Non-homologous end joining (NHEJ), and to a lesser extent, the error-free pathway known as homology-directed repair (HDR) are cellular mechanisms for recovery from double-strand DNA breaks (DSB) induced by RNA-guided programmable nuclease CRISPR/Cas. Since NHEJ is equivalent to using a duck tape to stick two pieces of metals together, the outcome of this repair mechanism is prone to error. Any out-of-frame mutations or premature stop codons resulting from NHEJ repair mechanism are extremely handy for loss-of-function studies. Substitution of a mutation on the genome with the correct exogenous repair DNA requires coordination via an error-free HDR, for targeted transgenesis. However, several practical limitations exist in harnessing the potential of HDR to replace a faulty mutation for therapeutic purposes in all cell types and more so in somatic cells. In germ cells after the DSB, copying occurs from the homologous chromosome, which increases the chances of incorporation of exogenous DNA with some degree of homology into the genome compared with somatic cells where copying from the identical sister chromatid is always preferred. This review summarizes several strategies that have been implemented to increase the frequency of HDR with a focus on somatic cells. It also highlights the limitations of this technology in gene therapy and suggests specific solutions to circumvent those barriers.
The discovery of restriction enzymes enabled researchers to effectively manipulate DNA
Double-strand breaks (DSBs) are unavoidable consequences accompanying cellular life. Living organisms have evolved elaborate repair machineries to fix DSBs whether the source of the break is natural or experimentally induced by Cas9. DSB repair mechanisms are grouped under two major categories: Non-Homologous End Joining (NHEJ) and Homology-directed Repair (HDR) (15). The choice of NHEJ versus HDR as a preferred repair machinery is not arbitrary and several factors contribute to the decision-making. Strategies to manipulate either of these repair mechanisms can affect the outcome of gene editing and should be considered carefully before proceeding to experiment. Resembling the scenario in classical Robert Frost’s poem,
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The current review focuses on several strategies that have been implemented to increase the frequency of HDR in somatic cells and provides a perspective on the challenges that lie ahead for potential application of this technology for gene therapy in humans.
After the generation of DSBs by Cas9, they need to recognized and repaired by evolutionarily conserved cellular DNA repair mechanisms namely NHEJ and HDR. NHEJ is the predominant form of mammalian DNA repair mechanism that successfully joins broken pieces of DNA together (16). Based on the molecular players involved, NHEJ pathway is further divided into canonical non-homologous end joining (c-NHEJ) and alternative non-homologous end joining (alt-NHEJ) also referred to as microhomology-mediated end-joining (MMEJ). The major molecular players in c-NHEJ are ku70/ku80 heterodimer and DNA protein kinase catalytic subunit (DNA-PKcs) (17). The KU70/KU80 heterodimer binds to DSBs along with DNA-PKcs. This molecular assembly protects the DNA wound site and maintains the ends in close proximity, which is critical for rejoining the pieces of DNA together. The auto kinase activity of DNA-PKcs or transphosphorylation by ataxia telangiectasia mutated (ATM) kinase results in the recruitment of Artemis and DNA pol
Alt-NHEJ pathway works independently of KU70/KU80 resulting in larger deletions and chromosome translocations (20). The major molecular player in this form of repair is poly [ADP-ribose] polymerase 1 (PARP1) that competes with KU70/KU80 for binding to DNA ends and thus is refractory to the c-NHEJ. PARP1 facilitates stabilization of γH2AX (phosphorylated form of H2AX) via its ADP-ribosylation activity on nucleosome exchange factor SUPT16H and results in the formation of MRE11-Rad50-NBS1 (MRN) complex tilting the balance of repair machinery away from c-NHEJ in favor of alt-NHEJ (21). Mechanistically, alt-NHEJ resembles HDR because it is favored in the S and G2 phases of cell cycle unlike c-NHEJ that is active in all stages of mitosis. Moreover, the choice of DNA ligase in c-NHEJ is LIG4, whereas in alt-NHEJ, LIG1 and 3 are utilized (22).
Accuracy of DNA repair is strongly enhanced by the utilization of the sequence from the sister chromatid or homologous chromosome, and constitutes the basis of HDR. Since sister chromatids are available at the S/G2, HDR is restricted to these phases of cell cycle (23). The first step in the HDR pathway is phosphorylation of H2AX by ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and Rad3-related protein (ATR) in conjunction with the mediator of DNA damage checkpoint 1 (MDC1) that results in the localized accumulation of γH2AX at the DNA damage site (24). Next, the MRN complex localizes to the DSB and plays a stabilizing role to inhibit chromosomal breaks. Following the stabilization of the initial DSB, the 5′ exonuclease activity by either MRE11/CtIP (short resection) or Exo1/BLM (long resection) results in the generation of 3′ single strand (3′SS) overhangs that are covered by human replication protein A (RPA) (25). Rad51 in conjunction with breast cancer 1 and 2 (BRCA1 and 2) along with partner and localizer of BRCA2 (PALB2), subsequently replaces RPA to form filaments on the DNA. The coating of 3′ overhang by Rad51 initiates the search for the repair template and invasion of homologous DNA/sister chromatid for the start of recombination process (26). When the invading strand coated with Rad51 filaments infiltrates its homologous partner, it results in the formation of displacement loops (D-loops). DNA polymerase delta attaches to the DNA with the help of proliferating cell nuclear antigen (PCNA) and synthesizes the missing piece of DNA. The formation of the new DNA strand results in the formation of Holliday junctions that are resolved by nickases and finally ligated to complete the HDR process restoring the original DNA sequence (27).
Depending on the nature of experimental design, the DNA repair molecular machinery can be tweaked to either favor NHEJ or HDR. In loss-of-function studies, the presence of a guide RNA that dictates Cas9 to cleave at a particular site is sufficient to induce NHEJ. Since NHEJ is error-prone, in many instances the end-product of NHEJ includes missing or added DNA sequences resulting in nonfunctional coding sequence (28). In addition, NHEJ is constitutively active in all stages of the cell cycle and is the predominant form of repair process, thus, animal or cell-based models of loss of function studies can be generated with relative ease. For these studies, the proper choice of guide RNAs to target a specific sequence and elimination of off-target effect should be of critical consideration.
HDR machinery should be utilized for “knock in” studies warranting insertion of a particular base pair or a definite stretch of DNA for enhanced therapeutic potential. However, taming endogenous cellular HDR machinery to insert a desired DNA sequence has proven to be a tricky business and more so in somatic cells (29). The problem is attributed to HDR occurring only in S/G2 phages unlike NHEJ that bears the major proportion of repair load and is active in all stages of cell cycle. Thus, the timing of DSB generation and the presence of repair template at the right time in the right location is of critical importance to create the best scenario for HDR. Generation of germ-line-based “knock-in” animal model has benefited tremendously from Cas9-based activation of HDR pathways compared to classical genome engineering with an infinitesimally small probability of incorporation of donor DNA simply based on sequence homology (30). The relative ease of HDR-mediated transgenesis in germ cells compared to somatic cells stems from the fact that during meiosis copying of the information occurs between homologous chromosomes that might contain specific sequence differences (31). Thus, for the generation of germ-line edited organisms, a piece of donor DNA with sequence homology around the cut site has reasonable possibility (albeit closer to or less than 1%) to be attacked by a genomic 3′ invading strand as a repair template (32). Researchers have used this special condition to generate germ-line edited organisms from flies to pigs (33). However, the scenario is completely different in somatic cells where copying occurs from closely attached identical sister chromatid. Thus, the probability of donor DNA to be used as a repair template after DSB is extremely low in somatic cells (34). Furthermore, in non-dividing cells, the possibility of utilizing HDR in transgenesis is tricky, due to the higher experimental barrier posed by genome editing in these types of cells (35). The major strategies that have been employed to increase HDR include chemical and genetic activation of HDR and suppression of NHEJ, stopping cells at the S/G2 stage of cell-cycle to allow more time for HDR, allowing Cas9-induced DSB only at S/G2 phase by attaching degron to Cas9 for degradation at all stages of the cell cycle where HDR is absent, enrichment of correctly edited cells with a selection marker, and increasing the concentration of donor DNA near the cut site so that the probability of the utilization of donor DNA as a repair template is increased.
It was previously reported that inhibition of NHEJ results in the activation of HDR as a compensatory mechanism after the generation of Cas9-mediated DSB (36). Several researchers have cleverly utilized this knowledge to inhibit NHEJ by blocking the function of key proteins involved either by using chemicals or siRNA. One of the promising studies demonstrated Scr7-mediated inhibition of NHEJ-specific LIG4 that resulted in nearly 20-fold enhancement of HDR (37). However, several follow-up studies showed that the use of Scr7 is not as promising in increasing HDR as previously described suggesting that results varied depending on the experimental systems (38). Another alternative explanation is the observation that LIG4 might not be a specific target of Scr7 (39). Nonetheless, choking other critical molecules involved in NHEJ pathway remains an attractive strategy to increase HDR. Some examples of this strategy include the use of molecules such as NU7441 (40) and Ku-0060648 (41) that inhibit DNA-PKcs. A more direct approach to increase HDR is to utilize Rad51 activator RS1 that increases CRISPR/Cas9 and TALEN-mediated knock-in efficiency (42).
Similarly, the ectopic expression of HDR molecules such as Rad51 enhances HDR (43). A recent study also demonstrated that fusing Cas9 with a domain of CtIP resulted in enhanced HDR probably by rapid positioning of the components of HDR machinery around the cut site (44). This study demonstrates that an appropriate experimental design to increase HDR should consider the optimized space and time for cutting and delivering the donor DNA.
Another approach to increase HDR is to use siRNA or shRNA to knock down key NHEJ effectors. In one such case, shRNA-mediated knockdown of Ku70, Ku80, and Lig4 individually or in combination resulted in suppression of NHEJ and 2-to-5-fold increase in HDR (37). Additional molecular players such as DNA-PK were silenced by Robert
Since HDR occurs in S/G2 phase, a straightforward strategy to increase the probability of HDR would be to ensure that all the events such as Cas9-mediated splicing at the target site occur at these stages of cell cycle. This strategy is particularly important because the continued activity of Cas9 in other stages of cell cycle increases the chances of error-prone NHEJ, basically excluding the target site from integrating the correct repair sequence. Based on this strategy, the Doudna laboratory demonstrated that G2/M synchronization of cells using nocodazole for 24 h followed by nucleofection of Cas9–gRNA ribonucleoprotein (RNP) and single-strand oligo deoxynucleotide (ssODN) repair template at the
Transgenesis is extremely challenging in cells that are terminally differentiated because of lack of HDR activity. While studying the mechanism of HDR suppression in these cells, Orthwein
Even though NHEJ is vilified in the world of precise genome engineering as being error-prone, it is actually an overstatement. Cas9 cleavage of target site is invariably followed by NHEJ repair, and the process is repeated until there is an error, which prevents Cas9-mediated DNA cleavage (53). Thus, the end-product of NHEJ observed might be an error, although NHEJ intrinsically does not result in such errors. This basic idea has been exploited to generate Homology-Independent Targeted Integration (HITI) and Obligate Ligation-Gated Recombination (ObLiGaRe) by utilizing NHEJ for precise gene repair in non-dividing cells. HITI is a knock-in strategy that utilizes NHEJ-based ligation of donor DNA (54). In this strategy, the donor sequence is flanked by a single circular donor or two linear donor sgRNA cleavage sites. A third identical sequence in the target locus present in the reverse orientation basically prevents reverse integration of the donor DNA.
ObLiGaRe utilizes mutant variants of FokI domains fused to ZFNs or TALENs that induce cleavage only when paired as heterodimers but not as homodimers, and facilitates the ligation of donor DNA into the genomic cleavage site (55). In this system the target site in circular donor plasmid is identical to genomic DNA, but in reverse orientation. This strategy excludes the possibility of repeated cleavage of donor DNA after accurate insertion.
After the 3′SS DNA is coated with Rad51, the homologous DNA sequence is used as a repair template (32). In somatic cells, the copying occurs from the sister chromatid whereas in germ cells it starts in the homologous chromosome. By supplying the donor DNA with homology-arms around the target site, a researcher can hope that the donor DNA externally supplied is used as a repair template. Since the probability of invading genomic 3′SS DNA to attack donor DNA is very low, several recent studies have positioned donor DNA near the target site instead of randomly floating them in the nucleoplasm and this strategy has been successful to increase the HDR efficiency. Following this theory, a recent report demonstrated that the fusion of Cas9 with the donor DNA using SNAP-tag resulted in a 24-fold increase in HDR (56). A similar study conjugated donor DNA with Cas9 via biotin-mono-avidin resulted in 2-to-5-fold increase in knock-in mice generation (57).
A recent study performed the localization of donor DNA near the target site using biotin-avidin conjugation to another level by delivering the components of genome editing (streptavidin Cas9, guide RNA, and biotinylated donor DNA) in the two-cell stage of mouse development, the stage at which G2 phase is the longest, and demonstrated increased HDR in most of the loci tested (58). Collectively, increasing the local concentration of donor DNA at the cut site increases the probability of use as a repair template, hence increase in HDR frequency.
Another approach to obtain higher percentage of cells that have undergone correct HDR entails a classical gene targeting strategy: to enrich the cells by positively selecting them via insertion of an antibiotic resistance gene or any selectable marker, and landing them in a safe-harbor locus such as
Several strategies have been implemented to increase the efficiency of HDR in general and with some urgency in somatic cells. Since germline editing raises ethical concerns associated, our ability to rewrite the erroneous genetic code in somatic cells with the correct genetic information holds enormous therapeutic potential. Elegant studies conducted within a span of several years have pinpointed several criteria such as inhibition of NHEJ, activation of HDR, controlled editing in HDR permissive stages of cell cycle, and increasing local concentration of donor DNA near the target cleavage site as playing an important role to boost HDR in somatic cells. Further studies should focus on finding the optimum combination of conditions that is non-lethal in order to maximize HDR.
Several elegant studies have pinpointed the potential shortcomings of CRISPR-Cas9-mediated genome-engineering technology in general, and more specifically for therapeutic applications. In addition to the lower efficiency of HDR in CRISPR-Cas-mediated transgenesis, three critical challenges need to be addressed to ensure a giant transition of this technology from ‘bench to bed’. The first challenge is the immune reaction that is already activated in humans against the most widely used forms of Cas9. A recent study demonstrated that majority of humans have already developed immune reaction against Cas9 from
Even though 6 years is a relatively short time frame to judge the success of a technology, there is no doubt that CRISPR/Cas system has revolutionized genome engineering. As it is with every promising technology in the history of mankind, the hope CRISPR-Cas9 has generated must be matched with rigorous quality control experiments to increase its efficiency and reduce side effects before its potential application in gene therapy can be fully realized.
The authors have no conflicting interests.