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Modulating DNA repair pathways to improve precision genome engineering. Katherine S Pawelczak, Navnath S. Gavande, Pamela S. VanderVere-Carozza, and John J. Turchi ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.7b00777 • Publication Date (Web): 06 Dec 2017 Downloaded from http://pubs.acs.org on December 7, 2017

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Modulating DNA repair pathways to improve precision genome engineering. Katherine S. Pawelczak1, Navnath S. Gavande2, Pamela S. VanderVere-Carozza2 and John J. Turchi1,2,3 1

NERx Biosciences, 212 W 10th Street, Suite A480, Indianapolis, IN 46202

2

Department of Medicine and 3 Department of Biochemistry and Molecular Biology

Indiana University School of Medicine 980 W Walnut Street R3 C560 Indianapolis, IN 46202

ABSTRACT Programmable nucleases like the popular CRISPR/Cas9 system allow for precision genome engineering by inducing a site-specific DNA double strand break (DSB) within a genome. The DSB is repaired by endogenous DNA repair pathways, either non-homologous end joining (NHEJ) or homology directed repair (HDR). The predominant and error-prone NHEJ pathway often results in small nucleotide insertions or deletions that can be used to construct knockout alleles. Alternatively, HDR activity can result in precise modification incorporating exogenous DNA fragments into the cut site.

However, genetic recombination in mammalian systems

through the HDR pathway is an inefficient process and requires cumbersome laboratory methods to identify the desired accurate insertion events. This is further compromised by the activity of the competing DNA repair pathway, NHEJ, which repairs the majority of nuclease induced DNA DSBs and also is responsible for mutagenic insertion and deletions events at off-target locations throughout the genome. Various methodologies have been developed to increase the efficiency of designer nuclease-based HDR mediated gene editing.

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towards modulating the activities of the two critical DNA repair pathways, HDR and NHEJ, to enhance precision genome engineering.

Programmable Nuclease Technology The development of programmable nucleases has greatly enhanced the world of precision genome engineering, and the ability to make targeted modifications has opened up a wide variety of options for scientists in both therapeutic and biotechnology disciplines in industry and academia. Several classes of nucleases have been developed, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and the more recent and popular CRISPR/Cas9 tools. CRISPR/Cas9 is particularly popular due to the ease of design and readily available reagents. However, all of these tools share the common modality of being capable of inducing a site-specific DNA double strand break (DSB) anywhere in the genome


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(1;2).

Following creation of the DNA DSB, independent of the mechanism and nuclease

employed, one of two competing endogenous cellular pathways is engaged to remedy the break. The genetic alteration that occurs in each of the individual cells is a function of the pathway that cell utilized to repair the DSB. (3-5)

Figure 1. Non homologous end joining. Following creating of a DSB by a programmable nuclease like TALEN, ZFN or CRISPR/Cas9, the NHEJ pathway is initiated by the binding of the Ku heterodimeric complex. A synaptic complex is formed, bringing the two ends into close proximity, and DNA-PKcs is activated. Processing of the DNA termini by polymerases and/or nucleases occurs and small insertions or deletions are formed at the DNA ends. Following end processing, the Ligase IV/XRCC4/XLF complex is recruited to DNA termini and catalyzes ligation of the DSB, resulting in a repaired dsDNA that contain small mutagenic changes at the location of the nuclease induced DSB.



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NHEJ catalyzed DSB repair Following induction of a DSB the NHEJ pathway is initiated by the binding of the Ku heterodimeric complex (Ku 70/80) to the DNA termini (Figure 1). Once bound, Ku binds and activates the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). DNA-PK activation is dependent on a physical interaction with Ku at the DNA DSB, and DNA termini structure and sequence influence activity (6;7). DNA-PK coordinates NHEJ through autophosphorylation and phosphorylation of other target proteins, leading to DNA end processing by nucleases (Artemis), polymerases (Pol X family), polynucleotide kinase (PNK), and tyrosyl-DNA phosphodiesterase (TDP1).

Following end processing, the DNA Ligase IV/ x-Ray Cross Complementing-4

(XRCC-IV)/XRCC4-like factor (XLF) complex is recruited to DNA termini and catalyzes ligation of the DNA DSB (8;9).

HDR catalyzed DSB repair and gene insertion The canonical HDR pathway is considerably more complicated and requires numerous biochemical factors (Figure 2). HDR is initiated by the MRN complex binding to the DNA DSB. Resection by endonucleases (CtIP) occurs, resulting in long 3’ single stranded DNA (ssDNA) fragments that are coated by replication protein A (RPA). In a critical step to HDR, Rad51 replaces RPA bound to ssDNA and forms a nucleoprotein presynaptic filament and facilitates a search for a homologous donor. A heteroduplex DNA structure is formed and dissociation of Rad51 accompanies the synthesis and final ligation step (10;11).


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Figure 2. HDR mediated gene insertion.

Following the generation of a site-specific DSB the

CtIP (depicted in grey) and MRN nucleases engage the break and resect the DNA in the 5-3 direction leaving a single strand DNA overhang that is bound by RPA (Blue). In a BRCA2 dependent manner, Rad51 (Pink) replaces RPA and initiates the homology search. Left and right homology arms designed to flank the gene of interest (GOI) provide the site of strand invasion. DNA synthesis (indicated by dotted lines) and ligation creates a double Holliday junction



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molecule that upon branch migration and resolution results in the accurate insertion of the GOI into the site of the DNA DSB. Exploiting NHEJ and HDR for genetic modification NHEJ can be an error-prone pathway that often results in nucleotide insertions or deletions at the site of the DSB and these genetic alterations are useful for generating targeted knockouts. NHEJ does not require a homologous donor molecule and is the dominant pathway for repair of DNA DSB in mammalian cells (12). This extremely efficient, relatively simple pathway is active throughout the entire cell cycle, unlike HDR which is typically restricted to S and G2 phase of the cell cycle. In the presence of a homologous donor sequence, HDR results in accurate insertion of the donor molecule at the DSB site.

Exploiting the HDR pathway following

induction of DNA DSB by CRISPR/Cas9 is another popular method that has significant advantages in precision genome engineering, most importantly because it can be used to accurately insert exogenous pieces of DNA into a genome (13). This technique has opened up a large range of technical capabilities for a variety of industries, allowing researchers to create precise insertions or deletions, generate SNPs, insert sequences for epitope tags, and even insert entire genes to create genetically modified organisms. Despite the relatively straight-forward CRISPR/Cas9 and HDR method to create a site-specific modification, the activity of this DNA repair pathway is extremely low, and this directly and negatively impacts the efficiency of precision genome engineering applications.

Low HDR mediated gene targeting efficiency

necessitates extensive experimentation to identify a single modified clonal cell. Increasing the efficiency of HDR would render CRISPR/Cas9 genome engineering a faster, easier and more accurate process and render CRISPR/Cas9 based technologies more useful to industry and academia alike.


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Modulation of HDR activity to increase precision genome engineering: As it has become increasingly obvious that enhancing HDR activity would greatly increase the efficiency of nuclease-based genome engineering tools, several methods have been adopted to elevate DNA repair activity. Numerous groups have produced variants of the Cas9 enzyme that modulate genome editing activity and impact gene insertion efficiency (14).

Studies show that

electroporation of a ribonucleoprotein (RNP) made up of Cas9 protein and gRNA can increase NHEJ mediated gene editing events in a variety of organisms (15-17) as well as increase HDR mediated gene insertion (18;19) by 2-6 fold. Furthermore, new genetic information can be incorporated site-specifically and with high efficiency by increasing HDR activity using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes with the various cell cycle arrest drugs (Figure 3). As HDR is restricted to S and G2 phases of the cell cycle, cell cycle synchronization using Nocodozole and ABT-751 (ABT) has been shown to enhance HDR mediated gene-insertion (20;21), with widely varying efficiencies dependent on conditions (Figure 3).



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Figure 3. Modulation of HDR mediated Cas9 genome editing through cell cycle synchronization: Nocodazole and ABT causes cell cycle arrest at G2/M phase; Lovastatin blocks at early G1 and partially at G2/M phase and L-Mimosine, Aphidicolin, Hydroxyurea and Thymidine arrest cells at the G1-S border prior to onset of DNA replication.

Alternatively, synchronization of Cas9 expression by creating a Cas9-Geminin fusion protein that decreases expression in G1 but increases Cas9 expression in G2/S/M has also been shown to increase HDR mediated gene insertion (22). Manipulation of donor structure, including utilizing varying homology arm lengths in a double-strand plasmid donor (23;24), using single-strand DNA donor molecules in the form of long oligonucleotides (25) or generated using rAAV (26), or linearizing a donor molecule in-vivo through a user-designed cut site (27), have all been suggested to increase HDR mediated gene insertion efficiency. Finally, some groups claim greater success when two or more of these described methods are combined (28). It is critical to


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recognize that while many of the methods described above may have a positive effect on HDR efficiency, design parameters for genome engineering experiments have continuously shown to be dependent on variable biological and experimental characteristics including cell and organism type, genomic location and experimental design. Because of the extensive erraticism observed in precision genome engineering experiments, a strong need exists for the development of a universal method to increase HDR that could be applied across a variety of experiments in a simplistic fashion.

Chemical modulation of DNA repair The use of small molecules provides a simple method for enhancing precision genome engineering that has several advantages. Small molecules can often have highly penetrant effects that result in a rapid as well as controlled response. Furthermore, titration experiments can easily be done to ensure optimal concentrations of inhibitors are delivered to the cell with highly effective results. This is particularly useful in gene editing experiments in different cell types, where repair pathways may have different levels of activity. Chemical inhibitors are often reversible, an optimal detail as the genetic background of the cells being used is unchanged, a point not guaranteed with genetic manipulation. This also reduces any potential toxicity concerns that may arise within a system, as the compound is reversible and can be removed before any cell viability effects occur. Finally, use of chemical inhibitors is applicable to nearly all model systems, as delivery is easy and chemical compounds readily penetrate a variety of cell types. This is appealing for use with CRISPR mediated gene editing, as this technology is applicable to a wide range of cellular and model systems.



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Figure 4. RS-1 increases HDR by stimulating Rad51 binding to DNA. Various attempts have been undertaken in the field to chemically enhance HDR. A reporter based screen (29) and a nuclear domain “knock-in” screen (30) were developed to identify small molecules enhancers of HDR. L755507 and Brefeldin A were identified as capable of enhancing CRISPR-mediated HDR in human induced pluripotent stem cells, although the mechanism of action remains unclear (29). An additional study with L75507 found it increased HDR mediated gene insertion by 2 fold in porcine fetal fibroblasts (31).

A particularly

interesting compound identified from the knock-in screen (30)was RS-1, a compound that stimulates the DNA binding activity of the critical homologous recombination protein Rad51 and thus enhances recombination activity (Figure 4). This compound was further explored and results show a modest increase in efficiency (2-5 fold) in rabbit embyros (32). Interestingly, several groups have reported minimal increase in efficiency when RS-1 was used in various other organism and cell types. One group showed no increase in HDR mediated gene insertion when Rad51 was overexpressed (27), suggesting that perhaps the biological target of RS-1 is not useful for increasing precision genome engineering applications.

Modulation of NHEJ activity to enhance HDR mediated genome engineering: Numerous studies have shown that HDR activity is severely limited by the competing activity of NHEJ, and furthermore that HDR activity can be enhanced by inhibiting NHEJ (33-35). This observation has played out in the genome engineering world as well, where proof of concept studies have


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shown that inhibiting critical NHEJ proteins through genetic modifications as well as siRNA can result in an increase in HDR mediated genome engineering (15;28;36-38), highlighting the utility of inhibiting NHEJ in the genome engineering field. Furthermore, NHEJ activity is responsible for the non-specific insertion of a donor DNA molecule into random DSBs that occur naturally throughout the genome. This is problematic and can result in an increase in background when screening for genetic modifications, as well as create off-target genetic events. These NHEJ mediated off-target events make it even more difficult to identify a single, accurately modified, clonal cell, and will inevitably result in even lower gene targeting efficiency. Finally, off-target mutagenic effects are a great object of concern in the field, particularly since a recent publication reported an alarmingly high rate (> 1,500 single nucleotide mutations observed) and are a product of NHEJ activity at Cas9 induced non-specific DSB sites (39). Inhibiting NHEJ holds the potential to not only increase the specificity of HDR mediated gene editing but also decrease random integration of donor molecules and decreases mutagenic off-target effects that occur from NHEJ mediated inaccurate rejoining of DNA DSBs.



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Figure 5. Structure of L189 and SCR7 pyrazine.

DNA ligation mediated by Ligase IV is a critical final step in the NHEJ pathway, and inhibition of this step should halt completion of NHEJ mediated DNA repair. This is evidenced by various Ligase IV deficient mutants, created by siRNA or proteosomal degradation, that have substantially reduced NHEJ activity (40;41). These studies also examined the effects of a popular proclaimed Ligase IV inhibitor, SCR7 (Figure 5), and results showed an increase in the efficiency of CRISPR/Cas9 and HDR mediated gene targeting in mammalian cells (40;41). Interestingly, multiple groups since then have reported negligible effects on genome engineering experiments in the presence of SCR7 (15;21;22;27;32). Albeit, some groups have reported seeing a modest increase in efficiency of HDR mediated gene insertion when SCR7 is combined with other genome engineering enhancing techniques like cell synchronization or optimized Cas9 delivery (36). However, inhibiting Ligase IV by overexpression of a protein complex that mediates ubiquitination and proteasomal degradation of Ligase IV resulted in a greater increase in targeting frequency (~35%) (40). This supports the concept of inhibiting NHEJ to increase genome engineering, but also highlights the limitation of the Ligase IV inhibitor which is not fully effective in inhibition of NHEJ. SCR7 was initially developed from compound L189 (42) by Raghavan and co-workers in 2012(43). Both compounds, L189 and SCR7 are non-specific ligase inhibitor that exhibits much greater potency and selectivity to Ligase III and Ligase I (44). Interestingly, a world renowned expert in the DNA ligase field reported that attempts to synthesize SCR7 by the published procedure were plagued with synthesis problems, and they discovered discrepancies in the original reported structure of SCR7. Very recently, Greco and co-workers carried out extensive structural determination experiments and confirmed that


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original SCR7 only exist in more stable cyclized SCR7 pyrazine form (Figure 5) and not in the bis-imine form (44). Surprisingly, SCR7 pyrazine and it’s derivatives failed to inhibit DNA ligase IV-dependent V(D)J recombination in a cell-based assay. These results could explain the reported nominal and variable effects observed with SCR7. Overall, it likely appears that SCR7 is causing above effects by mechanisms other than inhibition of LigIV and it is safe to rule it out as a specific LigIV inhibitor.

Figure 6. NU7441 and KU-0060648 increases HDR by inhibiting DNA-PK catalytic activity in the NHEJ pathway.

Other attempts to inhibit NHEJ have focused on targeting critical proteins upstream in the pathway from Ligase IV. Several commercial inhibitors are available targeting DNA-PK (45), a serine/threonine protein kinase responsible for initiating the NHEJ pathway (Figure 1). The DNA PK holoenzyme consists of the 469kDa catalytic subunit, DNA-PKcs, and the Ku70/80 DNA binding subunit. Activation of the kinase follows binding to a DNA terminus and is mediated by numerous protein-protein and protein-DNA interactions. It is well understood that NHEJ mediated repair relies on DNA-PK activity, (46-48) and it has been shown through both



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small molecule inhibition and studies with genetic variants of DNA-PKcs that inhibiting this kinase results in an increase in HDR.

More recent studies have shown that use of small

molecules NU7441 or KU-0060648 (Figure 6) reduce the frequency of NHEJ and increase the rate of HDR at a DSB induced by Cas9 cleavage. Efficiencies varied dependent on the donor structure used, but the most robust increase in insertion observed was 3-4 fold higher with singlestrand antisense oligonucleotides (38). Similar experiments performed with SCR7 revealed only a 1.5-2 fold increase in insertion efficiency. These studies suggest that targeting an NHEJ protein that lies upstream of the ligation step holds the potential for a greater increase in HDR mediated gene insertion. Many attempts have been made to chemically inhibit DNA-PK, with molecules showing varying degrees of success via targeting the ATP binding site of the kinase domain. Active site-targeted agents have limitations, including specificity due to conserved catalytic mechanisms across kinase families, similar structural features of active sites, and the high intracellular ATP concentrations relative to the cellular concentrations of kinase inhibitors (49). NU7441 and KU-0060648 show greater specificity than their predecessors, but it is likely that they still display off-target effects that could be contributing to the observed increases in genome engineering.

Another biological target in the NHEJ pathway, the heterodimeric Ku complex exhibits a high binding affinity for double-strand DNA termini and high resolution structural analysis reveals a ring-like shape that does not undergo any major conformational change upon binding DNA (50). This ring-like structure allows for Ku to thread onto the end of a DNA duplex with ~1.5–2 turns of DNA spanning the channel. This Ku-DNA interaction is essential for kinase activation and is a viable target for intervention. Ku is a particularly alluring target for genome


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engineering because it is responsible for initiation of the NHEJ pathway by binding to the termini of broken DNA (Fig 3).

Thus inhibiting the initial molecular event in the NHEJ

pathway, Ku interactions with DNA ends, is anticipated to efficiently block NHEJ catalyzed repair and drive the processing enzymes to allow HDR mediated recombination with the appropriate donor DNA molecule. This point has been demonstrated genetically by knocking down Ku expression and observing increased CRISPR/Cas9 mediated recombination (36;38;40). The advantage of targeting Ku lies in its early role in NHEJ. Inhibition of the final step, ligation, allows incomplete NHEJ processing of the DSB but renders the break unable to be repaired by HDR as well. The advantage of targeting Ku is that the DSB remains unprocessed and eligible for HDR engagement and thus represents a true increase in the number of cells that are capable of HDR activity. Therefore, cells that have not been repaired by NHEJ and have not engaged the HDR system are destined for cell death, resulting in an increased sensitivity to propagate cells with the desired genetically modified sequence. This in turn would significantly reduce the workload needed to identify the appropriately modified cell populations. Despite the crucial role of Ku early in the NHEJ pathway, the only record of an inhibitor targeted to the Ku protein results from an in silico screen of a commercial library (51) and identifies Vitas-M STL127705 compound as Ku70/80 and DNA-PK inhibitor in low micromolar range. This core scaffold might be useful in generating new drug-like agents, though the compound’s ability to block NHEJ catalyzed DNA DSB repair is not documented to date.



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Figure 7. Ku70/80 and DNA-PK inhibitor, Vitas-M STL127705.

Recently, our group has developed a class of compounds that abolish the Ku-DNA end binding activity in vitro at low micro-molar concentrations.

DNA-PK catalytic activity is

absolutely dependent on the Ku-DNA interaction and so Ku inhibition is expected to inhibit DNA-PK kinase activity as well.

A representative experiment is presented in Figure 8.

Compounds 5102 and 5135 differ by a single Cl group which has a modest effect of Ku binding (panel B and C, circles) and on kinase activity (Panel C, triangles).


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Figure 8. A) Structure of Ku inhibitory compounds. B) Inhibition of Ku binding as assessed by EMSA. Purified Ku heterodimer was incubated with the indicated concentrations of inhibitors and binding to a duplex DNA assessed as we have previously described (6;7;52;53) C) Analysis of DNA-PK catalytic activity and effect of Ku inhibitors. Purified heterotrimeric DNA-PK was incubated with the indicated concentrations of inhibitors and kinase activity measured as we have previously described (6;7;53). Interestingly, there is greater than 90% inhibition of kinase activity at 1 µM while Ku binding retains ~50% activity at 3 µM 5102. The apparent increase in potency observed in the kinase assay compared to the DNA binding assay is likely a function of the different assay systems. The DNA binding assay is an equilibrium binding assay while DNA-PK kinase assays are catalytic. Thus, blocking a single Ku-DNA interaction can influence the catalysis of many phosphoryl transfer reactions. Our lead molecules show low nanomolar inhibition of DNA-PK kinase activity. In addition, in vivo studies confirm decreased Ku foci formation in the presence of the inhibitors as well as decreased NHEJ mediated re-circularization of a plasmid molecule



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(unpublished data). Target engagement studies conclusively demonstrate that the cellular effects observed are a function of Ku inhibition, and this data supports the specificity and potency observed in in vitro experiments. CRISPR/Cas9 genome engineering experiments targeting the EMX1 gene with a donor molecule containing 800bp homology arms flanking a ~2 kb payload were conducted in the presence and absence of Ku inhibitor. Results showed a 6-fold increase in HDR mediated gene insertion in the presence of Ku inhibitor. It has not yet been determined if this increase in efficiency is a result of a direct increase in HDR or is simply a result of a decrease in NHEJ mediated off-target gene insertions that would otherwise increase the false positive and make screening considerably more difficult. Regardless, targeting Ku to increase HDR will be highly useful to the genome engineering community, as Ku is an extremely well conserved protein in the NHEJ pathway and is potentially applicable to various model systems in different species outside of humans including agricultural and biotechnology fields.

Conclusions and future directions: HDR mediated precision genome engineering provides scientists with a tool to precisely modify genomic DNA in a controlled fashion. Increasing the efficiency of this powerful tool would be useful all aspects of genome engineering applications. Increasing HDR while inhibiting NHEJ activity has further advantages, including decreasing non-specific gene editing events and reduce the downstream workload required to identify cells of interest. This is particularly impactful at the regulatory level for researchers using the technology to generate genetically modified products for use in the clinic. It has also been shown that NHEJ is at least in part responsible for random integration events that are observed when transfecting cells with donor DNA molecules (54-56). These events appear to increase in the presence of an engineered nuclease that induces


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DNA DSBs (REF). Decreasing these random integration events will allow for easier screening to identify rare targeting events, as researchers will not have to screen large populations of recombinants to eliminate the non-specific gene insertion events. Two groups recently reported the role of polymerase theta in off target integration of exogenous DNA (57;58). Both groups reported that in a genetic background deficient in pol theta and a NHEJ component (Ku or Ligase IV), random integration events were eliminated completely and gene targeting efficiency utilizing HDR was increased to 100%. These results strongly suggest that pharmacological approaches to inhibiting pol theta and critical NHEJ proteins like Ku could drastically shift the paradigm that exists with gene targeting efficiency as well as improve the safety of gene targeting methodologies for industry and health care.

Acknowledgements: We would like to thank T. Vernon for analysis of DNA-PK inhibition. Additionally, we would like to thank the members of NERx Biosciences and the Turchi lab at Indiana University for their editorial and intellectual contributions.

Key Words: CRISPR: Clustered regulatory interspersed short palindromic repeats; a bacterial family of DNA sequences that play an important role in the bacterial defense system. Cas9: a DNA endonuclease found in Type II CRISPR systems; utilizes CRISPR guided RNA to generate a DNA double strand break at precise locations. Homology Directed Repair (HDR): Cellular DNA repair pathway responsible for repairing DNA double strand breaks; repair is dependent on the presence of DNA containing sequence homologous to DNA flanking double strand break. Non homologous end joining (NHEJ): DNA repair pathway responsible for repairing DNA DSBs that are not repaired by HDR. Ku 70/80: Heterodimeric protein that binds DNA double strand termini and plays a critical role in the initiation of NHEJ mediated DSB repair. Ligase IV: Protein responsible for the NHEJ catalyzed ligation. Gene insertion: HDR mediated insertion of exogenous DNA molecules into a DNA cleavage site created by CRISPR/Cas9 technology. Chemical modulation: The use of small molecules to increase cellular activities.



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Modulating DNA Repair Pathways to Improve ... - ACS Publications

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