WO2024175696A1

WO2024175696A1 - Use of acetylsalicylic acid to accelerate genome repair and protect against genotoxic injury

Info

Publication number: WO2024175696A1
Application number: PCT/EP2024/054482
Authority: WO
Inventors: Nelson O. GEKARA
Original assignee: Albert Ludwigs Universitaet Freiburg
Current assignee: Albert Ludwigs Universitaet Freiburg
Priority date: 2023-02-23
Filing date: 2024-02-22
Publication date: 2024-08-29
Anticipated expiration: 2025-08-23

Abstract

The recent discovery of CRISPR/Cas9 editing technology has drastically improved the ability to modify the genome of any cell at precise locations. The present invention relates to the use of acetylsalicylic acid, a nonsteroidal anti-inflammatory agent known commercially as "aspirin", to accelerate homologous genome repair, protect against genotoxic injury, and thereby increase the efficiency and efficacy of genome editing in target eukaryotic cells or organisms. The discovery that acetylsalicylic acid ("ASA") is a histone acetylating agent capable of modulating of chromatin structure and accelerating genome repair via HDR represents a significant/ novel advance in the current understanding of the mechanism of ASA. As a clinically safe drug, ASA can be used to enhance and improve precision genome editing in cell culture and in live organisms with minimal toxicities.

Description

USE OF ACETYLSALICYLIC ACID TO ACCELERATE GENOME REPAIR AND PROTECT AGAINST GENOTOXIC INJURY

FIELD OF THE INVENTION

The present invention relates generally to the field of genomic injury and repair. More particularly, the present invention relates to the use of acetylsalicylic acid (a drug commercially known as “aspirin”) to accelerate and direct the resolution of DNA breaks through homology-directed recombination (HDR), as opposed to non-homologous endjoining (NHEJ), and thereby enhance the efficiency of precision gene editing systems.

BACKGROUND OF THE INVENTION

Precision genome editing systems as exemplified by the Nobel prize winning CRISPR/Cas9 editing technology have revolutionized genetics and molecular and cell biology, serving as a novel tool for basic research with the potential for treating a range of medical conditions that have a genetic component. The CRISPR/Cas9 system in particular has been widely adopted to edit genomes by cutting target DNA sequences at precise locations and letting natural DNA repair processes take over. More particularly, the introduction of DNA cut by Cas9 requires PAM (protospacer adjacent motif) sequence of 5’NGG3’ directly downstream of the target DNA sequence, on the non-target strand. Cas9 targeting to a specific site is guided by a bound guide RNA (gRNA) that is complementary to 20 nucleotides adjacent to the PAM site. Thus, the disruption of genes by CRISPR/Cas9 often requires the introduction of double-stranded breaks (“DSBs”) at two sites flanking the target region and, as such, generates two sites requiring repair.

In eukaryotic cells, the natural process for repair occurs via two competing pathways: Non-Homologous-End-Joining (“NHEJ”) and Homology-Directed Repair (“HR” or “HDR”). NHEJ, the predominant repair pathway that occurs regardless of the phase of the cell cycle, is referred to as “non-homologous” because the broken ends are directly ligated without the need for a homologous template. However, NHEJ is both error prone and mutagenic, often resulting in a genomic deletion or insertional mutation. On the other hand, HDR is a significantly more accurate repair process that occurs during the S and G2 cell cycle phases and uses the undamaged homologous sequence on sister chromatid as template to restore the damaged DNA to original DNA sequence (1, 2). In contrast to NHEJ, HDR in the presence of donor templates allows the introduction of defined genomic changes into the genome.

In addition to the intentionally generated DSBs that arise in the course of precision gene editing, DSBs in DNA can also arise inadvertently, for example, as a result of exposure to ionizing radiation or radiomimetic chemicals. In either case, whether intentional or inadvertent, endogenous or exogenous, DSBs can be deleterious to health. In addition to their potential to trigger cell death, the processing and repair of DSBs can, as noted above, lead to mutations, loss of heterozygosity, and chromosome rearrangements that result in cell death or cancer. In particular, if unrepaired or mis-repaired, DSBs can cause chromosome deletions and translocations leading to long-term detrimental effects, including cancer and hereditary disorders. DSBs are also the cause of inflammation (18), a key driver of radio/chemotherapy-induced tissue injuries such as fibrosis that significantly impact the quality of life of patients and survivors. An outstanding limitation of precision genome editing applications (and known side-effect of exposure-induced DSBs) is the low efficiency of HDR relative to NHEJ (3). Evidence suggest that skewing the repair of DSBs towards HDR, for example by synchronization of cells at S or G2 phases (4, 5), or inhibition of NHEJ repair using small molecule inhibitors or shRNA silencing (5-7) can increase the efficiency of precision genome editing. Accordingly, factors that influence the choice of pathways for DSB repair, and particularly those that direct toward the more accurate HDR repair process, can minimize individual’s mutation burden and risk of cancer as well as enhance the efficiency and minimize the risks that accompany precision gene editing in cell culture and in live organisms (25).

Another key determinant of genome editing efficiency is chromatin structure (8- 10). Tightly packaged (heterochromatin) is generally a barrier to eukaryotic genome editing (11). In addition to regulating the accessibility of target DNA sites to editing nuclease, the chromatin structure is a determinant of the balance between HDR and NHEJ. The N-terminal tail of histone H4 is vital for inter-nucleosome interaction. Acetylation of histone H4 at lysine KI 6 (Ac-H4K16) is an essential modification that decreases the nucleosome-nucleosome stacking and chromatin folding, thus fosters a relaxed chromatin environment conducive for recruitment of repair proteins to DNA damage sites (12). Ac- H4K16 also controls the DSB repair choice by supporting preferential recruitment of the HDR checkpoint protein BRAC1 over the NHEJ repair factor 53BP1 to damage sites (13). Hence, increasing Ac-H4K16 levels is a possible avenue both, for increasing the accessibility of chromatin to CRISPR/Cas9 and promoting HDR to achieve the desired precision genome editing.

SUMMARY OF THE INVENTION

Bearing in mind the need in the art for agents that promote HDR and/or chromatin modulation, it is accordingly an objective of the present invention to provide a novel method for accelerating homologous genome repair, protect against genotoxic injury, and thereby enhance the efficiency and efficacy of precision genome editing that involves the concomitant administration of a histone acetylating agent, namely acetylsalicylic acid, to the target cell, system, or organism.

Acetylsalicylic acid (herein abbreviated “ASA”) is, arguably the oldest drug in the history of medicine and has been used for over 4000 years for the treatment of pain, inflammation, and fever, and, more recently, for cardiovascular prophylaxis (14) and cancer prevention (15). Until now, the therapeutic effects of ASA have been presumed to be linked to its ability to suppress inflammation via the inhibition of COXI and COX2 (16, 17). However, the results herein demonstrate that outside its anti-inflammatory effects, ASA accelerates HDR-mediated DNA repair and protects against genotoxicity. In addition, the results herein show that mechanistically, ASA acetylates histone H4K16 and thereby induces chromatin relaxation and the recruitment of DNA repair factors to damage sites. The discovery of ASA’s ability to de-compact the chromatin and accelerate genome repair via HDR suggest its utility in enhancing the efficiency of precise genome editing in target eukaryotic cells or organisms. Accordingly, it is an objective of the present invention to provide a modified method of precision genome editing that involves the generation of double-stranded breaks (DSBs) in genomic DNA in living cells, wherein the method includes the step of concomitantly administering with the generation of DSBs an amount of ASA effective to induce chromatin decompaction and/or promote genome repair via homology-directed repair (HDR). In a preferred embodiment, the amount of ASA is effective to induce chromatin relaxation and recruitment DNA repair factors to the DSB site.

In the context of the present invention, the administration of the effective amount of ASA may occur prior to the generation of the DSBs, subsequent to the generation of the DSBs, or simultaneously with the generation of said DSBs.

In particularly preferred embodiments, the underlying method of precision genome editing being modified and enhanced is one that utilizes the CRISPR/Cas-9 system. In other embodiments, the method of precision genome editing may be selected from among bacterial retron library recombineering (RLR), prokaryotic argonautes (pAgos), transcription activator-like effector nuclease engineering (TALENs), or meganuclease engineering.

It is an objective of the present invention, to enhance precision genome editing of living cells, both in vitro, e.g., in the form of a cell culture, and in vivo, e.g., in situ in a host organism. In preferred embodiments, the host organism is a mammal, preferably a human, and the living cells comprise part of a mammalian organ, such as a liver or bone marrow.

These and other objects and features of the invention will become more fully apparent when the following detailed description is read in conjunction with the accompanying figures and examples. However, it is to be understood that both the foregoing summary of the invention and the following detailed description are of a preferred embodiment, and not restrictive of the invention or other alternate embodiments of the invention. In particular, while the invention is described herein with reference to a number of specific embodiments, it will be appreciated that the description is illustrative of the invention and is not constructed as limiting of the invention. Various modifications and applications may occur to those who are skilled in the art, without departing from the spirit and the scope of the invention, as described by the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 Aspirin protects against radiation sickness by promoting HR-DNA repair, a-c, Survival of bone marrow (BM) cells 10 hours post irradiation (9 Gy) of (a) WT mice, (b) Myd88 Trif^ Mavs^ (TKO) mice and (c) Sting" ' mice pretreated with aspirin (ASP) or dimethyl sulfoxide (DMSO). (a-c) are presented as mean ±SD, One-way ANOVA test. ***P< 0.001, ****P< 0.0001. d, e ASP promotes DNA repair. Images (d) and corresponding quantification (e) of the comet tails of BMDMos pre-treated with ASP or DMSO) then irradiated (9 Gy on ice) then incubated at 37°C to allow DNA repair, scale bar=50 pm. f, g BRCA1 foci (f) and 53BP1 foci (g) per nucleus 1 hour after irradiation (9 Gy) of HEK293 cells pre-treated with DMSO or ASP. Presented as mean ±SEM, n = 30, One-way ANOVA test. NS p>o.5,****P < 0.0001. h, i ASP induces chromatin decompaction. Confocal images of mCherry-LacR in ASP-treated AO3 cells (h) and corresponding quantification of the relative array size (surface of the mcherry-LacR array/surface (i). Presented as ± SEM, two-tailed Student’s t-test. ****P < 0.0001. scale bar=10 pm. j Histone acetylation marks in chromatin isolated from ASP-treated AO3 cells, k ASP induces Ac-H4K16 in vivo and is associated with reduced markers of DNA damage. Histone acetylation and y-H2A.X in chromatin from bone marrow cells of mice that were treated with ASP (or not) followed by irradiation. 1- o. ASP protects against radiation sickness and extends survival. Mortality (1) clinical severity (m), weight loss (n) and piloerection score (o) in ASA treated and untreated mice following y-irradiation (18 Gy). Differences in morbidity (1) were analyzed by log-rank test, ***P<0.001 and graphs in (m, n, o) were shown as mean ± SEM. ***P< 0.001 (oneway ANOVA test).

Fig. 2. Aspirin protects against irradiation-induced bone marrow injury in WT mice, a-d, Survival of the hematopoietic progenitors (a) monocytes (b), neutrophils (c) and B cells (d) in bone marrow 10 hours post irradiation (IR: 9 Gy) of WT mice pretreated with ASP (n=8) or DMSO (n=8). e-g, Transcripts of Ifn0 (e), Tnfa (f), and Mxl (g) in bone marrow cells isolated from ASP or DMSO pre-treated WT mice, 10 hours post irradiation (IR: 9 Gy). The data are presented as mean ± SD. Statistical significance is assessed using one-way ANOVA followed by Tukey’s multiple comparisons test.

Fig. 3. Aspirin inhibits irradiation-induced inflammatory gene expression. a-c, Bone marrow derived monocytes (BMDMos) were pre-treated with ASP or DMSO for 12 hours then analyzed for the transcripts of the indicated inflammatory mediators; Ifti (a), Mxl (b) and Tnfa (c) at indicated time points after irradiation (9 Gy). The data are presented as mean ± SD. Statistical significance are assessed using two-way ANOVA test.

Fig. 4. Aspirin inhibits multiple PRR pathways for inflammatory gene induction. a, Schematic overview of PRR pathways for inflammatory gene activation, b-f, Iftif induction in Ifnb ^"^luc BMDMos that were pre-treated with DMSO or ASP (2 mM) then stimulated (or not) with 1 pg/ml Pam3CSK4 (b), 10 pg/ml poly(I:C) (c), 2 pg/ml poly(I:C) transfection (d), 1 pg/ml poly(dA:dT) transfection (e) 1 pg/ml cGAMP (f) then analyzed for luciferase activity. The data are presented as mean ± SD, n=3. Statistical significance is assessed using two-way ANOVA test.

Fig. 5. Effect of aspirin on AIM2 inflammasome. a Schematic summary of AIM2 inflammasome mediated pyroptosis and IL- 10 secretion, b Immunoblot analysis of indicated proteins in supernatants or lysates of BMDMos that were pre-treated with ASP (0.5 or 1 mM) then primed with LPS and transfected with the AIM2 agonist poly(dA:dT). c Estimation of pyroptosis by LDH release in BMDMos pretreated with ASP then transfected with poly(dA:dT). The data are presented as mean ± SD, n=3. Statistical significance is assessed using one-way ANOVA test. NS P>0.05. Fig. 6. Aspirin protects against irradiation-induced bone marrow injury in TKO mice, a-d Survival of the hematopoietic progenitors (a) monocytes (b), neutrophils (c) and B cells (d) in bone marrow 10 hours post-irradiation (IR: 9 Gy) of TKO mice pretreated with ASP (n=10) or DMSO (n=10). e-g Transcripts of Ifti (e), Tnfa (f), and AA7(g) in bone marrow cells isolated from ASP or DMSO pre-treated TKO mice, 10 hours post irradiation (IR: 9 Gy). The data are presented as mean ± SD. Statistical significance is assessed using one-way ANOVA followed by Tukey’s multiple comparisons test.

Fig. 7. Aspirin protects against irradiation-induced bone marrow injury in Sting'' mice, a-d Survival of the hematopoietic progenitors (a) monocytes (b), neutrophils (c) and B cells (d) in bone marrow 10 hours post irradiation (IR: 9 Gy) of in Sting ' mice pretreated with ASP (n=10) or DMSO (n=10). e-g Transcripts of Ifh/3 (e), Tnfa (f), and Mxl g) in bone marrow cells isolated from ASP or DMSO pre-treated Sting'⁷' mice, 10 hours post irradiation (IR: 9 Gy). The data are presented as mean ± SD. Statistical significance is assessed using one-way ANOVA followed by Tukey’s multiple comparisons test.

Fig. 8. Aspirin inhibits DNA damage-induced micronuclei generation, a Confocal microscopic visualization of micronuclei (indicated by arrowhead) in HEK293 cells pretreated with DMSO or ASP before exposure to y-irradiation (9 Gy). Scale bar=10 pm. (b) Average MNs/cell in corresponding representative images. Bar graphs show mean values from five different microscopic fields with over 200 cells. Graphs show as mean ±SEM and statistical significance were assessed using One-way ANOVA followed by Tukey’s multiple comparisons test. ***P < 0.001.

Fig. 9. Aspirin protect against chemotherapy-induced DNA damage, a Comet tails in BMDMos pre-treated (or not) with ASP (1 mM) for 4 hours then stimulated with doxorubicin (1 pM) for indicated duration, b Corresponding quantification of the comet tail moments from 20 different fields with n > 200 comets of three independent experiments. Scale bar=50 pm. The data presented as mean ± SEM, n = 200. ***P< 0.001, 0.0001, NS P>0.05 (one-way ANOVA test).

Fig. 10. Aspirin promotes HR-DNA repair, a, b Schematics of (a) HR-GFP and (b) NHEJ-GFP reporter assays, c, d DNA repair efficiency in HR-GFP reporter (c) or NHEJ- GFP reporter cells (d) pretreated with DMSO or indicated concentrations of ASP. (e) Expression levels of HA-I-Scel (the DNA break-inducing nuclease) in corresponding samples in c, d that were pre-treated with (or not) 2 mM ASP.

Fig. 11. Aspirin acetylates H4K16 and promotes the recruitment of BRCA1 but not 53BP1 to DNA damage sites, a Immunofluorescence images of BRCA1 and Ac-H4K16 1 hour after irradiation (9 Gy) of HEK293 cells pre-treated with DMSO or ASP. Scale bar = 10 pm. b Immunofluorescence images of 53BP1 and Ac-H4K16 1 hour after irradiation (9 Gy) of HEK293 cells pre-treated with DMSO or ASP. Scale bar = 10 pm. c, d Time course quantification of BRCAl(c) and 53BPl(d) foci per nucleus in HEK293 cells pretreated (or not) with ASP. Graphs were shown as mean ± SEM, n = 40. ***P< 0.001, 0.0001, NS P>0.05 (one-way ANOVA test).

Fig. 12. Potentiation of DNA repair by aspirin requires BRCA1. a Representative comet tails of Non-Target Control (NTC) or BRCAT⁷' HEK293T that were pre-treated (or not) with ASP (2 mM), then irradiated (9 Gy) on ice, followed by incubation at 37°C to allow DNA repair to occur for the indicated duration. Scale bar=50 pm, b Immunoblot analysis of BRCA1 expression and Tubulin (control) in NTC and BRCAP~ HEK293T cells, c Quantification of the comet tail moments from 20 different fields with n > 200 comets of three independent experiments. Graphs were shown as mean ± SEM, n = 200.

0.0001, ns P>0.05 (one-way ANOVA test).

Fig. 13. Aspirin promotes DNA repair independently of the NHEJ checkpoint protein 53BP1. a Representative comet tails of Non-Target Control (NTC) or 53BPP~ HEK293T that were pre-treated (or not) with ASP (2 mM), then irradiated (9 Gy) on ice, followed by incubation at 37°C to allow DNA repair to occur for the indicated duration. Scale bar=50 pm. b, c Corresponding quantification of the comet tail moments from 20 different fields with n > 200 comets of three independent experiments. Graphs were shown as mean ± SEM, n = 200. ****P< 0.0001, ns P>0.05 (one-way ANOVA test), d, 53BP1 and Tubulin (control) levels in NTC and 53BP1 HEK293T cells.

Fig. 14. Aspirin promotes DNA repair independently of the NHEJ kinase DNA-PKC. a Representative comet tails of BMDMos pre-treated (or not) with ASP (2 mM) and DNA- PK inhibitor (DNA-PKi) Nu7026 (500 nM) alone or in combination, then irradiated (9 Gy) on ice, followed by incubation at 37°C to allow DNA repair for indicated duration. Scale bar=50 pm. b, c Corresponding quantification of the comet tail moments from 20 different fields with n > 200 comets of three independent experiments, (b) Kinetic graph of DNA repair, c Comparison of the DNA damage level in the indicated treatment groups 45 minutes after radiation, d Titrated effect of DNA-PK inhibitor (DNA-PKi) Nu7026 on DNA damage-induced yH2AX. H2A.X was used as loading control. Graphs were shown as mean ± SEM, n = 200. ***P< 0.001, ****P< 0.0001, NS P>0.05 (one-way ANOVA test).

Fig. 15. Aspirin acetylates H4-K16 directly by its acetyl group, a ASP acetylates H4K16 independently of Histone acetyl transferases. Immunoblot of Ac-H4K16 and total H4 in BMDMos treated for 6 hours with indicated concentrations of the histone acetyltransferase inhibitor (HATi) MG149. b Ac-H4K16 and total H4 in BMDMos treated with ASP (2 mM), HATi (20 pM) or both for 6 hours, c ASP directly donates acetyl group to H4K16. Chromatin fractions isolated from BMDMos pre-incubated with indicated concentrations of ASP at 37 °C for 1 hour then immunoblotted for Ac-H4K16 and total acetylated proteins.

Fig. 16. Aspirin promotes H4-K16 acetylation and DNA damage-associated chromatin decompaction, a Overview of a single nucleosome complex with a depiction of the histone H4-dsDNA interface and histone H4K16 location, b Schematic overview of the chromatin compaction assay and effect of ASP on chromatin. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Unless otherwise defined herein, scientific and technical terms used in the present disclosures shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. In case of conflict, the present specification, including definitions, will control.

The term “a” or “an” entity refers to one or more of that entity; for example, “a vector,” is understood to represent one or more vectors.

The term “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and/or” as used in a phrase such as “A and/or B” herein is intended to include “A and B,” “A or B,” “A” (alone), and “B” (alone). Likewise, the term “and/or” as used in a phrase such as “A, B, and/or C” is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

The terms “polypeptide,” “peptide,” “protein,” and their grammatical equivalents as used interchangeably herein refer to polymers of amino acids of any length, which can be linear or branched. It can include unnatural or modified amino acids or be interrupted by non-amino acids. A polypeptide, peptide, or protein can also be modified with, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification.

The abbreviation “DSB” is used herein to refer to “double-stranded breaks” in genomic DNA.

The abbreviation “NHET’ is used herein to refer to “non-homologous end joining”, the primary yet error- prone pathway for the repair of DSBs throughout the cell cycle. It is to be contrasted with the homologous recombination process known as “homology-directed repair” which is herein alternatively abbreviated as “HR” or “HDR”.

In the context of the present invention, the term “precision genome editing system” is used to refer to the class of genetic engineering techniques that involve the introduction of targeted cuts and splices to genomic DNA and, more particularly, the introduction of double-strand breaks (DSBs) into the genome. While the CRISPR/Cas9 system is the most popular tool for genome editing, promising alternatives are known in the art and thus contemplated by the present invention. Illustrative examples include, but are not limited to, bacterial retron library recombineering (RLR), prokaryotic argonautes (pAgos), transcription activator-like effector nucleases (TALENs), and meganucleases. The present invention also contemplates modifications to the conventional CRISPR/Cas9 approach that utilize novel Cas nucleases (such as Cpfl (i.e., Casl2), C2c2 (i.e., Cast 3a), and C2cl (i.e., Casl3b)) as well as specialized versions such as the CasMINI, aversion that utilizes Casl2f that is smaller than Cas9 and thus can be delivered via adeno-associated virus (AAV) techniques, and Cas-CLOVER, a version that utilizes two gRNAs as well as an endonuclease, Clo51, fused to Cas9 so as to increase specificity and avoid off-target effects.

References herein to the “CRISPR/Cas-9” genome editing system are used herein to broadly encompass the processes of recognition, cleavage and repair in which at least one CRISPR-associated (“Cas-9”) protein and at least one guide RNA (“gRNA”), the latter of which comprises an sgDNA fragment that recognizes the target sequence in a gene of interest through complementary base pair, shepherd the insertion, removal, or modification of genes in living cells, either in vitro or in vivo. In the context of the present invention, the phrase “CRISPR/Cas system” includes both Class I systems (including types I, III, and IV), i.e., systems that utilize multi-subunit Cas-protein complexes, as well as Class II systems (including type II, V, and VI), i.e., systems utilize a single Cas-protein (29).

The present invention makes reference to the use of acetylsalicylic acid (“ASA”), the structure of which is depicted below, to accelerate and direct the resolution of DNA breaks through homology-directed recombination (HDR), as opposed to non-homologous end-joining (NHEJ), and thereby enhance the efficiency of precision gene editing systems.

However, given the discovery herein of a mechanism of action in which the chromosome accessibility for the HDR machinery is enhanced by means of Histone H4 acetylation, the present invention contemplates the use of other histone acetylating agents having a similar effect as ASA, examples of which include, but are not limited to, histone acetyltransferases, also known as HATs, a family of enzymes that acetylate the histone tails of the nucleosome. The HAT family of acetylating agent encompasses General Control Non-Derepressible 5 (Gcn5) -related N-Acetyltransferases (GNATs), the family of p300/CREB-binding proteins (CBPs) , and the MYST family made up of MOZ (Monocytic Leukemia Zinc Finger Protein), Ybf2/Sas3, Sas2 and Tip60 (Tat Interacting Protein). The HAT family further encompasses other agents having acetylating abilities but differing in structure from those previously mentioned, examples of which include, but are not limited to, steroid receptor coactivator 1 (SRC1), ATF-2 (which contains a transcriptional activation (ACT) domain and a basic zipper DNA-binding (bZip) domain with a HAT domain in-between), and TAFII250 (which has a Kinase domain at the N-terminus region, two bromodomains located at the C-terminus region and a HAT domain located inbetween).

As used herein, the term “administer” and its grammatical equivalents refer to the act of delivering, or causing to be delivered, a therapeutic or a pharmaceutical composition, particularly one containing ASA, to the body of a subject by a method described herein or otherwise known in the art. The therapeutic can be in drug or pro-drug form and further take the form of a compound, a polypeptide, or a cell. Administering a therapeutic or a pharmaceutical composition includes prescribing a therapeutic or a pharmaceutical composition to be delivered into the body of a subject. Exemplary forms of administration include oral dosage forms, such as tablets, capsules, syrups, suspensions; injectable dosage forms, such as intravenous (IV), intramuscular (IM), or intraperitoneal (IP); subcutaneous (SC), transdermal dosage forms, including creams, jellies, powders, or patches; buccal dosage forms; inhalation powders, sprays, suspensions, and rectal suppositories.

As used herein, the terms “effective amount,” “therapeutically effective amount,” and their grammatical equivalents refer to the administration of an agent to a cell, system or subject, either alone or as a part of a pharmaceutical composition and either in a single dose or as part of a series of doses, in an amount that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease, disorder or condition when administered to the subject. The therapeutically effective amount can be ascertained by measuring relevant physiological effects. The exact amount required vary from subject to subject, system to system, depending on the age, weight, and general condition of the subject, the severity of the condition being treated, the judgment of the clinician, and the like. A therapeutically effective amount is also one in which any toxic or detrimental effects of the therapeutic agent are outweighed by the therapeutically beneficial effects. An appropriate “effective amount” in any individual case can vary according to factors such as the disease state, age, sex, and weight of the individual, and can be determined by one of ordinary skill in the art using routine experimentation. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, for example, the delay or prevention of the onset of a disease or disorder. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount is commonly less than the therapeutically effective amount.

In the context of the present invention, the “effective amount” of ASA would be an amount effective to induce the desirable chromatin modification and/or induce homology- directed repair of intentionally or inadvertently introduced double-stranded DNA breaks.

The present invention contemplates both in vitro and in vivo utilities. In the context of in vivo applications, the term “subject” is sued to refer to any animal (e.g., a mammal), including, but not limited to, humans, non-human primates, canines, felines, rodents, and the like, which is to be the recipient of a particular treatment. A subject can be a human. A subject can be a patient with a particular disease.

The methods of the instant invention can enhance the efficiency of precision gene editing techniques and thereby boost the efficacy of techniques such as CRISPR in treating virtually any disease or disorder with a genetic origin. Illustrative examples of some first line genetically-linked diseases contemplated by the present invention include, but are not limited to, cancer immunotherapy; blood disorders such as beta-thalassemia, sickle cell disease, and hemophilia; inherited childhood blindness; HIV and AIDS; cystic fibrosis; muscular dystrophy; Huntington’s disease; and COVID- 19.

Hereinafter, the present invention is described in more detail by reference to the following experimental examples. However, the following materials, methods and examples only illustrate aspects of the invention and in no way are intended to limit the scope of the present invention. As such, methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention,

EXAMPLES

Materials and Methods:

1. Mice

All mice in this study were on C57BL/6J background. Ticaml-/- (C57BL/6J- TicamlLps2/J, stock #005037) and Sting -I- (C57BL/6J- Tmeml73gt/J, stock #017537) mice were from Jackson Laboratory. Myd88-/- and Mavs-/-(Ipsl-/-) mice were from S. Akira's laboratory, Osaka University, Japan. Myd88-/-, Ticaml-/-, Mavs-/- mice were crossed with each other to generate the Myd88-/- Ticaml-/- Mavs-/- micel, 2. Mice were bred in specific pathogen-free animal facility at Umea center for comparative Biology (UCCB). Experiments were carried out according to the guidelines set out by the Umea Regional Animal Ethic Committee (Umea Regionala Djurfbrsbksetiska Namnd), Approval no. A25-19.

2. Aspirins Treatment and Irradiation of Mice

Mice were given a daily intraperitoneal (i.p.) injection of dimethyl sulfoxide (“DMSO”) (control) or aspirin (“ASP”) (50 mg/kg=0.277mM) for 7 days. To assess the effect of irradiation on the bone marrow, mice were placed in a Gammacell 40 irradiator (MDS Nordion) with a 137 Cs gamma-ray source and given total dose of 9 Gy. 10 hours later they were sacrificed and bone marrow cells were isolated, counted and analyzed by flow cytometry for the following populations: hematopoietic stem cells (HSCs; c-Kit Sca- 1), B cells (B220 ) and neutrophils (Grl Ly6G ). The total bone marrow cells or specified cell populations in the femur were calculated and expressed as relative (percentage) to nonirradiated controls. To assess the effect of aspirin on irradiation-induced sickness, following total body irradiation, control and aspirin-treated mice were monitored daily for weight and. clinical severity. Clinical severity scoring was based on an arbitrary scale of 1 to 4, where 1 represented mice with mild but visible symptoms such as slowed activity and 4 represented those with severe morbidity, i.e., with lethargy, loose fecal pellet, piloerection, >20% weight loss, difficulty in breathing and movement and hence had to be euthanized. Experiments were done using adult mice (8-14 weeks old).

3. Antibodies and Reagents

Aspirin (Catalog# A2093), Doxorubicin (Catalog# DI 515), DNA-PK inhibitor (DNA-PKi) Nu7026 (Catalog# N1537) and Histone acetyltransferase inhibitor (HATi) MG149 (Catalog# SML3011) were purchased from Sigma-Aldrich. The Ac-lysine antibody was from Santa Cruz (Catalog #sc-32268). Antibodies against H2A.X (Catalog #2595), y- H2A.X (Catalog #2577), Histone H3 (Catalog #4499) and Histone H4 (Catalog #13919) were from Cell Signaling Technology. Alexa488-Anti-Sca-1 (Catalog #11-5981-82) and Ac-H4K16 (Catalog # MA5-27794) were from Invitrogen and PECY7-Anti-cKit (Catalog #561681), Alexa Fluor® 700 Rat anti- Mouse CD45R (B220) (Catalog #557957) APC- Anti-CDl lb (Catalog #553312), FITC-Anti-GRl (Catalog #553126) were from BD Pharmingen. 2’,3’-cGAMP (Catalog #tlrl-nacga23), Pam3CSK4 (Catalog #tlrl-pms), Poly(I:C) (Catalog #tlrl-picwlv), Poly(dA:dT) (Catalog #tlrl-patn) were from InvivoGen. Ac-H4K16 (Catalog #abl 09463) and Ac-H3K27 (Catalog #ab4729) antibodies were from Abeam.

4. Plasmids pHPRT-DRGFP (# 26476) and pCBAScel (# 26477) ⁶, pimEJ5GFP (# 44026) ⁷ were obtained from Addgene. The mCherry-LacR-stop plasmid ⁸ was a gift from Nico Dantuma laboratory, Karolinska Institute, Stockholm, Sweden.

5. Microscopic Visualization of Chromatin Compaction

The AO3 reporter cells 9 cultured in a 1:1 mixture of DME/Ham’s F12 medium supplemented with antibiotics and 20% FCS to 70% density were transfected by lipofectamine with the 1 pg/ml mCherry-LacR-stop plasmid 8. After 4 hours, they were treated with DMSO or indicated concentrations of aspirin (1 or 2 mM). 18 hours later samples were fixed with 4% paraformaldehyde and analyzed my fluorescence microscopy as described previously 10.

6. HR and NHEJ Reporter Assays

To assess the effect of aspirin on homologous recombination (HR) and NHEJ repair, briefly, the pHPRT-DRGFP (HR-reporter plasmid)6and the pimEJ5GFP (NHEJ reporter plasmid) 7 were stably transfected into HEK293T cells. 0.5 x 106 HEK293T stable reporter cells seeded in 6-well plates were transfected with 2 pg HA-I-Scel expression plasmid (pCBASce) then treated with aspirin or DMSO. 48 hours later, cells were analyzed by flow cytometry for GFP expression. Standard Mean of Error (±SEM) was calculated from three independent experiments.

7. Cells and Cell Culture

HEK293 cell and HEK293T cells were cultured under 5% CO2 at 37 °C in Dulbecco’s modified Eagle medium (DMEM, high glucose, GlutaMAX) (Life Technologies) containing 10% (v/v) fetal bovin serum (FBS, GIBCO), 1% (v/v), penicillin (100 IU/ ml) + streptomycin (100 pg/ml). Bone-marrow- differentiating monocytes (BMDMos) were generated by culturing the mouse bone marrow cells in IMDM medium (GIBCO, Life Technologies) supplemented with 10% (v/v) FBS (GIBCO, Life Technologies), 1% (v/v) penicillin (100 U ml-l)/streptomycin (100 pg/ml), 2 mM glutamine (Sigma- Aldrich) and 10% (v/v) L929 conditional medium and maintained with 5% CO2 at 37 °C. The cells were used for experiment 4 days after start of differentiation. AO3 hamster cells, containing a 90-Mbp amplification of LacO sequences and flanking DNA6, were cultured in a 1: 1 mixture of DME/Ham’s F12 medium supplemented with antibiotics and 20% FBS. 8. Generation of Knockout Cells

53BP1-/-, BRCAl-/-and non-target control (NTC) HEK293T cell lines were generated by CRISPR/Cas9 gene editing technology. Cells were transfected with the following gRNAs cloned into lentiCRISPR v2-puro:

53BP1 gRNA: CAGAATCATCCTCTAGAACC (SEQ ID NO: 1);

NTC gRNA2: GTGTAGTTCGACCATTCGTG (SEQ ID NO: 2);

BRCA1 gRNAl : TGCTAGTCTGGAGTTGATCA (SEQ ID NO: 3); and

BRCA1 gRNA2: AAATCTTAGAGTGTCCCATC (SEQ ID NO: 4).

Cells were selected with 10 mg mL-1 puromycin and resistant cells cloned

9. Immunofluorescence

Cells were seeded and cultured on glass coverslips in 12 well plate and fixed in 4% paraformaldehyde (PF A) in PBS for 20 min at room temperature. Cells were permeabilized in 0.5% Triton X-100 for 10 min, blocked in 5% normal goat serum (NGS) then incubated with primary antibodies diluted in 1% NGS overnight at 4 °C, followed by incubation with indicated secondary antibodies diluted in 1% NGS at RT for 1 h then finally stained with DAPI for 15 min at room temperature. Coverslips were mounted using Dako Fluorescence Mounting Medium (Agilent) and imaged using Nikon confocal microscope (Eclipse Cl Plus). All scoring was performed under blinded conditions. yH2A.X, BRCA1, and 53BP1 foci were counted from 40 microscopic fields containing approx. 300 cells from 3 independent experiments.

10. Chromatin Fractionation and Immunoblotting

To isolate the chromatin, we used the Subcellular Protein Fractionation Kit (Thermo Fisher) according to the manufacturer's instructions. Proteins were quantified by BCA reagent (Thermo Fisher Scientific, Rockford, IL). Samples were resolved in SDS- PAGE, transferred to nitrocellulose membrane (Amersham Protran 0.45 pm NC) and immunoblotted with specific primary antibodies followed by HRP-conjugated secondary antibodies. Protein bands were detected by Supersignal West Pico or Femto Chemiluminescence kit (Thermo Fisher Scientific).

Alternatively, cells were lyzed in mild Nonidet P-40 lysis buffer (1% NP-40, 50 mM Tris-HCl, 150 mM NaCl, pH 7.5,1 mM NaF, 2 mM PMSF, protease inhibitor cocktail [Roche Applied Science], 1 mM sodium orthovanadate, and 10 mM sodiumpyrophosphate). After centrifugation at 10,000g for 15 min at 4°C, proteins in supernatants were quantified by BCA reagent (Thermo Fisher Scientific, Rockford, IL). Proteins were resolved in SDS-PAGE, transferred to nitrocellulose membrane (Amersham Protran 0.45 pm NC) and immunoblotted with specific primary antibodies. Protein bands were detected by SuperSignal West Pico or FemtoChemiluminescence Kit (Thermo Fisher Scientific). 11. Inflammasome Activation Analysis

BMDMos seeded in the density of 1.5 x 106 cells/well were treated with aspirin overnight and then primed with 500 ng/ml LPS for 4 h. Cells were then transfected with 1 pg/ml poly(dA:dT) for 1 h using Lipofectamine 2000 (Invitrogen). Supernatants were collected. Proteins were precipitated using chloroform: methanol extraction and resuspended in 2 x Laemmli buffer. Cells were lysed in 2 x Leammli buffer. Samples were separated on 13.5% SDS-PAGE gel and analysed for activation of Caspase-1 and IL-10 by immunoblotting, as described in the section above.

12. In Vitro Protein Acetylation Assay by Aspirin

Chromatins fractions isolated as described above were incubated with indicated concentration of aspirin in reaction buffer (40 mMTris-HCl, 5 mMMgC12, lOO mMNaCl) for 1 hour at 37 °C. The mixture was boiled in loading buffer and analyzed by immunoblotting.

13. Analysis ofDNA Repair by Comet Assay

Cells were subjected to the indicated doses of y-irradiation or doxorubicin and chromosome fragmentation was determined by comet assay. Briefly, during irradiation cells were kept on ice to stop the DNA repair process. Thereafter, cells were transferred to 37°C to allow DNA repair to occur for indicated duration. Cells were then harvested by brief centrifugation and resuspension in cold PBS. Cells were mixed with 1% low-melting agarose (40°C) at a ratio of 1:3 vol/vol) before pipetting onto CometSlides. Slides were then immersed in prechilled lysis buffer (1.2 M NaCl, 100 mM EDTA, 0.1% sodium lauryl sarcosinate, 0.26M NaOH PH>13) for overnight (18-20 h) lysis at 4°C in the dark. Slides were carefully removed and submerged in room temperature rinse buffer (0.03 M NaOH and 2 mM EDTA, pH > 12) for 20 min in the dark. This washing step was done 2 times. Slides were transferred to a horizontal electrophoresis chamber containing rinse buffer and separated for 25 min at voltage (0.6 V/cm). Finally, slides were washed with distilled water and stained with 10 pg/ml propidium iodide and analyzed by fluorescence microscopy. 20 fields with about 200 cells in each sample were evaluated and quantified by the Fiji software to determine the tail length (tail moment).

14. Determination of Micronuclei

HEK293 cells pre-treated with aspirin (1 mM) then exposed to y-irradiation (or not) were cultured for 24 hours, then fixed (4% PF A), permeabilized (0.5% Triton X- 100), DAPI stained then analyzed by microscopy. Micronuclei were defined as discrete DNA aggregates separate from the primary nucleus in cells where interphase primary nuclear morphology was normal. Cells with an apoptotic or necrotic appearance were excluded.

15. RT-gPCR

Total RNA was extracted using the Trizol (Thermo Fisher) according to the manufacturer’s protocol. cDNA was prepared using Maxima H Minus First Strand cDNA Synthesis Kit and random oligomer primers (Thermo Fisher Scientific). Real-time qPCR was performed by using QuantStudio 5. The results were normalized to 18s (reference gene) and expressed as fold change relative to untreated or mock-treated controls using the comparative CT method (AACT). The following TaqMan Gene Expression Assays (FAM) (Applied Biosystems, Thermo Fisher Scientific) in combination with the TaqMan Gene Expression Master Mix (#4369016; Applied Biosystems, Thermo Fisher Scientific) were applied: Ifn0 (Mm00439552_sl), Mxl (Mm00487796_ml), Tnfa (Mm00443258_ml) and Rnl8s (Mm03928990_g l ).

16. Statistical Analysis

Statistical analysis was performed by GraphPad Prism 5.0 software. All of the data shown in the histograms were the results of at least three independent experiments and are presented as the mean ± SEM or mean ± SD. The sample size (n) for each statistical analysis and statistical methods used to assess significant differences are indicated in figure legends. Differences between values were considered statistically significant when *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

Results and Discussion:

Bone marrow failure is the primary cause of mortality following irradiation. Hence, protecting the bone marrow is a primary goal in the development of radiation countermeasures. Inflammation is a key outcome and driver of irradiation-induced tissue injury (18). Given its anti-inflammatory effects, it was investigated whether aspirin could protect against radiation. When inoculated into mice, aspirin protected against irradiation- induced bone marrow ablation (Fig. la, and Fig. 2, a-d) and suppressed the induction of inflammatory genes including Ifnbl, Mxl and Tnfa in vivo and in bone marrow-derived monocytes (BMDMos) (Fig. 2, e-g and Fig. 3, a-c).

Pattern recognition receptors (PRRs) including Toll-like receptors (TLRs), the RIG-I-like receptors (RLRs), and the cytosolic DNA sensors (CDS) are central to the initiation of inflammation and cell death5. PRRs signal via key adaptors including MYD88 and TRIF (for TLRs), MAVS (for RLRs), and STING (for CDS) (Fig. 4a). To assess the impact of aspirin on PRR pathways BMDMos were stimulated with specific agonists for PRRs including the TLRs (TLR2: Pam3CSK4, TLR3: Poly(I:C)), RIG-I (Poly(I:C) transfection), cGAS-STING (poly(dA:dT) or cGAMP transfection) and AIM2 inflammasome (poly(dA:dT)). Aspirin inhibited inflammatory gene induction via all these PRRs (Fig. 4, b-f) but not the AIM2 inflammasome (Fig. 5, a-c). To assess if radioprotection by aspirin was due to suppression of PRR-driven inflammation, wild type mice were compared with those defective in PRR signaling. Similar to the wild type (Fig. la, and Fig. 2, a-d), aspirin protected the bone marrows of triple knockout (TKO) mice that are defective in both the TLR and RLR pathways (Myd88-/-Trif-/-Mavs-/-) (Fig. lb, and Fig. 6, a-d), or those defective in cytosolic DNA sensing (Sting-/-) against irradiation (Fig. 1c, and Fig. 7, a-d), and suppressed inflammatory gene expression (Fig. 6, e-g and Fig. 7, e-g). This implied that observed bone marrow suppression was independent of PRR-driven inflammation and that radioprotection by aspirin was uncoupled from its anti-inflammatory effects.

Double-stranded DNA breaks (DSBs) are the most deleterious outcomes of irradiation. Micronuclei are key aftereffects of DSBs6. HEK293 cells are defective in PRR signaling and lack prostaglandin-endoperoxide synthases (COXI and COX2) - also key mediators of inflammation and pain, and the best-known targets of aspirin (16, 21). Aspirin suppressed irradiation- induced micronuclei generation in HEK293 cells (Fig. 8, a, b), indicating that such effect was independent of its anti-inflammatory activity. When irradiated on ice (to prevent spontaneous repair), then transferred from ice to 37 C to allow DNA repair to occur, aspirin pre-treated cells repaired DSBs faster (Fig. Id, e). Aspirin also accelerated DSBs induced by the anti-cancer drug doxorubicin (Fig. 9, a, b).

DSB repair occurs via homologous recombination (HR) and Non-Homologous End Joining (NHEJ). GFP -based reporter systems revealed that aspirin promotes the HR but not the NHEJ (Fig. 10, a-e). BRCA1 and 53BP1 are key checkpoint proteins for the HR and NHEJ repair respectively. Aspirin enhanced recruitment of BRCA1 but not the NHEJ repair protein 53BP1 to DNA damage sites (Fig. If, g and Fig. 11). Accordingly, deletion of BRCA1 significantly blunted acceleration of DSB repair by aspirin (Fig. 12). In contrast ablation of 53BP1 (Fig. 13) or inhibition of the NHEJ kinase DNA-PKc did not (Fig. 14).

Chromatin decompaction is essential for the recruitment of DNA repair machinery to damage sites (22). The N-terminal tail of histone H4 is central for inter-nucleosome interaction (Fig. 16, a). Acetylation of histone H4 at lysine KI 6 (Ac-H4K16) is vital for decreasing the nucleosome-nucleosome stacking and chromatin folding, to permit the recruitment of repair proteins (12). Ac-H4K16 also supports the preferential recruitment of BRCA1 over 53BP1 to damage sites, thereby tipping the balance towards HR. Aspirin- treated cells exhibited elevated Ac-H4K16 and recruitment of BRCA1 but not 53BP1 to DNA damage sites (Fig. Ih, i and Fig. 11, a-d). Consistent with direct donation of acetyl groups to targets, aspirin increased Ac-H4K16 in cells treated with the histone acetyltransferase inhibitor (Fig 15, a, b) or when incubated directly with chromatin isolates (Fig. 15, c). In contrast, acetylated H3K27 was already high at steady state and remained largely unaltered following aspirin (Fig. Ij). Conceivably, given its location at the N- terminal tail of histone H4 where it functions as the first contact point anchoring the H4 tail on the adjacent nucleosome (Fig 16a), H4K16 is likely more accessible for direct acetylation by aspirin.

To examine whether aspirin modulates chromatin compaction, AO3 cells containing genomic insertions of multiple copies of the Escherichia coli (E. coli) lactose operon (LacO) sequence within a heterochromatic region were employed. Upon chromatin decompaction, this region expands and this can be visualized by expressing fluorescent (mCherry)-tagged E. coli lactose repressor protein (LacR) (Fig. 16, b). Aspirin increased Ac-H4K16 and expanded the LacO array (Fig. 1, h-j).

To interrogate further, chromatin from bone marrow cells of mice was isolated. Consistent with the ability to accelerate the resolution of DNA breaks in vivo, aspirin-treated mice had elevated Ac-H4K16 and upon irradiation, exhibited decreased levels of the y-H2A - a marker of DNA damage. When monitored further, aspirin-treated mice had a prolonged survival and exhibited less severe irradiation symptoms (Fig. Ik-o).

The therapeutic effects of aspirin are generally presumed to be due to its ability to suppress inflammation. Mechanistically, aspirin was originally reported to achieve this via the inhibition of COXI and COX27 (21). Here we show that while aspirin also suppresses inflammation by blocking multiple PRR pathways, outside its anti-inflammatory effects, aspirin is a potent amplifier of HR-mediated DNA repair. Our data support a model whereby by acetylating the H4K16, aspirin enhances chromatin de-condensation and thereby enhances the recruitment of HR - repair factors to damage sites.

Conclusion:

DSBs are deleterious to health. In addition to their potential to trigger cell death, if unrepaired or mis-repaired, DSBs can cause chromosome deletions and translocations leading to long-term deleterious effects, including cancer and hereditary disorders. DSBs are also the cause of inflammation (18) - a key driver of the radio/chemotherapy-induced tissue injuries such as fibrosis that significantly impact life-quality of survivors. In addition to accelerating DSB repair, aspirin can also indirectly contribute to genome protection by suppressing inflammation-induced DNA damage. Thus, the abilities of acetylsalicylic acid to inhibit inflammatory pathways and promote the repair of DSBs via HR underscores it potential in the management of inflammatory and genome-instability-driven health afflictions. Further, the discovery that aspirin modulates chromatin structure and repair via histone acetylation offers a new mechanism that may explain some of its many acclaimed health benefits including cancer prevention (15).

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All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety.

While the invention has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various changes and modifications can be made therein without departing from the spirit and scope of the invention.

Claims

What is claimed is:

1. A modified method of precision genome editing that involves the generation of double-stranded breaks (DSBs) in genomic DNA in living cells, said method comprising the step of are concomitantly administering with the generation of said DSBs an amount of a histone acetylating agent effective to induce chromatin decompaction and/or promote genome repair via homology-directed repair (HDR).

2. The method of claim 1, wherein said histone acetylating agent is acetylsalicylic acid.

3. The method of claim 2, wherein said amount of acetylsalicylic acid is effective to induce chromatin relaxation and recruitment DNA repair factors to the DSB site.

4. The method of claim 2, wherein said concomitant administration comprises administration of said effective amount of acetylsalicylic acid prior to the generation of said DSBs.

5. The method of claim 2, wherein said concomitant administration comprises administration of said effective amount of acetylsalicylic acid subsequent to the generation of said DSBs.

6. The method of claim 2, wherein said concomitant administration comprises administration of said effective amount of acetylsalicylic acid simultaneously with the generation of said DSBs.

7. The method of claim 1, wherein said precision genome editing is performed using the CRISPR/Cas-9 system.

8. The method of claim 1, wherein said precision genome editing is performed using bacterial retron library recombineering (RLR), prokaryotic argonautes (pAgos), transcription activator-like effector nucleases (TALENs), or meganucleases.

9. The method of claim 1 , wherein said living cells comprise a cell culture.

10. The method of claim 1, wherein said living cells are in situ in a host organism.

11. The method of claim 9, wherein said host organism is a mammal.

12. The method of claim 9, wherein said host organism is a human.

13. The method of claim 1 wherein said living cells comprise part of a mammalian organ.

14. The method of claim 12, wherein said organ is a human liver.

15. The method of claim 12, wherein said organ is a human bone marrow.