimidine-4-amine (108 g, 360 mmol, 1.00 equivalent) in EtOH (1080 mL), iron powder (201 g, 3.60 mol, 10 equivalents) and NH4Cl (23.2 g, 432 mmol, 1.2 equivalents) were added at room temperature under a nitrogen atmosphere. The reaction mixture was stirred at 80°C for 18 hours. The resulting mixture was filtered through Celite and eluted with EtOH. The filtrate was concentrated to obtain the crude product. The crude product was purified by flash silica chromatography using petroleum ether:ethyl acetate in a 10:1 to 1:3 ratio to obtain N4-benzyl-6-(1-methylcyclopropoxy)pyrimidine-4,5-diamine (77 g, 79%) as an off-white solid. 1 H NMR(300MHz,DMSO-d6):0.68(2H,t),0.85(2H,t),1.60(3H,s),4.12(2H,s),4 .59(2H,d),6.69(1H,t),7.05-7.41(5H,m),7.72(1H,s).m / z:ES+[M+H]+271.

[0585] (E)-N-benzyl-5-((2-chloro-4-(2-(4-methylpiperazine-1-yl)ethoxy)benzylidene)amino)-6-(1-methylcyclopropoxy)pyrimidine-4-amine

[0586] [ka]

[0587] To a stirred solution of N4-benzyl-6-(1-methylcyclopropoxy)pyrimidine-4,5-diamine (35 g, 129 mmol, 1.00 equivalent) in MeOH / AcOH (20V / 1V), 2-chloro-4-(2-(4-methylpiperazin-1-yl)ethoxy)benzaldehyde (47.6 g, 168 mmol, 1.30 equivalent) was added under a nitrogen atmosphere at room temperature. The reaction mixture was stirred at room temperature for 15 hours. The resulting mixture was concentrated under vacuum to obtain (E)-N-benzyl-5-((2-chloro-4-(2-(4-methylpiperazin-1-yl)ethoxy)benzylidene)amino)-6-(1-methylcyclopropoxy)pyrimidine-4-amine (82 g, crude) as a yellow oil, which was used directly without further purification. 1 H NMR(300MHz,CDCl3):0.69-0.81(2H,m),0.98-1.10(2H,m),1.70(3H,s),2.55(3H,s),2.73-3.10(10H,m),4.08-4.20(2H,m),4.75(2H,d) ,6.38-6.49(1H,m),6.77-6.88(1H,m),6.88-6.97(1H,m),7.24-7.38(5H,m),8.00(1H,d),8.23(1H,s),9.40(1H,s).m / z:ES+[M+H]+535.

[0588] The starting material used, 2-chloro-4-(2-(4-methylpiperazin-1-yl)ethoxy)benzaldehyde, was prepared as follows.

[0589] tert-butyl 4-(2-(3-chloro-4-formylphenoxy)ethyl)piperazine-1-carboxylate

[0590] [ka]

[0591] To a stirred solution of 2-chloro-4-hydroxybenzaldehyde (300 g, 1.92 mol, 1.00 equivalent) in DMF (3000 mL), tert-butyl 4-(2-chloroethyl)piperazine-1-carboxylate (643.4 g, 2.59 mol, 1.35 equivalent), K2CO3 (528.8 g, 3.84 mol, 2.00 equivalent), and KI (63.6 g, 0.38 mol, 0.20 equivalent) were added under a nitrogen atmosphere. The reaction mixture was stirred at 80°C for 14 hours. The resulting mixture was diluted with 2 L of water. The resulting mixture was filtered, and the filtrate cake was washed with H2O. Next, the filtrate cake was dried under vacuum to obtain tert-butyl 4-(2-(3-chloro-4-formylphenoxy)ethyl)piperazine-1-carboxylate (390 g, 55%) as a yellow solid, which was used in the next step without further purification. 1 H NMR(400MHz,DMSO-d6):1.39(9H,s),2.43(4H,t),2.73(2H,t),3.30(4H,m),4.23( 2H,t),7.08(1H,dd),7.20(1H,d),7.81(1H,d),10.19(1H,s).m / z:ES+[M+H]+369.

[0592] 2-Chloro-4-(2-(4-methylpiperazine-1-yl)ethoxy)benzaldehyde

[0593] [ka]

[0594] To a stirred solution of tert-butyl 4-(2-(3-chloro-4-formylphenoxy)ethyl)piperazine-1-carboxylate (390 g, 1.06 mol, 1.00 equivalent) in formic acid (1850 mL), formaldehyde (48.5 g, 1.28 mol, 1.20 equivalent) was added under a nitrogen atmosphere. The reaction mixture was stirred at 70 °C for 14 hours. The reaction mixture was diluted with 1000 mL of H₂O and extracted with MTBE (1500 mL x 3). The aqueous layer was adjusted to pH=10 with NaOH and extracted with DCM (1000 mL x 3). The combined organic layers were washed with saturated brine (1000 mL x 5), dried over Na₂SO₄, and concentrated. The residue was purified by flash silica chromatography eluting with DCM / ammonia solution (3.5 M in MeOH) (1:100~1:30). This yielded 176 g (42%) of 2-chloro-4-(2-(4-methylpiperazin-1-yl)ethoxy)benzaldehyde as a yellow oily substance. 1 H NMR(300MHz,DMSO-d6):2.14(3H,s),2.39(8H,d),2.70(2H,t),4.22(2H,t) ,7.10(1H,dd),7.22(1H,d),7.82(1H,d),10.20(1H,d).m / z:ES+[M+H]+283.

[0595] Synthesis Example 5 9-Benzyl-8-(2-chloro-4-((1-methylpiperidine-4-yl)methoxy)phenyl)-6-(1-methylcyclopropoxy)-9H-purine

[0596] [ka]

[0597] DIAD (143 μL, 0.74 mmol) was added dropwise to 4-(9-benzyl-6-(1-methylcyclopropoxy)-9H-purine-8-yl)-3-chlorophenol (150 mg, 0.37 mmol), (1-methylpiperidine-4-yl)methanol (95 mg, 0.74 mmol), and Ph3P (193 mg, 0.74 mmol) in THF (5 mL) under nitrogen at 0°C. The resulting mixture was stirred at room temperature for 18 hours. The reaction mixture was concentrated, diluted with HCl (50 mL), and sequentially washed with saturated NH4Cl (2 × 15 mL) and saturated brine (2 × 15 mL). The organic layer was dried over Na2SO4, filtered, and evaporated to obtain the crude product. The residue was purified by preparative TLC (HCl) to obtain the crude product as a yellow gum-like substance. The crude product was purified by preparative HPLC (XBridge Shield RP18 OBD column, 5 μm silica, 30 mm diameter, 150 mm length) using water (containing 0.05% NH3 water) and a mixture that reduces the polarity of MeCN as the eluent. The fraction containing the desired compound was evaporated to dryness to obtain 9-benzyl-8-(2-chloro-4-((1-methylpiperidine-4-yl)methoxy)phenyl)-6-(1-methylcyclopropoxy)-9H-purine (5.0 mg, 2.6%) as a white solid. 1 H NMR(400MHz,DMSO-d6):0.80-0.88(2H,m),0.98-1.06(2H,m),1.21-1.37(2H,m),1.64-1.78(6H,m),1.80-1.95(2H,m),2.17(3H,s),2.79( 2H,dd),3.94(2H,d),5.29(2H,s),6.86-6.95(2H,m),7.00-7.06(1H,m),7.16-7.25(4H,m),7.40(1H,d),8.61(1H,s).m / z:ES+[M+H]+518.

[0598] 4-(9-benzyl-6-(1-methylcyclopropoxy)-9H-purine-8-yl)-3-chlorophenol, used as a starting material, was prepared as follows.

[0599] 9-benzyl-6-chloro-9H-purine

[0600] [ka]

[0601] (Bromomethyl)benzene (12.2 g, 71.2 mmol) was added dropwise to 6-chloro-9H-purine (10 g, 64.70 mmol) and potassium carbonate (10.73 g, 77.64 mmol) in acetonitrile (300 mL) over 10 minutes at 25°C. The resulting suspension was stirred at 25°C for 16 hours. The reaction mixture was filtered through Celite, and the filtrate was concentrated. The crude product was purified by flash silica chromatography with an elution gradient of 0-50% siRNA in petroleum ether. The pure fraction was evaporated to dryness to obtain 9-benzyl-6-chloro-9H-purine (9.00 g, 57%) as a white solid. 1 H NMR(400MHz, CDCl3):5.48(2H,s),7.29-7.47(5H,m),8.13(1H,s),8.81(1H,s).m / z:ES+[M+H]+245.

[0602] 9-benzyl-6-(1-methylcyclopropoxy)-9H-purine

[0603] [ka]

[0604] 1-Methylcyclopropan-1-ol (6.52 g, 90.4 mmol) was added to 9-benzyl-6-chloro-9H-purine (8.81 g, 36.0 mmol) in THF (72 mL). The reaction mixture was stirred at 0°C, and then sodium hydride (60% in mineral oil, 3.62 g, 90.5 mmol) was slowly added to the mixture. The reaction mixture was stirred at room temperature for 17 hours. Water (50 mL) was slowly added, and the reaction mixture was extracted with DCM (3 × 50 mL). The organic matter was combined and concentrated under vacuum. The crude substance was purified by column chromatography using silica gel (330 g, 0-50% ethyl phosphate in heptane) to obtain 9-benzyl-6-(1-methylcyclopropoxy)-9H-purine (7.18 g, 71%) as a yellow gum-like substance. 1 H NMR (500MHz, CDCl3):0.8-0.86(2H,m),1.1-1.17(2H,m),1.78(3H,s),5.41(2H,s),7.26-7.39(5H,m),7.88(1H,s),8.64(1H,s).

[0605] m / z:ES+[M+H]+281.

[0606] 9-benzyl-8-bromo-6-(1-methylcyclopropoxy)-9H-purine

[0607] [ka]

[0608] Lithium bis(trimethylsilyl)amide (28.6 mL, 28.6 mmol) was added to 9-benzyl-6-(1-methylcyclopropoxy)-9H-purine (5 g, 17.84 mmol) and 1,2-dibromotetrachloroethane (8.72 g, 26.8 mmol) in THF (29 mL) under nitrogen at 0°C. The resulting mixture was stirred at 25°C for 16 hours. The reaction mixture was quenched with water (50 mL) and extracted with toluene (3 × 100 mL) and saturated brine (1 × 100 mL). The organic layer was dried over Na₂SO₄, filtered, and evaporated to obtain the crude product. The crude product was purified by flash silica chromatography with an elution gradient of 0–30% toluene in petroleum ether. The pure fraction was evaporated to dryness to obtain 9-benzyl-8-bromo-6-(1-methylcyclopropoxy)-9H-purine (5.70 g, 89%) as a yellow gum-like substance. 1 H NMR (300MHz, CDCl3):0.76-0.87(2H,m),1.06-1.16(2H,m),1.76(3H,s),5.44(2H,s),7.27-7.37(5H,m),8.60(1H,s);m / z:ES+[M+H]+359.

[0609] 4-(9-benzyl-6-(1-methylcyclopropoxy)-9H-purine-8-yl)-3-chlorophenol

[0610] [ka]

[0611] Dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium(II) (907 mg, 1.39 mmol) was added to 9-benzyl-8-bromo-6-(1-methylcyclopropoxy)-9H-purine (5 g, 13.9 mmol), 3-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (7.09 g, 27.8 mmol), and cesium carbonate (9.07 g, 27.8 mmol) in 1,4-dioxane (20 mL) and water (2 mL) at 25 °C under nitrogen. The resulting mixture was stirred at 90 °C for 3 hours. The reaction mixture was diluted with ELISA (50 mL) and sequentially washed with water (3 × 50 mL). The organic layer was dried over Na₂SO₄, filtered, and evaporated to obtain the crude product. The crude product was purified by flash silica chromatography under an elution gradient of 0-100% siRNA in petroleum ether. The pure fraction was evaporated to dryness to obtain 4-(9-benzyl-6-(1-methylcyclopropoxy)-9H-purine-8-yl)-3-chlorophenol (5.60 g, 99%) as a yellow solid. 1 1H NMR (400MHz, DMSO-d6): 0.83(2H,t), 1.02(2H,t), 1.73(3H,s), 5.29(2H,s), 6.83(1H,dd), 6.88-6.92(2H,m), 6.99(1H,d), 7.18-7.22(3H,m), 7.29(1H,d), 8.61(1H,s) - 1 H atom not observed. m / z: ES-[MH]-405.

[0612] Synthesis Example 6 9-Benzyl-8-(2-chloro-4-(2-(piperazin-1-yl)ethoxy)phenyl)-6-(1-methylcyclopropoxy)-9H-purine

[0613] [ka]

[0614] 12.05 mL, 52.5 mmol of tert-butyldimethylsilyltrifluoromethanesulfonate was added to 6.5 g, 10.50 mmol of tert-butyl 4-(2-(4-(9-benzyl-6-(1-methylcyclopropoxy)-9H-purine-8-yl)-3-chlorophenoxy)ethyl)piperazine-1-carboxylate in 130 mL of DCM. The resulting solution was stirred at room temperature for 10 minutes. The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography with an elution gradient of 30-90% MeOH in water (containing 0.1% NH4HCO3). The pure fraction was evaporated to dryness to obtain 9-benzyl-8-(2-chloro-4-(2-(piperazin-1-yl)ethoxy)phenyl)-6-(1-methylcyclopropoxy)-9H-purine (3.10 g, 57%) as a white foam. 1 ¹H NMR (400MHz, DMSO-d6): 0.72-0.9 (2H,m), 0.95-1.12 (2H,m), 1.73 (3H,s), 2.25-2.49 (4H,m), 2.59-2.92 (6H,m), 4.19 (2H,t), 5.30 (2H,s), 6.91 (2H,dd), 7.04 (1H,dd), 7.13-7.29 (4H,m), 7.41 (1H,d), 8.63 (1H,s). No protons were observed. m / z: ES+[M+H]+519.

[0615] The tert-butyl 4-(2-(4-(9-benzyl-6-(1-methylcyclopropoxy)-9H-purine-8-yl)-3-chlorophenoxy)ethyl)piperazine-1-carboxylate used as a starting material was prepared as follows.

[0616] tert-butyl 4-(2-(4-(9-benzyl-6-chloro-9H-purine-8-yl)-3-chlorophenoxy)ethyl)piperazine-1-carboxylate

[0617] [ka]

[0618] Iron(III) chloride solution (20.73 g, 128 mmol) was added to N4-benzyl-6-chloropyrimidine-4,5-diamine (30 g, 128 mmol, Synthesis Example 1) and tert-butyl 4-(2-(3-chloro-4-formylphenoxy)ethyl)piperazine-1-carboxylate (51.9 g, 141 mmol) in EtOH (500 mL). The resulting mixture was stirred at 60°C for 2 days. The reaction mixture was evaporated to dryness, redissolved with ELISA (100 mL), and washed with water (3 × 100 mL). The organic layer was dried over Na₂SO₄, filtered, and evaporated to obtain the crude product. The crude product was purified by flash C18 chromatography with an elution gradient of 40–70% MeCN in water. The pure fraction was evaporated to dryness to obtain tert-butyl 4-(2-(4-(9-benzyl-6-chloro-9H-purine-8-yl)-3-chlorophenoxy)ethyl)piperazine-1-carboxylate (12.00 g, 16%) as a yellow solid.

[0619] 1 H NMR(300MHz,DMSO-d6):1.17(2H,t),1.40(9H,s),1.99(2H,s),2.45(4H,t),2.74(2H,t),4.22(2H,t),5.38(2H,s ),6.89-6.99(2H,m),7.09(1H,dd),7.17-7.25(3H,m),7.29(1H,d),7.51(1H,d),8.86(1H,s).m / z:ES+[M+H]+583.

[0620] tert-butyl 4-(2-(4-(9-benzyl-6-(1-methylcyclopropoxy)-9H-purine-8-yl)-3-chlorophenoxy)ethyl)piperazine-1-carboxylate

[0621] [ka]

[0622] Sodium hydride (6.17 g, 257 mmol) was added to 1-methylcyclopropan-1-ol (5.56 g, 77.12 mmol) and tert-butyl 4-(2-(4-(9-benzyl-6-chloro-9H-purine-8-yl)-3-chlorophenoxy)ethyl)piperazine-1-carboxylate (30 g, 51.4 mmol) in THF (200 mL) under nitrogen at 0°C. The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was poured into ice water. The reaction mixture was evaporated, diluted with ELISA (250 mL), and sequentially washed with water (3 × 200 mL) and saturated brine (2 × 200 mL). The organic layer was dried over Na₂SO₄, filtered, and evaporated to obtain the crude product. The crude product was purified by flash C18-flash chromatography with an elution gradient of 30–80% MeCN in water (containing 0.1% NH₄HCO₃). The pure fraction was evaporated to dryness to obtain tert-butyl 4-(2-(4-(9-benzyl-6-(1-methylcyclopropoxy)-9H-purine-8-yl)-3-chlorophenoxy)ethyl)piperazine-1-carboxylate (13.00 g, 41%) as a yellow solid. 1 H NMR(300MHz,DMSO-d6):0.8-0.88(2H,m),0.98-1.06(2H,m),1.40(9H,s),1.74(3H,s),2.39-2.48(4H,m),2.69-2.82(2H,m),3.33( 4H,s),4.20(2H,t),5.30(2H,s),6.85-6.96(2H,m),7.05(1H,d),7.14-7.29(4H,m),7.42(1H,d),8.62(1H,s).m / z:ES+[M+H]+619.

[0623] The tert-butyl 4-(2-(3-chloro-4-formylphenoxy)ethyl)piperazine-1-carboxylate used as a starting material was prepared as described in Synthesis Example 4.

[0624] Synthesis Example 7 4-((8-(2-chloro-4-(2-(4-methylpiperazine-1-yl)ethoxy)phenyl)-6-(1-methylcyclopropoxy)-9H-purine-9-yl)methyl)-2-methylthiazole

[0625] [ka]

[0626] A solution of sodium hydrosulfite (282 mg, 1.62 mmol) in water (1 mL) was added to a stirred mixture of 6-(1-methylcyclopropoxy)-N-((2-methylthiazole-4-yl)methyl)-5-nitropyrimidine-4-amine (130 mg, 0.40 mmol) and 2-chloro-4-(2-(4-methylpiperazin-1-yl)ethoxy)benzaldehyde (137 mg, 0.49 mmol, starting material for Synthesis Example 4) in NMP (3 mL). The resulting mixture was stirred at 100°C for 16 hours, then at 110°C for a further 16 hours. The crude product was purified by flash C18-flash chromatography with an elution gradient of 5-40% MeCN in water (containing 5% TFA). The fraction was evaporated to dryness to obtain the crude product. The crude product was further purified by preparative HPLC (XBridge Prep OBD C18 column, 5 μm silica, 30 mm diameter, 150 mm length) using water (containing 0.1% NH3 water and 10 mmol / L NH4HCO3) and a mixture that reduces the polarity of MeCN as the eluent. The fraction containing the desired compound was evaporated to dryness to obtain 4-((8-(2-chloro-4-(2-(4-methylpiperazine-1-yl)ethoxy)phenyl)-6-(1-methylcyclopropoxy)-9H-purine-9-yl)methyl)-2-methylthiazole (55 mg, 24%) as a white solid. 1 H NMR(400MHz,DMSO-d6):0.81-0.88(2H,m),1.01(2H,d),1.73(3H,s),2.15(3H,s),2.33(4H,s),2.49(3H,s),2.52(4H,s),2. 69(2H,t),4.17(2H,t),5.30(2H,s),6.96(1H,s),7.02(1H,dd),7.22(1H,d),7.43(1H,d),8.60(1H,s).m / z:ES+[M+H]+554.

[0627] The starting material used, 6-(1-methylcyclopropoxy)-N-((2-methylthiazole-4-yl)methyl)-5-nitropyrimidine-4-amine, was prepared as follows.

[0628] 6-Chloro-N-((2-methylthiazole-4-yl)methyl)-5-nitropyrimidine-4-amine

[0629] [ka]

[0630] A solution of (2-methylthiazole-4-yl)methaneamine (330 mg, 2.58 mmol) in DCM (10 mL) was added at 0°C to a stirred mixture of 4,6-dichloro-5-nitropyrimidine (500 mg, 2.58 mmol) and N,N-diisopropylethylamine (1.35 mL, 7.73 mmol) in DCM (10 mL). The resulting mixture was stirred at room temperature for 1 hour. The reaction mixture was diluted with water (25 mL) and extracted with DCM (3 × 20 mL). The organic layer was dried over Na₂SO₄, filtered, and evaporated to obtain 6-chloro-N-((2-methylthiazole-4-yl)methyl)-5-nitropyrimidine-4-amine (0.4 g) as a yellow gum. The product was used directly in the next step without further purification.

[0631] 6-(1-methylcyclopropoxy)-N-((2-methylthiazole-4-yl)methyl)-5-nitropyrimidine-4-amine

[0632] [ka]

[0633] LHMDS (2.66 mL, 2.66 mmol) was added at 0°C to 6-chloro-N-((2-methylthiazole-4-yl)methyl)-5-nitropyrimidine-4-amine (0.38 g, 1.33 mmol) and 1-methylcyclopropan-1-ol (0.192 g, 2.66 mmol) in THF (20 mL). The resulting mixture was stirred at room temperature for 16 hours. The reaction mixture was quenched with saturated NH4Cl (25 mL), and the THF solvent was removed under reduced pressure. The reaction mixture was extracted with siRNA (3 × 20 mL), the organic layer was dried over Na2SO4, filtered, and evaporated to obtain a yellow gum-like substance. The crude product was purified by flash C18-flash chromatography with an elution gradient of 5–100% MeCN in water. The pure fraction was evaporated to dryness to obtain 6-(1-methylcyclopropoxy)-N-((2-methylthiazole-4-yl)methyl)-5-nitropyrimidine-4-amine (0.150 g, 35%) as a yellow solid. 1 H NMR(300MHz,DMSO-d6):0.76(2H,dd),0.88-0.96(2H,m),1.61(3H,s),2.61(3H ,s),4.71(2H,dd),7.18(1H,d),8.33(1H,s),8.80(1H,t).m / z:ES+[M+H]+322.

[0634] Synthesis Example 8 1-Benzyl-2-(2-chloro-4-(2-(piperazine-1-yl)ethoxy)phenyl)-5-isopropoxy-1H-imidazo[4,5-b]pyridine

[0635] [ka]

[0636] tert-butyl 4-(2-(4-(1-benzyl-5-isopropoxy-1H-imidazo[4,5-b]pyridine-2-yl)-3-chlorophenoxy)ethyl)piperazine-1-carboxylate (44 mg, 0.07 mmol) was added to trifluoroacetic acid (0.5 ml, 6.49 mmol) in dichloromethane (2 mL). The resulting solution was stirred at room temperature for 1 hour, and then the solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography with an elution gradient of 30-80% MeOH in water (0.1% NH4HCO3). The pure fraction was evaporated to dryness to obtain 1-benzyl-2-(2-chloro-4-(2-(piperazin-1-yl)ethoxy)phenyl)-5-isopropoxy-1H-imidazo[4,5-b]pyridine (0.026 g, 71%) as a white solid. 1 ¹H NMR (300MHz, DMSO-d6): 1.32(6H,d), 2.40(4H,t), 2.64-2.72(6H,m), 4.18(2H,t), 5.24-5.34(3H,m), 6.63(1H,d), 6.94-6.99(2H,m), 7.07(1H,dd), 7.21-7.29(4H,m), 7.49(1H,d), 7.80(1H,d) - 1 proton was not observed. m / z: ES+[M+H]+506.

[0637] The tert-butyl 4-(2-(4-(1-benzyl-5-isopropoxy-1H-imidazo[4,5-b]pyridine-2-yl)-3-chlorophenoxy)ethyl)piperazine-1-carboxylate used as a starting material was prepared as follows.

[0638] 6-amino-5-nitropyridine-2(1H)-one

[0639] [ka]

[0640] 6-Chloro-3-nitropyridine-2-amine (10 g, 57.62 mmol) was added to NaOH (23.1 g, 57.6 mmol) in EtOH (50 mL) and water (16.6 mL). The resulting solution was stirred at 80°C for 30 minutes. The reaction mixture was acidified with concentrated HCl, and the resulting precipitate was filtered to obtain 6-amino-5-nitropyridine-2(1H)-one (8.00 g, 90%) as a yellow solid. The product was used directly in the next step without further purification. 1 H NMR (300MHz, DMSO-d6): 11.51 (s, 1H), 8.53 (s, 2H), 7.98 (d, 1H), 5.66 (d, 1H). m / z: ES-[MH]-154.

[0641] 6-Isopropoxy-3-nitropyridine-2-amine

[0642] [ka]

[0643] 6-amino-5-nitropyridine-2(1H)-one (4 g, 25.8 mmol) was added at 80°C to potassium carbonate (10.7 g, 77.4 mmol) and 2-iodopropane (13.1 g, 77.4 mmol) in DMF (80 mL). The resulting solution was stirred overnight at 80°C. After cooling the reaction mixture, the solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography with an elution gradient of 0-20% siRNA in petroleum ether. The pure fraction was evaporated to dryness to obtain 6-isopropoxy-3-nitropyridine-2-amine (3.20 g, 63%) as a yellow solid. 1 H NMR(300MHz,DMSO-d6):1.18(6H,d),5.12-5.24(1H,m),5.95(1H,d),7.97(2H,s),8.10(1H,d).m / z:ES+[M+H]+198.

[0644] 6-Isopropoxypyridine-2,3-diamine

[0645] [ka]

[0646] 6-Isopropoxy-3-nitropyridine-2-amine (3.1 g, 15.72 mmol) and palladium carbon (310 mg, 0.29 mmol) in MeOH (60 mL) were stirred at room temperature under a hydrogen atmosphere for 2 hours. The reaction mixture was filtered through filter paper to obtain 6-isopropoxypyridine-2,3-diamine (2.60 g, 99%) as a purple oil. The product was used directly in the next step without further purification. 1 H NMR(300MHz,DMSO-d6):1.18(6H,d),3.92-4.69(2H,m),4.9-4.97(1H,m),4.97-5.51(2H,m),5.73(1H,d),6.73(1H,d).m / z:ES+[M+H]+168.

[0647] N3-benzyl-6-isopropoxypyridine-2,3-diamine

[0648] [ka]

[0649] 6-Isopropoxypyridine-2,3-diamine (1.5 g, 8.97 mmol) was added to benzaldehyde (0.909 ml, 8.97 mmol) and acetic acid (0.051 ml, 0.90 mmol) in dichloromethane (30 mL). The reaction mixture was stirred at room temperature for 5 hours. Then, sodium triacetoxyborohydride (5.70 g, 26.91 mmol) was added to the reaction mixture. The resulting solution was stirred at room temperature for a further 16 hours. The reaction mixture was concentrated under reduced pressure. The crude product was purified by flash silica chromatography with an elution gradient of 0-20% siRNA in petroleum ether. The pure fraction was evaporated to dryness to obtain N3-benzyl-6-isopropoxypyridine-2,3-diamine (1.10 g, 48%) as a purple oil. 1¹H NMR (300MHz, DMSO-d6): 1.17(6H,d), 4.20(2H,s), 4.94(1H,p), 5.45(2H,s), 5.73(1H,d), 6.57(1H,d), 7.12-7.25(1H,m), 7.25-7.46(4H,m). No protons were observed. m / z: ES+[M+H]+258.

[0650] tert-butyl 4-(2-(4-(1-benzyl-5-isopropoxy-1H-imidazo[4,5-b]pyridine-2-yl)-3-chlorophenoxy)ethyl)piperazine-1-carboxylate

[0651] [ka]

[0652] N3-benzyl-6-isopropoxypyridine-2,3-diamine (50 mg, 0.19 mmol) was added to tert-butyl 4-(2-(3-chloro-4-formylphenoxy)ethyl)piperazine-1-carboxylate (86 mg, 0.23 mmol, starting material for Synthesis Example 6) and copper(II) acetate monohydrate (7.76 mg, 0.04 mmol) in acetic acid (2 mL). The resulting solution was stirred at 100°C for 1 hour. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography with an elution gradient of 0-10% MeOH in dichloromethane. The pure fraction was evaporated to dryness to obtain tert-butyl 4-(2-(4-(1-benzyl-5-isopropoxy-1H-imidazo[4,5-b]pyridine-2-yl)-3-chlorophenoxy)ethyl)piperazine-1-carboxylate (118 mg, 100%) as a yellow solid. 1H NMR(300MHz,DMSO-d6):1.2-1.35(6H,m),1.39(9H,d),2.44(4H,s),2.73(2H,s),3.17(8H,d),4.08(3H,q),4.19(1H,d),5 .30(1H,d),6.82(1H,t),6.97(1H,d),7.07(1H,d),7.2-7.25(1H,m),7.27(1H,d),7.46-7.56(1H,m).m / z:ES+[M+H]+606.

[0653] Synthesis Example 9 2-(2-chloro-4-(2-(4-methylpiperazine-1-yl)ethoxy)phenyl)-1-(3-chlorobenzyl)-1H-benzo[d]imidazole-5-ol

[0654] [ka]

[0655] tert-butyl 4-(2-(4-(1-benzyl-4-isopropoxy-1H-imidazo[4,5-c]pyridine-2-yl)-3-chlorophenoxy)ethyl)piperazine-1-carboxylate (90 mg, 0.15 mmol) was added to trifluoroacetic acid (1 mL, 12.98 mmol) in dichloromethane (4 mL). The resulting solution was stirred at room temperature for 1 hour. The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography with an elution gradient of 30-80% MeOH in water (0.1% NH4HCO3). The pure fraction was evaporated to dryness to obtain 1-benzyl-2-(2-chloro-4-(2-(piperazin-1-yl)ethoxy)phenyl)-4-isopropoxy-1H-imidazo[4,5-c]pyridine (0.029 g, 38%) as a white solid. 1¹H NMR (400MHz, DMSO-d6): 1.37(6H,d), 2.36-2.44(4H,m), 2.64-2.72(6H,m), 4.18(2H,t), 5.28(2H,s), 5.44-5.55(1H,m), 6.92-6.96(2H,m), 7.05(1H,dd), 7.12(1H,d), 7.21-7.28(4H,m), 7.47(1H,d), 7.85(1H,d) - 1 proton was not observed. m / z: ES+[M+H]+506.

[0656] The tert-butyl 4-(2-(4-(1-benzyl-4-isopropoxy-1H-imidazo[4,5-c]pyridine-2-yl)-3-chlorophenoxy)ethyl)piperazine-1-carboxylate used as a starting material was prepared as follows.

[0657] 4-Chloro-2-isopropoxy-3-nitropyridine

[0658] [ka]

[0659] 4-Chloro-3-nitropyridine-2(1H)-one (5.0 g, 28.6 mmol) was added to sodium hydride (2.06 g, 85.9 mmol) in DMF (100 mL) at 0°C. The reaction mixture was heated to room temperature over 30 minutes, and then 2-iodopropane (24.3 g, 143.2 mmol) was added. The resulting solution was stirred at room temperature for a further 12 hours. The reaction mixture was quenched with saturated NH4Cl (50 mL), extracted with ELISA (3 × 100 mL), the organic layer was dried over Na2SO4, filtered, and evaporated to obtain the crude product. The crude product was purified by flash C18-flash chromatography with an elution gradient of 5–80% MeOH in water (0.1% NH4HCO3). The pure fraction was evaporated to dryness to obtain 4-chloro-2-isopropoxy-3-nitropyridine (2.10 g, 34%) as a yellow oily substance. 1H NMR (300MHz, DMSO-d6): 1.30 (6H, d), 5.29-5.44 (1H, m), 7.43 (1H, d), 8.37 (1H, d).

[0660] N-benzyl-2-isopropoxy-3-nitropyridine-4-amine

[0661] [ka]

[0662] 4-Chloro-2-isopropoxy-3-nitropyridine (1.0 g, 4.62 mmol) was added to benzylamine (0.504 mL, 4.62 mmol) and TEA (6.43 mL, 46.2 mmol) in DMSO (20 mL). The resulting solution was stirred at 90°C for 5 hours. The solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography with an elution gradient of 0-10% siRNA in petroleum ether. The pure fraction was evaporated to dryness to obtain N-benzyl-2-isopropoxy-3-nitropyridine-4-amine (1.20 g, 90%) as a yellow solid. 1 H NMR(300MHz,DMSO-d6):1.25(6H,d),4.49(2H,d),5.21-5.35(1H,m),6.39(1H,d), 7.21-7.28(1H,m),7.3-7.35(4H,m),7.75(1H,d),7.87(1H,t).m / z:ES-[MH]-286.

[0663] N4-benzyl-2-isopropoxypyridine-3,4-diamine

[0664] [ka]

[0665] N-benzyl-2-isopropoxy-3-nitropyridine-4-amine (1 g, 3.48 mmol) was added to iron (0.972 g, 17.40 mmol) and ammonium chloride (1.862 g, 34.80 mmol) in EtOH (16 mL) and water (1.6 mL). The resulting solution was stirred at 80°C for 12 hours. The reaction mixture was filtered through filter paper, and the solvent was removed under reduced pressure. The crude product was purified by flash silica chromatography with an elution gradient of 0-20% siRNA in petroleum ether. The pure fraction was evaporated to dryness to obtain N4-benzyl-2-isopropoxypyridine-3,4-diamine (0.518 g, 58%) as a yellow solid. 1 H NMR(300MHz,DMSO-d6):1.25(6H,d),4.04(2H,s),4.35(2H,d),5.09-5.19(1H,m),5.91(1H,t),6.1 0(1H,d),7.18(1H,d),7.2-7.25(1H,m),7.28-7.32(1H,m),7.32-7.37(3H,m).m / z:ES+[M+H]+258.

[0666] tert-butyl 4-(2-(4-(1-benzyl-4-isopropoxy-1H-imidazo[4,5-c]pyridine-2-yl)-3-chlorophenoxy)ethyl)piperazine-1-carboxylate

[0667] [ka]

[0668] N4-benzyl-2-isopropoxypyridine-3,4-diamine (50 mg, 0.19 mmol) was added to tert-butyl 4-(2-(3-chloro-4-formylphenoxy)ethyl)piperazine-1-carboxylate (86 mg, 0.23 mmol) and copper(II) acetate monohydrate (7.8 mg, 0.04 mmol) in AcOH (2 mL). The resulting solution was stirred at 100°C for 1 hour. The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography with an elution gradient of 10-80% MeOH in water (0.1% NH4HCO3). The pure fraction was evaporated to dryness to obtain tert-butyl 4-(2-(4-(1-benzyl-4-isopropoxy-1H-imidazo[4,5-c]pyridine-2-yl)-3-chlorophenoxy)ethyl)piperazine-1-carboxylate (95 mg, 81%) as a yellow solid. 1 H NMR(400MHz,DMSO-d6):1.37(6H,d),1.39(9H,s),2.44(4H,t),2.74(2H,t),3.28-3.3(2H,m),3.32-3.34(2H,m),4.20(2H,t),5.28(2H,s),5. 47-5.53(1H,m),6.92-6.96(2H,m),7.05(1H,dd),7.12(1H,d),7.21-7 .24(3H,m),7.27(1H,d),7.47(1H,d),7.85(1H,d).m / z:ES+[M+H]+606.

[0669] Synthesis Example 10 8-(2-chloro-4-(2-(piperazine-1-yl)ethoxy)phenyl)-9-((4-chloropyridine-2-yl)methyl)-6-(1-methylcyclopropoxy)-9H-purine

[0670] [ka]

[0671] A solution of tert-butyl 4-(2-(3-chloro-4-(9-((4-chloropyridine-2-yl)methyl)-6-(1-methylcyclopropoxy)-9H-purine-8-yl)phenoxy)ethyl)piperazine-1-carboxylate (2.07 g, 3.16 mmol) in acetonitrile was cooled to 0°C in an ice / water bath. 4.0 M hydrogen chloride in dioxane (8.0 mL, 32.0 mmol) was added, and the ice bath was removed. After stirring at room temperature for 1 hour, an additional 4.0 M hydrogen chloride in dioxane (8.0 mL, 32.0 mmol) was added, and stirring was continued for a further 30 minutes. The reaction mixture was evaporated to obtain the crude product as a yellow solid. The crude product was purified by ion-exchange chromatography using an SCX column. The desired product was eluted from the column using 1 M NH3 / MeOH, dried, and obtained the crude free base. The crude free base was purified by flash inactivated alumina chromatography with an elution gradient of 0-10% MeOH in DCM to obtain 8-(2-chloro-4-(2-(piperazin-1-yl)ethoxy)phenyl)-9-((4-chloropyridine-2-yl)methyl)-6-(1-methylcyclopropoxy)-9H-purine (1.50 g, 86%) as a yellow foam.

[0672] 1 ¹H NMR (500MHz, CDCl3): 0.8-0.87 (2H,m), 1.14-1.2 (2H,m), 1.81 (3H,s), 2.55 (4H,s), 2.80 (2H,t), 2.92 (4H,t), 4.12 (2H,t), 5.44 (2H,s), 6.80 (1H,dd), 6.88 (1H,dd), 7.02 (1H,d), 7.12 (1H,dd), 7.25 (1H,d), 8.30 (1H,dd), 8.64 (1H,s). 1H was not observed. m / z: ES+[M+H]+554.

[0673] The starting material used, tert-butyl 4-(2-(3-chloro-4-(9-((4-chloropyridine-2-yl)methyl)-6-(1-methylcyclopropoxy)-9H-purine-8-yl)phenoxy)ethyl)piperazine-1-carboxylate, was prepared as follows.

[0674] tert-butyl 4-(2-(3-chloro-4-(6-chloro-9H-purine-8-yl)phenoxy)ethyl)piperazine-1-carboxylate

[0675] [ka]

[0676] tert-butyl 4-(2-(3-chloro-4-formylphenoxy)ethyl)piperazine-1-carboxylate (1 g, 2.71 mmol) and 6-chloropyrimidine-4,5-diamine (0.431 g, 2.98 mmol) were dissolved in IPA (38.7 mL). Iron(III) chloride (0.088 g, 0.54 mmol) was added, and the reaction mixture was stirred at 80°C under air for 2.5 days. The reaction mixture was cooled to room temperature, diluted with DCM (50 mL), and water (50 mL) was added. The mixture was filtered through a small Celite plug to aid separation, and the Celite was washed with DCM (50 mL). The mixture was then extracted with DCM (50 mL x 3), the combined organic matter was washed with saturated NaHCO3 (20 mL), separated, dried over MgSO4, filtered, and evaporated to obtain the crude product. The crude product was purified by flash silica chromatography with an elution gradient of 0-100% siRNA in heptane, followed by elution of the product with a 3:1 siRNA / EtOH elution gradient in siRNA. The pure fraction was evaporated to dryness to obtain tert-butyl 4-(2-(3-chloro-4-(6-chloro-9H-purine-8-yl)phenoxy)ethyl)piperazine-1-carboxylate (1.050 g, 78%) as a pale yellow solid. 1 ¹H NMR (500MHz, DMSO-d6): 1.40 (9H, s), 2.44-2.48 (4H, m), 2.76 (2H, t), 3.26-3.38 (4H, m), 4.23 (2H, t), 7.15 (1H, dd), 7.30 (1H, d), 7.81 (1H, d), 8.75 (1H, s). 1H was not observed. m / z: ES + [M + H] + 493.

[0677] The tert-butyl 4-(2-(3-chloro-4-formylphenoxy)ethyl)piperazine-1-carboxylate used as a starting material was prepared as described in Synthesis Example 4.

[0678] tert-butyl 4-(2-(3-chloro-4-(6-chloro-9-((4-chloropyridine-2-yl)methyl)-9H-purine-8-yl)phenoxy)ethyl)piperazine-1-carboxylate

[0679] [ka]

[0680] To a solution of tert-butyl 4-(2-(3-chloro-4-(6-chloro-9H-purine-8-yl)phenoxy)ethyl)piperazine-1-carboxylate (1.62 g, 3.28 mmol) and 4-chloro-2-(chloromethyl)pyridine hydrochloride (0.977 g, 4.93 mmol) in anhydrous DMF (16.4 mL), cesium carbonate (3.21 g, 9.85 mmol) was added. The reaction mixture was inactivated by applying three cycles of vacuum and nitrogen filling, and then stirred under nitrogen at 60°C for 19 hours. The reaction was quenched with ice water (20 mL), and ethyl acetate (20 mL) was added. The organic layer was removed, and the aqueous layer was further extracted with ethyl acetate (10 mL x 6). The combined organic layers were washed with saturated aqueous solution of LiCl (10 mL x 3), filtered through an islute phase separation cartridge, and evaporated to obtain the crude product as a brown foam. The crude product was purified by flash silica chromatography with an elution gradient of 0-35% and then ~70% 3:1 siRNA:EtOH in n-heptane. The pure fraction was evaporated to dryness to obtain tert-butyl 4-(2-(3-chloro-4-(6-chloro-9-((4-chloropyridine-2-yl)methyl)-9H-purine-8-yl)phenoxy)ethyl)piperazine-1-carboxylate (1.13 g, 56%) as a pale brown foam. 1H NMR(500MHz,CDCl3):1.47(9H,s),2.5-2.54(4H,m),2.83(2H,t),3.44-3.48(4H,m),4.14(2H,t),5.47(2H,s),6 .84(1H,dd),6.98(1H,d),7.05(1H,d),7.14(1H,dd),7.31(1H,d),8.28(1H,d),8.77(1H,s).m / z:ES+[M+H]+618.

[0681] tert-butyl 4-(2-(3-chloro-4-(9-((4-chloropyridine-2-yl)methyl)-6-(1-methylcyclopropoxy)-9H-purine-8-yl)phenoxy)ethyl)piperazine-1-carboxylate

[0682] [ka]

[0683] To a suspension of sodium hydride (0.889 g, 22.2 mmol) in tetrahydrofuran (15 mL) cooled to 0°C under nitrogen, a solution of 1-methylcyclopropan-1-ol (0.802 g, 11.1 mmol) and tert-butyl 4-(2-(3-chloro-4-(6-chloro-9-((4-chloropyridine-2-yl)methyl)-9H-purine-8-yl)phenoxy)ethyl)piperazine-1-carboxylate (3.44 g, 5.56 mmol) in tetrahydrofuran (30 mL) was added at a steady flow rate. The reaction mixture was stirred at 0°C for 20 minutes, then the ice bath was removed, and the reaction mixture was stirred under nitrogen at room temperature for 19 hours. The reaction mixture was cooled in an ice / water bath and carefully quenched with saturated ammonium chloride solution (15 mL). After gas generation subsided, the mixture was diluted with water (150 mL) and ethyl acetate (150 mL). The aqueous phase was extracted with ELISA (150 mL). The combined organic phase was washed with brine, dried, and evaporated. The crude product was purified by flash silica chromatography under an elution gradient of 0-100% 3:1 ELISA / EtOH in heptane to obtain tert-butyl 4-(2-(3-chloro-4-(9-((4-chloropyridine-2-yl)methyl)-6-(1-methylcyclopropoxy)-9H-purine-8-yl)phenoxy)ethyl)piperazine-1-carboxylate (2.07 g, 57%) as a pale yellow foam. 1 H NMR(500MHz,CDCl3):0.8-0.87(2H,m),1.14-1.2(2H,m),1.47(9H,s),1.81(3H,s),2.45-2.59(4H,m),2.82(2H,t),3.38-3.53(4H,m),4.12( 2H,t),5.44(2H,s),6.80(1H,dd),6.89(1H,d),7.01(1H,d),7.12(1H,dd),7.24-7.28(1H,m),8.30(1H,d),8.64(1H,s).m / z:ES+[M+H]+654.

[0684] Synthesis Example 11 9-Benzyl-8-(2-chloro-4-(2-(piperazine-1-yl)ethoxy)phenyl)-6-((1,1,1-trifluoro-2-methylpropane-2-yl)oxy)-9H-purine

[0685] [ka]

[0686] tert-butyl 4-(2-(4-(9-benzyl-6-((1,1,1-trifluoro-2-methylpropane-2-yl)oxy)-9H-purine-8-yl)-3-chlorophenoxy)ethyl)piperazine-1-carboxylate (120 mg, 0.18 mmol) was added to trifluoroacetic acid (0.2 mL, 2.60 mmol) in dichloromethane (2 mL). The resulting solution was stirred at room temperature for 2 hours. The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography with an elution gradient of 50-100% MeOH in water (0.1% NH4HCO3), and subsequently purified by preparative HPLC (XBridge Prep OBD C18 column, 30 × 150 mm, 5 μm) using water (containing 10 mmol / L NH4HCO3) and a mixture that reduces the polarity of MeCN as the eluent. The fraction containing the desired compound was evaporated to dryness to obtain 9-benzyl-8-(2-chloro-4-(2-(piperazin-1-yl)ethoxy)phenyl)-6-((1,1,1-trifluoro-2-methylpropan-2-yl)oxy)-9H-purine (30.0 mg, 29%) as a white solid. 1 ¹H NMR (300MHz, DMSO-d6): 1.95 (6H,s), 2.3-2.45 (4H,m), 2.58-2.79 (6H,m), 4.18 (2H,t), 5.30 (2H,s), 6.91 (2H,dd), 7.04 (1H,dd), 7.15-7.23 (3H,m), 7.27 (1H,d), 7.43 (1H,d), 8.61 (1H,s) - 1H was not observed. m / z: ES+[M+H]+575.

[0687] The starting material used, tert-butyl4-(2-(4-(9-benzyl-6-((1,1,1-trifluoro-2-methylpropane-2-yl)oxy)-9H-purine-8-yl)-3-chlorophenoxy)ethyl)piperazine-1-carboxylate, was prepared as follows.

[0688] N-benzyl-5-nitro-6-((1,1,1-trifluoro-2-methylpropane-2-yl)oxy)pyrimidine-4-amine

[0689] [ka]

[0690] LHMDS (11.3 mL, 11.3 mmol) was added at 0°C to N-benzyl-6-chloro-5-nitropyrimidine-4-amine (1 g, 3.78 mmol) and 1,1,1-trifluoro-2-methylpropan-2-ol (1.24 mL, 11.3 mmol) in THF (20 mL). The resulting mixture was stirred at 60°C for 12 hours. The reaction mixture was diluted with HCl (100 mL) and sequentially washed with saturated brine (3 × 100 mL). The organic layer was dried over Na₂SO₄, filtered, and evaporated to obtain the crude product. The crude product was purified by flash silica chromatography with an elution gradient of 0–5% HCl in petroleum ether. The fraction was evaporated to dryness to obtain N-benzyl-5-nitro-6-((1,1,1-trifluoro-2-methylpropan-2-yl)oxy)pyrimidine-4-amine (800 mg, 59%) as a pale yellow oily substance. 1 H NMR(300MHz,DMSO-d6):1.81(6H,s),4.69(2H,d),7.07-7.47(5H,m),8.31(1H,s),8.90(1H,t).m / z:ES+[M+H]+357.

[0691] N4-benzyl-6-((1,1,1-trifluoro-2-methylpropan-2-yl)oxy)pyrimidine-4,5-diamine

[0692] [ka]

[0693] Iron (549 mg, 9.82 mmol) was added to a mixture of N-benzyl-5-nitro-6-((1,1,1-trifluoro-2-methylpropan-2-yl)oxy)pyrimidine-4-amine (700 mg, 1.96 mmol) and ammonium chloride (1.05 g, 19.6 mmol) in ethanol (15 mL). The resulting mixture was stirred at 80°C for 4 hours. The solvent was then removed under reduced pressure, and the crude product was purified by flash silica chromatography with an elution gradient of 0-30% siRNA in petroleum ether. The pure fraction was evaporated to dryness to obtain N4-benzyl-6-((1,1,1-trifluoro-2-methylpropan-2-yl)oxy)pyrimidine-4,5-diamine (400 mg, 63%) as a white solid. 1 H NMR(300MHz,DMSO-d6):1.72(6H,s),4.16(2H,s),4.61(2H,d),6.95(1H,t),7.11-7.46(5H,m),7.72(1H,s).m / z:ES+[M+H]+327.

[0694] tert-butyl4-(2-(4-(9-benzyl-6-((1,1,1-trifluoro-2-methylpropane-2-yl)oxy)-9H-purine-8-yl)-3-chlorophenoxy)ethyl)piperazine-1-carboxylate

[0695] [ka]

[0696] Iron(III) chloride (49.7 mg, 0.31 mmol) was added to N4-benzyl-6-((1,1,1-trifluoro-2-methylpropane-2-yl)oxy)pyrimidine-4,5-diamine (100 mg, 0.31 mmol), tert-butyl 4-(2-(3-chloro-4-formylphenoxy)ethyl)piperazine-1-carboxylate (124 mg, 0.34 mmol), and AcOH (1.7 μL, 0.03 mmol) in IPA (2 mL). The resulting mixture was stirred at 80°C for 4 hours. The solvent was then removed under reduced pressure, and the crude residue was purified by C18-flash chromatography using a 40-90% MeOH elution gradient in water (0.1% NH4HCO3). The pure fraction was evaporated to dryness to obtain tert-butyl 4-(2-(4-(9-benzyl-6-((1,1,1-trifluoro-2-methylpropane-2-yl)oxy)-9H-purine-8-yl)-3-chlorophenoxy)ethyl)piperazine-1-carboxylate (140 mg, 68%) as a white foam. 1 H NMR(300MHz,DMSO-d6):1.38(9H,s),1.94(6H,s),2.43(4H,t),2.72(2H,t),3.24-3.32(4H,m),4.19(2H,t),5.29( 2H,s),6.90(2H,dd),7.03(1H,dd),7.12-7.24(3H,m),7.26(1H,d),7.43(1H,d),8.60(1H,s).m / z:ES+[M+H]+675.

[0697] Synthesis Example 12 9-Benzyl-8-(2-chloro-4-(2-(piperazin-1-yl)ethoxy)phenyl)-6-(1-methylcyclobutoxy)-9H-purine

[0698] [ka]

[0699] Pd(PPh3)4 (31.0 mg, 0.03 mmol) was added under nitrogen to 1,4-dioxane (2 mL) and water (0.4 mL) containing 9-benzyl-8-bromo-6-(1-methylcyclobutoxy)-9H-purine (100 mg, 0.27 mmol), 1-(2-(3-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)ethyl)piperazine (196 mg, 0.54 mmol) and Cs2CO3 (262 mg, 0.80 mmol). The resulting solution was stirred at 100°C for 1 hour. The solvent was removed under reduced pressure. The crude product was purified by flash C18-flash chromatography with an elution gradient of 50-100% MeOH in water (0.1% NH4HCO3). Subsequently, it was purified by preparative HPLC (Phenomenex Gemini-NX axia Prep C18 OBD column, 5 μm silica, 19 mm diameter, 100 mm length) using water (containing 0.1% NH3) and a mixture that reduced the polarity of MeCN as the eluent. The fraction containing the desired compound was evaporated to dryness to obtain 9-benzyl-8-(2-chloro-4-(2-(piperazin-1-yl)ethoxy)phenyl)-6-(1-methylcyclobutoxy)-9H-purine (16 mg, 11%) as a white solid. 1 ¹H NMR (300MHz, DMSO-d6): 1.62-1.96 (5H,m), 2.26-2.49 (8H,m), 2.61-2.78 (6H,m), 4.18 (2H,t), 5.28 (2H,s), 6.85-6.95 (2H,m), 6.98-7.11 (1H,m), 7.15-7.25 (3H,m), 7.26 (1H,d), 7.41 (1H,d), 8.51 (1H,s). No H atoms were observed. m / z: ES+[M+H]+533.

[0700] The starting material used, 1-(2-(3-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)ethyl)piperazine, was prepared as follows.

[0701] tert-butyl 4-(2-(4-bromo-3-chlorophenoxy)ethyl)piperazine-1-carboxylate

[0702] [ka]

[0703] Potassium carbonate (6.66 g, 48.2 mmol) was added to 4-bromo-3-chlorophenol (5 g, 24.1 mmol) and tert-butyl 4-(2-chloroethyl)piperazine-1-carboxylate (7.19 g, 28.92 mmol) in DMF (100 mL) under nitrogen at 25 °C. The resulting suspension was stirred at 80 °C for 3 hours. The reaction mixture was diluted with water (300 mL), and the aqueous layer was extracted with ethyl acetate (3 × 100 mL). The organic layer was dried over Na₂SO₄, filtered, and evaporated to obtain the crude product. The crude product was purified by crystallization from ethyl acetate / petroleum ether to obtain tert-butyl 4-(2-(4-bromo-3-chlorophenoxy)ethyl)piperazine-1-carboxylate (5.50 g, 54%) as a white solid. 1 H NMR(300MHz,CDCl3):1.46(9H,s),2.51(4H,s),2.81(2H,t),3.45(4H,t),4.07(2H,t),6.70(1H,dd),7.02(1H,d),7.47(1H,d).m / z:ES+[M+H]+419。

[0704] tert-butyl 4-(2-(3-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)ethyl)piperazine-1-carboxylate

[0705] [ka]

[0706] PdCl2(dppf) (0.523 g, 0.71 mmol) was added under nitrogen to tert-butyl 4-(2-(4-bromo-3-chlorophenoxy)ethyl)piperazine-1-carboxylate (3 g, 7.15 mmol), bis(pinacorato)diboron (3.63 g, 14.29 mmol), and potassium acetate (2.10 g, 21.4 mmol) in 1,4-dioxane (60 mL). The resulting solution was stirred at 100 °C for 2 hours. The reaction mixture was diluted with ethyl acetate (250 mL) and sequentially washed with water (3 × 250 mL) and saturated brine (3 × 250 mL). The organic layer was dried over Na₂SO₄, filtered, and evaporated to obtain the crude product. The crude product was purified by flash silica chromatography with an elution gradient of 0–30% ethyl acetate in petroleum ether. The pure fraction was evaporated to dryness to obtain tert-butyl 4-(2-(3-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)ethyl)piperazine-1-carboxylate (3.00 g, 90%) as a pale yellow gum-like substance. 1 H NMR(300MHz,DMSO-d6):1.16(9H,s),1.27(12H,s),2.42(4H,m),2.58(2H,t),3.17-3 .20(4H,m),4.01(2H,t),6.79(1H,dd),6.88(1H,d),7.46(1H,d).m / z:ES+[M+H]+467.

[0707] 1-(2-(3-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)ethyl)piperazine

[0708] [ka]

[0709] Trifluoroacetic acid (20 mL) was added to tert-butyl 4-(2-(3-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)ethyl)piperazine-1-carboxylate (2 g, 4.28 mmol) in dichloromethane (20 mL). The resulting solution was stirred at 25°C for 2 hours, and then the solvent was removed under reduced pressure. The crude product was purified by preparative HPLC (XBridge Prep C18 OBD column, 30 × 150 mm, 5 μm) using water (containing 10 mmol / L NH4HCO3 and 0.1% aqueous ammonia) and a mixture that reduced the polarity of MeCN as the eluent. The fraction containing the desired compound was evaporated to dryness to obtain 1-(2-(3-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenoxy)ethyl)piperazine (1.05 g, 67%) as a white solid. 1 1H NMR (300MHz, DMSO-d6): 1.29 (12H,s), 3.20 (4H,t), 3.30 (6H,t), 4.30 (2H,t), 6.95 (1H,dd), 7.05 (1H,d), 7.62 (1H,d) - 1 proton was not observed. m / z: ES+[M+H]+367.

[0710] The 9-benzyl-8-bromo-6-(1-methylcyclobutoxy)-9H-purine used as a starting material was prepared as follows.

[0711] 9-benzyl-6-(1-methylcyclo...

Claims

1. A method for inserting a target polynucleotide into the genome of a eukaryotic cell, wherein the method is a. Adding an inhibitor of the microhomology-mediated end-joining (MMEJ) pathway to the composition containing the eukaryotic cells. b. Adding Cas effector protein to the composition, c. Adding the aforementioned polynucleotide to the composition, The aforementioned polynucleotide is inserted into the genome by homologous recombination repair (HDR) or single-strand template repair (SSTR). The inhibitor of the MMEJ pathway is a compound of formula (I): 【Chemistry 1】 Or an inhibitor of PolQ selected from any stereoisomer thereof or a pharmaceutically acceptable salt thereof, During the ceremony, R １ and R ２ are each independently H, halo, C １ to C ３ alkyl, C １ to C ３ alkoxy, C １ to C ３ haloalkyl, C １ to C ３ hydroxyalkyl, -CN, C ２ to C ４ alkyne, or C ２ to C ６ alkoxyalkyl, and Q １ Q ２ , and Q ３ However, independently, N, C-L-R, or CR ｘ Q １ Q ２ , and Q ３ One or fewer of these are C-L-R, L is a bond, -O-, -C(O)-, -O(CH ２ ), ｐ C(O)-, -C(O)NR ｙ -, -O(CH ２ ), ｐ C(O)NR ｙ -, -O(CH ２ ), ｐ NR ｙ -, -NR ｙ -, -(CH ２ ), ｐ～ -, -(CH ２ ), ｐ NR ｙ -, -(CH ２ ), ｐ O-, -(CH ２ ), ｐ C(O)-, -(CH ２ ), ｐ C(O)O-, -O(CH ２ ), ｐ -, and p is independently 1, 2, or 3. R is H, R ａ , R ｂ , R ｃ , or R ｄ And, R ａ However, amino, carboxy, halo, hydroxy, oxo, -CN, -S(O) ２ OH, C1-C4 alkylamino, C1-C5 alkoxy, C ２ ~C ５ Alkoxyalkyl groups, 4-6 membered heterocycles, and C １ ~C ７ A 3- to 10-membered heterocycle optionally substituted with 1 to 4 substituents selected from alkyl groups, wherein the C1-C7 alkyl group is amino, carboxy, halo, hydroxy, oxo, -CN, C ２ ~C ８ Esters, and C １ ~C ５ It is optionally substituted with 1 to 4 substituents selected from alkoxys. R ｂ However, C １ ~C ７ Alkyl, and the C １ ~C ７ One or two methylene groups from alkyl are NR ｅ Alternatively, it is optionally replaced with O, and C ２ ~C ７ One or two single bonds in the alkyl chain are optionally replaced by double or triple bonds, and the C １ ~C ７ Alkyl is a compound of halo, oxo, hydroxy, carboxyl, amino, -CN, and C. ２ ~C ４ Alkinyl, C ２ ~C ６ Carbamate, C １ ~C ８ Amido, C １ ~C ４ Sulfonyl, C １ ~C ４ Sulfonamide, C １ ~C ４ Alkylamino, C １ ~C ５ Alkoxy, C ３ ~C ６ It is optionally substituted with 1 to 4 substituents selected from carbon rings and 3 to 10-membered heterocycles. Said C ３ ~C ６ The carbon ring is optionally substituted with 1 to 4 substituents selected from hydroxyl, halo, and carboxyl groups. The 3- to 10-membered heterocyclic ring is optionally substituted with 1 to 4 substituents selected from amino, carboxy, halo, hydroxy, oxo, -CN, -S(O) ２ OH, C １ ~C ４ alkylamino, C １ ~C ５ alkoxy, C ２ ~C ５ alkoxyalkyl, 4- to 6-membered heterocyclic ring, and C １ ~C ７ alkyl, and the C １ ~C ７ alkyl is optionally substituted with 1 to 4 substituents selected from amino, carboxy, halo, hydroxy, oxo, -CN, C ２ ~C ８ ester, and C １ ~C ５ alkoxy, R ｃ However, C is optionally substituted with 1 to 4 substituents selected from hydroxyl, halo, and carboxyl. ３ ~C ６ It is a carbon ring, R ｄ However, C １ ~C ４ Sulfonyl or C １ ~C ４ It is a sulfonamide, R ｙ However, H, C １ ~C ３ Alkyl or C1-3 haloalkyl, R ｘ However, H, halo, hydroxy, -CN, -NH ２ , C １ ~C ３ Alkoxy, C １ ~C ３ Alkyl, or C １ - ３ It is a haloalkyl, R ｅ However, H, Haro, C １ ~C ８ Alkyl, or C １ ~C ８ It is a haloalkyl, X is C １ ~C ４ It is alkylene, Y is phenyl or a 5-6 member heteroaryl, and the phenyl or heteroaryl is a halo, C １ ~C ３ Alkyl, C １ ~C ３ Alkoxy, -CN, C １ ~C ３ It is optionally substituted with 1 to 3 substituents selected from haloalkyl and cyclopropyl groups. G is either N or CH, G ａ and G ｂ However, N, CH, or CR ５ G ａ and G ｂ Only one of them is N or CH, and G ａ and G ｂ Only one of them is CR ５ And, R ５ but, 【Chemistry 2】 And, Z ａ and Z ｂ However, independently, C １ ~C ３ Alkyl or C １ ~C ３ It is a haloalkyl or Z ａ and Z ｂ However, it forms a 3-6 membered carbon ring or heteroring, Z ｃ However, H, -CN, C １ ~C ３ Alkyl, C １ ~C ３ Haloalkyl, or C ２ ~C ４ Alkynes, or combinations thereof, or methods.

2. The method according to claim 1, further comprising (a) adding an inhibitor of the non-homologous end-joining (NHEJ) pathway.

3. (d) The method according to claim 1 or 2, further comprising adding a polynucleotide comprising an RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof to the composition.

4. The method according to any one of claims 1 to 3, wherein the Cas effector protein is added in (b) by adding a Cas polynucleotide encoding the Cas effector protein.

5. The method according to any one of claims 1 to 4, wherein one or more of the following are encoded on the vector: (i) the target polynucleotide, (ii) the polynucleotide of (d), or (iii) the Cas polynucleotide.

6. The method according to any one of claims 1 to 4, wherein (i) the target polynucleotide, (ii) the polynucleotide of step (d), and (iii) the Cas polynucleotide are encoded on a single vector.

7. The method according to any one of claims 1 to 6, wherein the aforementioned polynucleotide is added as DNA.

8. The method according to any one of claims 1 to 6, wherein the polynucleotide of step (d) is added as DNA.

9. The method according to any one of claims 1 to 6, wherein the polynucleotide in step (d) is added as RNA.

10. The method according to any one of claims 1 to 6, wherein a Cas effector polynucleotide is added as DNA.

11. The method according to any one of claims 1 to 6, wherein the Cas polynucleotide is added as RNA.

12. The method according to any one of claims 1 to 6, wherein the Cas polynucleotide is added as mRNA.

13. The method according to claim 5 or 6, wherein the vector is a viral vector.

14. The method according to claim 13, wherein the viral vector is a retrovirus, lentivirus, adenovirus, or adeno-associated virus (AAV).

15. The method according to claim 3, wherein the Cas effector protein and the polynucleotide of (d) are added in the form of ribonucleoprotein (RNP).

16. The method according to any one of claims 1 to 15, wherein the Cas effector protein, the target polynucleotide, and the polynucleotide of (d) are added to the cells by microinjection, electroporation, or via lipid nanoparticles, liposomes, exosomes, gold nanoparticles, or DNA nanoclues.

17. The method according to claim 5 or 9, wherein the vector is added to the composition by transfecting the eukaryotic cells.

18. The method according to any one of claims 1 to 17, wherein the Cas effector protein is Cas9 nuclease, Cas12a nuclease, or Cas12f nuclease.

19. The method according to claim 18, wherein the Cas effector protein is Cas9 nuclease.

20. The method according to claim 19, wherein the Cas9 nuclease is a Cas9 nuclease fused to reverse transcriptase, a Cas9 nuclease fused to DNA polymerase, a Cas9 nuclease fused to DN1S, a Cas9 nicasse, a Cas9 fused to a geminin deglon domain, or a Cas9 nuclease fused to CTIP.

21. The method according to any one of claims 1 to 20, wherein the polynucleotide of the objective is added via a vector.

22. The method according to claim 21, wherein the vector is a viral vector.

23. The method according to claim 22, wherein the viral vector is a retrovirus, lentivirus, adenovirus, or adeno-associated virus (AAV).

24. The method according to any one of claims 1 to 23, wherein the target polynucleotide comprises the target gene.

25. The method according to any one of claims 1 to 23, wherein the target polynucleotide has a length of 1 to 50 base pairs.

26. The method according to any one of claims 1 to 23, wherein the target polynucleotide has a length of 50 to 5000 base pairs.

27. The method according to any one of claims 1 to 23, wherein the polynucleotide for the purpose is single-stranded.

28. The method according to any one of claims 1 to 23, wherein the polynucleotide for the purpose is double-stranded.

29. The method according to any one of claims 1 to 23, wherein the target polynucleotide is a hybrid polynucleotide comprising a single-stranded region and a double-stranded region.

30. The method according to claim 29, wherein the hybrid polynucleotide comprises a double-stranded sequence at the 5' end and the 3' end, and an internal single-stranded sequence.

31. The method according to any one of claims 1 to 28, wherein the target polynucleotide is a double-stranded molecule having blunt ends.

32. The method according to any one of claims 1 to 30, wherein the target polynucleotide is a double-stranded molecule having a 3' overhang.

33. The method according to any one of claims 1 to 30, wherein the target polynucleotide is a double-stranded molecule having a 5' overhang.

34. The method according to any one of claims 1 to 29, wherein the target polynucleotide is a cyclic polynucleotide.

35. The method according to any one of claims 1 to 34, wherein the target polynucleotide comprises a chemical modification that enhances the stability, activity, distribution, or uptake of the polynucleotide.

36. The inhibitor of PolQ is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein Q １ Q ２ , or Q ３ However, C-L-R ｂ And R ｂ The method according to any one of claims 1 to 35, wherein the heterocycle is substituted with a 3- to 10-membered heterocycle, for example, an N-heterocycle (not limited to), for example, a 4- to 7-membered N-heterocycle (not limited to), and the heterocycle is substituted with a C1- to C7 alkyl, oxo, and / or halo, or a combination thereof.

37. The method according to claim 36, wherein the inhibitor of PolQ is one of the compounds listed in Tables I, II, and III, a pharmaceutically acceptable salt thereof, or a combination thereof.

38. The method according to claim 1, wherein the PolQ inhibitor is 9-benzyl-8-(2-chloro-4-(2-(4-methylpiperazine-1-yl)ethoxy)phenyl)-6-(1-methylcyclopropoxy)-9H-purine (compound 1), or a pharmaceutically acceptable salt thereof.

39. The method according to any one of claims 1 to 38, wherein the concentration of the inhibitor of the MMEJ pathway in the composition is about 0.01 mM to about 1 mM.

40. The method according to any one of claims 1 to 38, wherein the concentration of the inhibitor of the MMEJ pathway in the composition is about 0.1 mM to about 100 mM.

41. The method according to any one of claims 2 to 38, wherein the inhibitor of the NHEJ pathway is an inhibitor of DNA-dependent protein kinase (DNA-PK).

42. The method according to claim 41, wherein the DNA-PK inhibitor is M3814, M9831 / VX984, Nu7441, Nu7026, KU0060648, AZD7648, or a combination thereof.

43. The method according to claim 42, wherein the DNA-PK inhibitor is AZD7648.

44. The method according to claim 41, wherein the DNA-PK inhibitor is a peptide.

45. The method according to any one of claims 2 to 44, wherein the concentration of the inhibitor of the NHEJ pathway in the composition is about 0.01 μM to about 1 mM.

46. The method according to any one of claims 2 to 44, wherein the concentration of the inhibitor of the NHEJ pathway in the composition is about 0.1 mM to about 100 mM.

47. The method according to any one of claims 1 to 46, wherein the inhibitor of the MMEJ pathway is added to the composition from 0 minutes to about 48 hours before the Cas effector protein is added to the composition.

48. The method according to any one of claims 1 to 46, wherein the inhibitor of the MMEJ pathway is added to the composition from 0 minutes to about 24 hours before the Cas effector protein is added to the composition.

49. The method according to any one of claims 1 to 46, wherein the inhibitor of the MMEJ pathway is added to the composition 0 minutes to about 6 hours before the Cas effector protein is added to the composition.

50. The method according to any one of claims 1 to 46, wherein the inhibitor of the MMEJ pathway is added to the composition 0 minutes to about 1 hour after the Cas effector protein is added to the composition.

51. The method according to any one of claims 2 to 50, wherein the inhibitor of the NHEJ pathway is added to the composition from 0 minutes to about 48 hours before the Cas effector protein is added to the composition.

52. The method according to any one of claims 2 to 50, wherein the inhibitor of the NHEJ pathway is added to the composition from 0 minutes to about 24 hours before the Cas effector protein is added to the composition.

53. The method according to any one of claims 2 to 50, wherein the inhibitor of the NHEJ pathway is added to the composition 0 minutes to about 6 hours before the Cas effector protein is added to the composition.

54. The method according to any one of claims 2 to 50, wherein the inhibitor of the NHEJ pathway is added to the composition 0 minutes to about 1 hour after the Cas effector protein is added to the composition.

55. The method according to any one of claims 2 to 54, wherein the inhibitor of the MMEJ pathway and the inhibitor of the NHEJ pathway are added simultaneously to the composition.

56. The method according to any one of claims 2 to 54, wherein the inhibitor of the MMEJ pathway and the inhibitor of the NHEJ pathway are added to the composition at different time points.

57. The method according to any one of claims 2 to 54, wherein the inhibitor of the MMEJ pathway, the inhibitor of the NHEJ pathway, and the Cas effector protein are added simultaneously to the composition.

58. The method according to any one of claims 1 to 57, wherein the inhibitor of the MMEJ pathway is present in the composition for about 1 to about 300 hours.

59. The method according to any one of claims 1 to 57, wherein the inhibitor of the MMEJ pathway is present in the composition for about 10 to about 100 hours.

60. The method according to any one of claims 1 to 57, wherein the inhibitor of the MMEJ pathway is added at least once, at least twice, or at least three times.

61. The method according to any one of claims 2 to 60, wherein the inhibitor of the NHEJ pathway is present in the composition for about 1 to about 300 hours.

62. The method according to any one of claims 2 to 60, wherein the inhibitor of the NHEJ pathway is present in the composition for about 10 to about 100 hours.

63. The method according to any one of claims 2 to 60, wherein the inhibitor of the NHEJ pathway is added at least once, at least twice, or at least three times.

64. The method according to any one of claims 1 to 63, wherein the composition containing the eukaryotic cells is a cell culture.

65. The method according to claim 64, wherein the cell culture is an in vitro cell culture or an ex vivo cell culture.

66. The method according to any one of claims 1 to 65, wherein the eukaryotic cells are in vivo.

67. The method according to claim 64, wherein the cell culture comprises a cell extract.

68. The method according to any one of claims 1 to 67, wherein the eukaryotic cell is a lymphocyte.

69. The method according to claim 68, wherein the lymphocyte comprises a chimeric antigen receptor (CAR) or a T cell receptor (TCR).

70. The method according to any one of claims 1 to 67, wherein the eukaryotic cell is a pluripotent stem cell.

71. The method according to claim 70, wherein the pluripotent stem cells are induced pluripotent stem cells.

72. The method according to claim 64, wherein the cell culture is a mammalian cell culture.

73. A method for inserting a target polynucleotide into the genome of a eukaryotic cell, wherein the method is a. Adding an inhibitor of the microhomology-mediated end-joining (MMEJ) pathway to the composition containing the eukaryotic cells. b. Adding the aforementioned polynucleotide to the composition, The genome comprises a genomically integrated Cas polynucleotide, and the target polynucleotide is inserted into the genome by homologous recombination repair (HDR) or single-strand template repair (SSTR). The inhibitor of the MMEJ pathway is a compound of formula (I): 【Transformation 3】 Or an inhibitor of PolQ selected from any stereoisomer thereof or a pharmaceutically acceptable salt thereof, During the ceremony, R １ and R ２ However, each is independent of H, Haro, and C. １ ~C ３ Alkyl, C １ ~C ３ Alkoxy, C １ ~C ３ Haloalkyl, C １ ~C ３ Hydroxyalkyl, -CN, C ２ ~C ４ Alkynes, or C ２ ~C ６ It is an alkoxyalkyl, Q １ Q ２ , and Q ３ However, independently, N, C-L-R, or CR ｘ Q １ Q ２ , and Q ３ One or fewer of these are C-L-R, L is selected from the group consisting of a bond, -O-, -C(O)-, -O(CH ２ ), ｐ C(O)-, -C(O)NR ｙ -, -O(CH ２ ), ｐ C(O)NR ｙ -, -O(CH ２ ), ｐ NR ｙ -, -NR ｙ -, -(CH ２ ), ｐ～ -(CH ２ ), ｐ NR ｙ -, -(CH ２ ), ｐ O-, -(CH ２ ), ｐ C(O)-, -(CH ２ ), ｐ C(O)O-, -O(CH ２ ), ｐ -; p is independently 1, 2, or 3. R is H, R ａ , R ｂ , R ｃ , or R ｄ And, R ａ However, amino, carboxy, halo, hydroxy, oxo, -CN, -S(O) ２ OH, C1-C4 alkylamino, C1-C5 alkoxy, C ２ ~C ５ Alkoxyalkyl groups, 4-6 membered heterocycles, and C １ ~C ７ A 3- to 10-membered heterocycle optionally substituted with 1 to 4 substituents selected from alkyl groups, wherein the C1-C7 alkyl group is amino, carboxy, halo, hydroxy, oxo, -CN, C ２ ~C ８ Esters, and C １ ~C ５ It is optionally substituted with 1 to 4 substituents selected from alkoxys. R ｂ However, C １ ~C ７ Alkyl, and the C １ ~C ７ One or two methylene groups from alkyl are NR ｅ Alternatively, it is optionally replaced with O, and C ２ ~C ７ One or two single bonds in the alkyl chain are optionally replaced by double or triple bonds, and the C １ ~C ７ Alkyl is a compound of halo, oxo, hydroxy, carboxyl, amino, -CN, and C. ２ ~C ４ Alkinyl, C ２ ~C ６ Carbamate, C １ ~C ８ Amido, C １ ~C ４ Sulfonyl, C １ ~C ４ Sulfonamide, C １ ~C ４ Alkylamino, C １ ~C ５ Alkoxy, C ３ ~C ６ It is optionally substituted with 1 to 4 substituents selected from carbon rings and 3 to 10-membered heterocycles. Said C ３ ~C ６ The carbon ring is optionally substituted with 1 to 4 substituents selected from hydroxyl, halo, and carboxyl groups. The aforementioned 3- to 10-membered heterocycles are amino, carboxy, halo, hydroxy, oxo, -CN, -S(O) ２ OH, C １ ~C ４ Alkylamino, C １ ~C ５ Alkoxy, C ２ ~C ５ Alkoxyalkyl groups, 4-6 membered heterocycles, and C １ ~C ７ The C is optionally substituted with 1 to 4 substituents selected from alkyl groups. １ ~C ７ Alkyl is amino, carboxy, halo, hydroxy, oxo, -CN, C ２ ~C ８ Esters, and C １ ~C ５ It is optionally substituted with 1 to 4 substituents selected from alkoxys. R ｃ However, C is optionally substituted with 1 to 4 substituents selected from hydroxyl, halo, and carboxyl. ３ ~C ６ It is a carbon ring, R ｄ However, C １ ~C ４ Sulfonyl or C １ ~C ４ It is a sulfonamide, R ｙ However, H, C １ ~C ３ Alkyl or C1-3 haloalkyl, R ｘ However, H, halo, hydroxy, -CN, -NH ２ , C １ ~C ３ Alkoxy, C １ ~C ３ Alkyl, or C １ - ３ It is a haloalkyl, R ｅ However, H, Haro, C １ ~C ８ Alkyl, or C １ ~C ８ It is a haloalkyl, X is C １ ~C ４ It is alkylene, Y is phenyl or a 5-6 member heteroaryl, and the phenyl or heteroaryl is a halo, C １ ~C ３ Alkyl, C １ ~C ３ Alkoxy, -CN, C １ ~C ３ It is optionally substituted with 1 to 3 substituents selected from haloalkyl and cyclopropyl groups. G is either N or CH, G ａ and G ｂ However, N, CH, or CR ５ G ａ and G ｂ Only one of them is N or CH, and G ａ and G ｂ Only one of them is CR ５ And, R ５ but, 【Chemistry 4】 And, Z ａ and Z ｂ However, independently, C １ ~C ３ Alkyl or C １ ~C ３ It is a haloalkyl or Z ａ and Z ｂ However, it forms a 3-6 membered carbon ring or heteroring, Z ｃ However, H, -CN, C １ ~C ３ Alkyl, C １ ~C ３ Haloalkyl, or C ２ ~C ４ Alkynes, or combinations thereof, or methods.

74. The method according to claim 73, further comprising (a) adding an inhibitor of the non-homologous end-joining (NHEJ) pathway to the composition.

75. (c) The method according to claim 73 or 74, further comprising adding a polynucleotide comprising an RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof to the composition.

76. The method of claim 75, wherein (i) the target polynucleotide and (ii) the polynucleotide of (c) are encoded on a vector.

77. The method according to any one of claims 73 to 76, wherein the aforementioned polynucleotide is added as DNA.

78. The method according to any one of claims 75 to 77, wherein the polynucleotide in (c) is added as DNA.

79. The method according to any one of claims 75 to 77, wherein the polynucleotide in (c) is added as RNA.

80. The method according to claim 76, wherein the vector is a viral vector.

81. The method according to claim 80, wherein the viral vector is a retrovirus, lentivirus, adenovirus, or adeno-associated virus (AAV).

82. The method according to claim 76, wherein the vector is added to the composition by transfecting the eukaryotic cells.

83. The method according to any one of claims 73 to 82, wherein the genomically integrated Cas polynucleotide is inducible.

84. The method according to any one of claims 73 to 83, wherein the Cas effector protein is Cas9 nuclease, Cas12a nuclease, or Cas12f nuclease.

85. The method according to claim 84, wherein the Cas effector protein is Cas9 nuclease.

86. The method according to claim 85, wherein the Cas9 nuclease is a Cas9 nuclease fused to reverse transcriptase, a Cas9 nuclease fused to DNA polymerase, a Cas9 nuclease fused to DN1S, a Cas9 nicasse, a Cas9 fused to a geminin deglon domain, or a Cas9 nuclease fused to CTIP.

87. The method according to any one of claims 73 to 86, wherein the target polynucleotide is added via a vector.

88. The method according to claim 87, wherein the vector is a viral vector.

89. The method according to claim 88, wherein the viral vector is a retrovirus, lentivirus, adenovirus, or adeno-associated virus (AAV).

90. The method according to any one of claims 73 to 89, wherein the target polynucleotide comprises the target gene.

91. The method according to any one of claims 73 to 90, wherein the target polynucleotide is 1 to 50 base pairs long.

92. The method according to any one of claims 73 to 90, wherein the target polynucleotide has a length of 50 to 5000 base pairs.

93. The method according to any one of claims 73 to 90, wherein the polynucleotide for the purpose is single-stranded.

94. The method according to any one of claims 73 to 90, wherein the polynucleotide for the purpose is double-stranded.

95. The method according to any one of claims 73 to 90, wherein the target polynucleotide is a hybrid polynucleotide comprising a single-stranded region and a double-stranded region.

96. The method according to claim 95, wherein the hybrid polynucleotide comprises a double-stranded sequence at the 5' end and the 3' end, and an internal single-stranded sequence.

97. The method according to any one of claims 73 to 90, wherein the target polynucleotide is a double-stranded molecule having blunt ends.

98. The method according to any one of claims 73 to 90, wherein the target polynucleotide is a double-stranded molecule having a 3' overhang.

99. The method according to any one of claims 73 to 90, wherein the target polynucleotide is a double-stranded molecule having a 5' overhang.

100. The method according to any one of claims 73 to 90, wherein the polynucleotide is a cyclic polynucleotide.

101. The method according to any one of claims 73 to 100, wherein the polynucleotide comprises a chemical modification that enhances the stability, activity, distribution, or uptake of the polynucleotide.

102. The inhibitor of PolQ is a compound of formula (I), or any stereoisomer thereof, or a pharmaceutically acceptable salt thereof, wherein Q １ Q ２ , or Q ３ However, C-L-R ｂ And R ｂ The method according to any one of claims 73 to 101, wherein the heterocycle is substituted with a 3- to 10-membered heterocycle, for example, an N-heterocycle (not limited to), for example, a 4- to 7-membered N-heterocycle (not limited to), and the heterocycle is substituted with a C1- to C7 alkyl, oxo, and / or halo, or a combination thereof.

103. The method according to claim 102, wherein the inhibitor of PolQ is one of the compounds listed in Tables I, II, and III, a pharmaceutically acceptable salt thereof, or a combination thereof.

104. The method according to claim 102, wherein the PolQ inhibitor is 9-benzyl-8-(2-chloro-4-(2-(4-methylpiperazine-1-yl)ethoxy)phenyl)-6-(1-methylcyclopropoxy)-9H-purine (compound 1), or a salt thereof.

105. The method according to any one of claims 73 to 104, wherein the concentration of the inhibitor of the MMEJ pathway in the composition is about 0.01 mM to about 1 mM.

106. The method according to any one of claims 73 to 104, wherein the concentration of the inhibitor of the MMEJ pathway in the composition is about 0.1 mM to about 100 mM.

107. The method according to any one of claims 74 to 106, wherein the inhibitor of the NHEJ pathway is an inhibitor of DNA-dependent protein kinase (DNA-PK).

108. The method according to claim 107, wherein the DNA-PK inhibitor is M3814, M9831 / VX984, Nu7441, Nu7026, KU0060648, AZD7648, or a combination thereof.

109. The method according to claim 107, wherein the DNA-PK inhibitor is a peptide.

110. The method according to claim 109, wherein the DNA-PK inhibitor is AZD7648.

111. The method according to any one of claims 74 to 110, wherein the concentration of the inhibitor of the NHEJ pathway in the composition is about 0.01 μM to about 1 mM.

112. The method according to any one of claims 74 to 110, wherein the concentration of the inhibitor of the NHEJ pathway in the composition is about 0.1 mM to about 100 mM.

113. The method according to any one of claims 73 to 112, wherein the inhibitor of the MMEJ pathway is added to the composition 0 minutes to about 48 hours before the induction of the genomically integrated Cas polynucleotide.

114. The method according to any one of claims 73 to 112, wherein the inhibitor of the MMEJ pathway is added to the composition 0 minutes to about 24 hours before the induction of the genomically integrated Cas polynucleotide.

115. The method according to any one of claims 73 to 112, wherein the inhibitor of the MMEJ pathway is added to the composition 0 minutes to about 6 hours before the induction of the genomically integrated Cas polynucleotide.

116. The method according to any one of claims 73 to 115, wherein the inhibitor of the NHEJ pathway is added to the composition 0 minutes to about 24 hours before the induction of the genomically integrated Cas polynucleotide.

117. The method according to any one of claims 74 to 115, wherein the inhibitor of the NHEJ pathway is added to the composition 0 minutes to about 24 hours before the induction of the genomically integrated Cas polynucleotide.

118. The method according to any one of claims 74 to 115, wherein the inhibitor of the NHEJ pathway is added to the composition 0 minutes to about 6 hours before the induction of the genomically integrated Cas polynucleotide.

119. The method according to any one of claims 74 to 118, wherein the inhibitor of the MMEJ pathway and the inhibitor of the NHEJ pathway are added simultaneously to the composition.

120. The method according to any one of claims 74 to 118, wherein the inhibitor of the MMEJ pathway and the inhibitor of the NHEJ pathway are added to the composition at different time points.

121. The method according to any one of claims 74 to 120, wherein the inhibitor of the MMEJ pathway and the inhibitor of the NHEJ pathway are added to the composition at the same time as the induction of the genomically integrated Cas polynucleotide.

122. The method according to any one of claims 73 to 121, wherein the inhibitor of the MMEJ pathway is present in the composition for about 1 to about 300 hours.

123. The method according to any one of claims 73 to 121, wherein the inhibitor of the MMEJ pathway is present in the composition for about 10 to about 100 hours.

124. The method according to any one of claims 73 to 123, wherein the inhibitor of the MMEJ pathway is added at least once, at least twice, or at least three times.

125. The method according to any one of claims 74 to 124, wherein the inhibitor of the NHEJ pathway is present in the composition for about 1 to about 300 hours.

126. The method according to any one of claims 74 to 124, wherein the inhibitor of the NHEJ pathway is present in the composition for about 10 to about 100 hours.

127. The method according to any one of claims 74 to 126, wherein the inhibitor of the NHEJ pathway is added at least once, at least twice, or at least three times.

128. The method according to any one of claims 73 to 127, wherein the composition containing the eukaryotic cells is a cell culture.

129. The method according to claim 128, wherein the cell culture is an in vitro cell culture or an ex vivo cell culture.

130. The method according to any one of claims 73 to 129, wherein the eukaryotic cells are in vivo.

131. The method according to claim 130, wherein the cell culture comprises a cell extract.

132. The method according to any one of claims 73 to 131, wherein the eukaryotic cell is a lymphocyte.

133. The method according to claim 132, wherein the lymphocyte comprises a chimeric antigen receptor or a T cell receptor (TCR).

134. The method according to any one of claims 73 to 131, wherein the eukaryotic cell is a pluripotent stem cell.

135. The method according to claim 134, wherein the pluripotent stem cells are induced pluripotent stem cells.

136. The method according to claim 131, wherein the cell culture is a mammalian cell culture.

137. A method for inserting polynucleotides into the genome of a eukaryotic cell, wherein the method is a. Adding an inhibitor of the microhomology-mediated end-joining (MMEJ) pathway to the composition containing the eukaryotic cells. b. The eukaryotic cells i. A vector encoding a Cas effector protein, ii. A vector containing the target polynucleotide, iii. Transfecting with a vector containing a polynucleotide including an RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof, The vectors (i), (ii), and (iii) may be on the same vector or on different vectors, and the target polynucleotide is inserted into the genome by homologous recombination repair (HDR) or single-strand template repair (SSTR). The inhibitor of the MMEJ pathway is a compound of formula (I): 【Transformation 5】 Or an inhibitor of PolQ selected from any stereoisomer thereof or a pharmaceutically acceptable salt thereof, During the ceremony, R １ and R ２ However, each is independent of H, Haro, and C. １ ~C ３ Alkyl, C １ ~C ３ Alkoxy, C １ ~C ３ Haloalkyl, C １ ~C ３ Hydroxyalkyl, -CN, C ２ ~C ４ Alkynes, or C ２ ~C ６ It is an alkoxyalkyl, Q １ Q ２ , and Q ３ However, independently, N, C-L-R, or CR ｘ Q １ Q ２ , and Q ３ One or fewer of these are C-L-R, L is selected from the group consisting of a bond, -O-, -C(O)-, -O(CH ２ ), ｐ C(O)-, -C(O)NR ｙ -, -O(CH ２ ), ｐ C(O)NR ｙ -, -O(CH ２ ), ｐ NR ｙ -, -NR ｙ -, -(CH ２ ), ｐ～ -(CH ２ ), ｐ NR ｙ -, -(CH ２ ), ｐ O-, -(CH ２ ), ｐ C(O)-, -(CH ２ ), ｐ C(O)O-, -O(CH ２ ), ｐ -; and p is independently 1, 2, or 3. R is H, R ａ , R ｂ , R ｃ , or R ｄ And, R ａ However, amino, carboxy, halo, hydroxy, oxo, -CN, -S(O) ２ OH, C1-C4 alkylamino, C1-C5 alkoxy, C ２ ~C ５ Alkoxyalkyl groups, 4-6 membered heterocycles, and C １ ~C ７ A 3- to 10-membered heterocycle optionally substituted with 1 to 4 substituents selected from alkyl groups, wherein the C1-C7 alkyl group is amino, carboxy, halo, hydroxy, oxo, -CN, C ２ ~C ８ Esters, and C １ ~C ５ It is optionally substituted with 1 to 4 substituents selected from alkoxys. R ｂ However, C １ ~C ７ Alkyl, and the C １ ~C ７ One or two methylene groups from alkyl are NR ｅ Alternatively, it is optionally replaced with O, and C ２ ~C ７ One or two single bonds in the alkyl chain are optionally replaced by double or triple bonds, and the C １ ~C ７ Alkyl is a compound of halo, oxo, hydroxy, carboxyl, amino, -CN, and C. ２ ~C ４ Alkinyl, C ２ ~C ６ Carbamate, C １ ~C ８ Amido, C １ ~C ４ Sulfonyl, C １ ~C ４ Sulfonamide, C １ ~C ４ Alkylamino, C １ ~C ５ Alkoxy, C ３ ~C ６ It is optionally substituted with 1 to 4 substituents selected from carbon rings and 3 to 10-membered heterocycles. Said C ３ ~C ６ The carbon ring is optionally substituted with 1 to 4 substituents selected from hydroxyl, halo, and carboxyl groups. The aforementioned 3- to 10-membered heterocycles are amino, carboxy, halo, hydroxy, oxo, -CN, -S(O) ２ OH, C １ ~C ４ Alkylamino, C １ ~C ５ Alkoxy, C ２ ~C ５ Alkoxyalkyl groups, 4-6 membered heterocycles, and C １ ~C ７ The C is optionally substituted with 1 to 4 substituents selected from alkyl groups. １ ~C ７ Alkyl is amino, carboxy, halo, hydroxy, oxo, -CN, C ２ ~C ８ Esters, and C １ ~C ５ It is optionally substituted with 1 to 4 substituents selected from alkoxys. R ｃ However, C is optionally substituted with 1 to 4 substituents selected from hydroxyl, halo, and carboxyl. ３ ~C ６ It is a carbon ring, R ｄ However, C １ ~C ４ Sulfonyl or C １ ~C ４ It is a sulfonamide, R ｙ However, H, C １ ~C ３ Alkyl or C1-3 haloalkyl, R ｘ However, H, halo, hydroxy, -CN, -NH ２ , C １ ~C ３ Alkoxy, C １ ~C ３ Alkyl, or C １ - ３ It is a haloalkyl, R ｅ However, H, Haro, C １ ~C ８ Alkyl, or C １ ~C ８ It is a haloalkyl, X is C １ ~C ４ It is alkylene, Y is phenyl or a 5-6 member heteroaryl, and the phenyl or heteroaryl is a halo, C １ ~C ３ Alkyl, C １ ~C ３ Alkoxy, -CN, C １ ~C ３ It is optionally substituted with 1 to 3 substituents selected from haloalkyl and cyclopropyl groups. G is either N or CH, G ａ and G ｂ However, N, CH, or CR ５ G ａ and G ｂ Only one of them is N or CH, and G ａ and G ｂ Only one of them is CR ５ And, R ５ but, 【Transformation 6】 And, Z ａ and Z ｂ However, independently, C １ ~C ３ Alkyl or C １ ~C ３ It is a haloalkyl or Z ａ and Z ｂ However, it forms a 3-6 membered carbon ring or heteroring, Z ｃ However, H, -CN, C １ ~C ３ Alkyl, C １ ~C ３ Haloalkyl, or C ２ ~C ４ Alkynes, or combinations thereof, or methods.

138. The method according to claim 137, further comprising adding a non-homologous end-joining (NHEJ) pathway inhibitor to the composition comprising the eukaryotic cells.

139. The method according to claim 137 or 138, wherein the Cas effector protein is encoded by a Cas polynucleotide.

140. The method according to any one of claims 137 to 139, wherein (i) the Cas effector protein and (ii) the target polynucleotide are encoded on a vector.

141. The method according to any one of claims 137 to 139, wherein the Cas effector protein and the polynucleotide of (iii) are encoded on a vector.

142. The method according to any one of claims 137 to 139, wherein the Cas effector protein, the target polynucleotide, and the polynucleotide of (iii) are encoded on a single vector.

143. The method according to claim 137 or 138, wherein the Cas effector protein and the polynucleotide of (iii) are added in the form of ribonucleoprotein (RNP).

144. A method for increasing the efficiency of homologous recombination repair (HDR) and single-strand template repair (SSTR) gene insertion in eukaryotic cells, wherein the method comprises adding a microhomology-mediated end-joining (MMEJ) pathway inhibitor when performing CRISPR / Cas-mediated gene insertion in the eukaryotic cells, wherein the MMEJ pathway inhibitor is a PolQ inhibitor selected from the compound of formula (I), any stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a combination thereof.

145. The method according to claim 144, further comprising adding an inhibitor of the non-homologous end-joining (NHEJ) pathway.

146. The method according to claim 144 or 145, wherein the CRISPR / Cas-mediated gene insertion is a CRISPR / Cas9-mediated gene insertion.

147. A method for reducing recombination of the microhomology-mediated end-joining (MMEJ) pathway during CRISPR / Cas-mediated gene insertion in cells, wherein the method comprises adding an inhibitor of the microhomology-mediated end-joining (MMEJ) pathway to the cells when performing Cas-mediated gene insertion, wherein the inhibitor of the MMEJ pathway is a PolQ inhibitor selected from the compound of formula (I), any stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a combination thereof.

148. The method according to claim 147, further comprising reducing non-homologous end joining (NHEJ) recombination during CRISPR / Cas-mediated gene insertion in the cells, comprising adding an inhibitor of the non-homologous end joining (NHEJ) pathway to the cells.

149. The method according to claim 147 or 148, wherein the CRISPR / Cas-mediated gene insertion is a CRISPR / Cas9-mediated gene insertion.

150. A composition, a. Cas effector protein, or a vector encoding a Cas effector protein, b. A composition comprising an inhibitor of the microhomology-mediated end-binding (MMEJ) pathway, wherein the inhibitor of the MMEJ pathway is an inhibitor of PolQ selected from a compound of formula (I), any stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a combination thereof.

151. The composition according to claim 150, further comprising an inhibitor of the non-homologous end-joining (NHEJ) pathway.

152. The composition according to claim 150 or 151, further comprising a polynucleotide comprising at least one RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof.

153. The composition according to any one of claims 150 to 152, wherein the Cas effector protein is Cas9 nuclease, Cas12a nuclease, or Cas12f nuclease.

154. The method according to claim 153, wherein the Cas effector protein is Cas9 nuclease.

155. The method according to claim 154, wherein the Cas9 nuclease is a Cas9 nuclease fused to a reverse transcriptase, a Cas9 fused to a DNA polymerase, a Cas9 fused to DN1S, a Cas9 nickase, a Cas9 fused to a geminin deglon domain, or a Cas9 nuclease fused to CTIP.

156. The composition according to any one of claims 150 to 155, wherein the vector encoding the Cas effector protein is a viral vector.

157. The composition according to any one of claims 150 to 155, wherein the polynucleotide comprising at least one guide RNA sequence, a Cas-binding region, a DNA template sequence, or a combination thereof is encoded on a vector.

158. The composition according to claim 157, wherein the vector encoding the polynucleotide, which includes at least one guide RNA sequence, a Cas-binding region, a DNA template sequence, or a combination thereof, is a viral vector.

159. The composition according to claim 150 or 151, wherein the Cas effector protein and the polynucleotide comprising at least one guide RNA sequence, a Cas binding region, a DNA template sequence, or a combination thereof are in the form of a ribonucleoprotein (RNP).

160. The composition according to any one of claims 150 to 159, further comprising a pharmaceutically acceptable carrier, diluent, or excipient.

161. It's a kit, a. Cas effector protein, or a vector encoding a Cas effector protein, b. A kit comprising an inhibitor of the microhomology-mediated end-binding (MMEJ) pathway, wherein the inhibitor of the MMEJ pathway is an inhibitor of PolQ selected from a compound of formula (I), any stereoisomer thereof, a pharmaceutically acceptable salt thereof, or a combination thereof.

162. The kit according to claim 161, further comprising an inhibitor of the non-homologous end-joining (NHEJ) pathway.

163. The kit according to claim 161 or 162, further comprising a polynucleotide having at least one RNA guide sequence, a Cas binding region, a DNA template sequence, or a combination thereof.

164. The kit according to any one of claims 161 to 163, wherein the Cas effector protein is Cas9 nuclease, Cas12a nuclease, or Cas12f nuclease.

165. The kit according to claim 164, wherein the Cas effector protein is Cas9 nuclease.

166. The kit according to claim 165, wherein the Cas9 nuclease is a Cas9 nuclease fused to a reverse transcriptase, a Cas9 fused to a DNA polymerase, a Cas9 fused to DN1S, a Cas9 nickase, a Cas9 fused to a geminin deglon domain, or a Cas9 nuclease fused to CTIP.

167. The kit according to any one of claims 161 to 166, wherein the vector encoding the Cas effector protein is a viral vector.

168. The kit according to any one of claims 161 to 167, wherein a guide polynucleotide is encoded on the vector.

169. The kit according to claim 168, wherein the vector encoding the guide polynucleotide is a viral vector.

170. The kit according to claim 161 or 162, wherein the Cas effector protein and guide polynucleotide are in the form of ribonucleoprotein (RNP).

171. A method, compound, or kit according to any one of claims 1 to 170, wherein Y is a phenyl or a 5-6 membered heteroaryl, and the phenyl or heteroaryl is optionally substituted with 1 to 3 substituents selected from halo, C1-C3 alkyl, C1-C3 alkoxy, -CN, and C1-C3 haloalkyl.

172. The method, compound, or kit according to any one of claims 1 to 170, wherein the PolQ, the inhibitor of PolQ, is a compound listed in Table I, a pharmaceutically acceptable salt thereof, or a combination thereof.

173. The method, compound, or kit according to any one of claims 1 to 170, wherein the PolQ, the inhibitor of PolQ, is a compound listed in Table II, Table III, a pharmaceutically acceptable salt thereof, or a combination thereof.

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