Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery

Reviewer #1:

The CRISPR/Cas9 system is by now a widely used system for site-directed genome editing. Upon site directed cleavage by an RNA-guided CRISPR/Cas9 protein a double strand break (DSB) is introduced. The DSB can be sealed either by error-prone non-homologous end joining (NHEJ) or homology-directed repair (HDR). Typically NHEJ is strongly favored in cells and thus integration of desired DNA into a given genome location by HDR is difficult to achieve. Accordingly, people have tried in the past to identify experimental parameters that can be modified to increase HDR efficiency.

The article under review is very well written and the figures are well designed and clear. However, the Results section of this article contains many numbers. Maybe the authors can identify sections where they can make general statements rather than only listing all data points. The text in its current form is hard to read due to the excessive listing of data.

We agree with the reviewer that excessive listing of numerical data makes reading difficult, and have now clarified the manuscript by removing several data listings and keeping only the numbers that are essential to the discussion.

The manuscript under review focused on optimization of the experimental parameters of CRISPR/Cas9-mediated genome editing in human cells to achieve higher HDR frequencies. The authors take advantage of the fact that the ratio of HDR versus NHEJ differs in different cell cycles. They use a set of chemicals that induce arrest within defined cell cycles. As mentioned by the authors, this idea has been used before in combination with TAL-effector based nucleases (TALENs) (Rivera-Torres, 2014, PLoS ONE 9: e96483). However, it seems that TALENs work is less efficient in combination with cell-cycle arresting chemicals as CRISPR/Cas9 nucleases. The judgment of the authors seems correct. Yet, the fact remains that the conceptual idea is not novel but has been published before.

In addition to the use of cell-cycle arresting chemicals, the authors tested other experimental parameters (e.g. concentration of CRISPR/Cas9, guide RNA and length of the HDR repair template) to improve HDR efficiency. In their studies, they identify various parameters like the CRISPR/Cas9 concentration, which significantly improve HDR efficiency. They also provide data indicating that experimental conditions that improve HDR do not cause increased off-target activity. I believe that the identified experimental conditions will be of use for the huge community of scientists that want to use the CRISPR/Cas9 system for HDR in human cells. Yet, while the improvements are typically significant they remain in most cases marginal. Furthermore, the authors did not test if the used chemicals will have non-desirable side-effects (e.g. high overall mutation rate, increased cell-death frequencies, etc.). I assume the cellular consequences that are linked to the use of these chemicals are in many cases known and thus should be tested experimentally. It should be straightforward to test at least the effect on viability of treated cells. I am convinced that these studies would increase the value of the studies.

We thank the reviewer for offering critical evaluation of our work and we appreciate the potential for side effects when using cell cycle inhibitors. First, we would like to emphasize that the enhancement in editing efficiency is not marginal. We chose to present the direct measure of %HDR, which is capped at 100%, instead of converting the percent readouts into percent increase. Although in some cases the enhancement may appear modest, these changes in %HDR could make a significant difference when deciding to proceed with single cell isolation to obtain homozygous clones.

Second, we now include cell cycle analysis of the HEK293T cells, fibroblasts and hES cells following release from cell cycle inhibitors in Figure 1–figure supplement 1. In all cases, synchronized cells rapidly return to a normal cell cycle. Viability of synchronized hES cells relative to unsynchronized cells when passaged and nucleofected was comparable when sub-cultured at high density, while viability was reduced when sub-cultured at low density. Survival of synchronized cells sub-cultured at low cell density required ROCK apoptosis inhibitor. Importantly, the hES colonies that formed from the synchronized cultures had no apparent changes in colony morphology; all colonies expressed high levels of alkaline phosphatase, a marker for pluripotency.

Reviewer #2:

The CRISPR/Cas9 system is rapidly revolutionizing the field of genome engineering, allowing researchers to manipulate both coding and non-coding genomic sequences at will in a constantly growing number of biological systems. This system creates double-strand breaks (DSBs) at target loci, which can be repaired through one of two cellular mechanisms: non-homologous end joining (NHEJ) or homology-directed repair (HDR). The ability of a cell to repair a DSB generated by Cas9 through HDR-mediated incorporation of exogenous DNA templates has recently been exploited to engineer several modifications to endogenous loci, such as novel knock-in alleles, point mutations and fluorescent tags, among others. However, the frequency of NHEJ is usually higher than HDR due to the fact that NHEJ does not require any homologous or exogenous DNA molecules to repair the DSB. Therefore, developing experimental methods that increase the frequency of HDR is important in order for this technology to fulfill its full potential in various laboratory studies as well as in clinical applications.

Lin et al. have addressed whether cell cycle synchronization might affect the relative use of these two repair pathways in an effort to define conditions that lead to more efficient HDR. The authors tested whether reversible treatment of cells with drugs reported to arrest cells in the S and late G2 cell cycle phases could increase the rate of HDR when combined with timed delivery of Cas9-sgRNA ribonucleoprotein complexes (RNPs) and various exogenous DNA templates. Using six pharmacological agents and further narrowing the list down to two (nocodazole, which is reported to block cells at late G2/M phase, and aphidicolin, which blocks cells at S phase), the authors convincingly demonstrate that these treatments, coupled with timed delivery of Cas9 RNPs and exogenous DNA templates, significantly increased the rate of HDR across two loci in two different cell types. Importantly, they also convincingly demonstrate that off-target editing is negligible using this approach.

This is a significant extension of this groups previous efforts aimed at establishing the CRISPR/Cas9 genome editing system in mammalian cells for inducing both NHEJ and HDR at specific genomic loci (Jinek et al., 2013). The ability to increase the rate of HDR through cell synchronization coupled with timed delivery of Cas9 RNPs will undoubtedly have a significant impact in the field of genome editing, particularly for applications aimed at engineering specific mutations of interest into a variety of cell types, such as human ES and iPS cells.

Major comments:

1) The key observation here is that cells treated and released from different chemical inhibitors of cell cycle progression undergo increased CRISPR/Cas9-mediated HDR compared to untreated cells. While this is clearly shown, the cell cycle effects that are associated with this treatment are not well characterized. Although the drugs employed are commonly used in the field, it is important to characterize them in the particular cell lines studied. The authors show cell cycle analysis in of HEK293T cells in Figure 1–figure supplement 1. However, for both nocodazole and aphidicolin treatment, the data appear to show significant 2n as well as 4n peaks. Thus, it is unclear what cell cycle phase might be associated with the increased HDR observed following release from these treatments. Regarding the experiments with H9 human ES cells, in which a combination of nocodazole and aphidicolin was used, there is no cell cycle analysis shown at all. It will be important to address both of these issues prior to publication.

We now include a complete panel of cell cycle analysis for HEK293T, hES cells and the newly added primary neonatal fibroblasts in Figure 1–figure supplement 1. We also performed alkaline phosphatase assay to ensure the hES cells remain undifferentiated after synchronization. Regarding the HEK293T nocodazole cell cycle block, we have redone the cell cycle analysis and find that indeed the majority of cells are 4N. The same is true for primary fibroblasts and hES cells. Some 4N HEK293T cells are slipping through the M-phase checkpoint and initiating S-phase. This mitotic slippage phenomenon has been observed before in various cell lines treated with nocodazole and other anti-microtubule drugs (Riffell et al., 2009). No mitotic slippage was observed with nocodazole treatment of primary fibroblasts or the hES cells that have intact cell cycle checkpoint regulation.

2) With the exception of the experiments presented in Figure 2C using the H9 cells, most of the experiments were carried out with HEK293T cells, which are readily transfectable with nucleofection methods. Importantly, the rate of HDR reported was significantly lower in drug-treated H9 cells compared to drug-treated HEK293T cells. Moreover, induction of HDR in ES cells required a modification of the protocol to incorporate a 16-hour pulse of nocodazole followed by a 3-hour pulse of aphidicolin before Cas9 RNP nucleofection. One wonders how generalizable these methods will be to other cell types. Therefore, the manuscript would be strengthened with the addition of analysis of a panel of cell lines.

We agree that analysis of other cell lines is important to show that this method is broadly applicable. We now include data for primary neonatal fibroblasts, a cell type with low transfection efficiency. In this cell type, we observed enhanced total editing and HDR with aphidicolin synchronization, in contrast to enhancement with nocodazole treatment as observed in HEK293T and hES cells. Although these findings indicate some variability according to cell type, the cell cycle synchronization procedure itself is often not generalizable across different cell types. Due to variations in physiology, growth rate and duration of cell cycle phases, one needs to determine and optimize the synchronization protocol empirically. Nonetheless, the results presented here establish the feasibility of timed delivery of Cas9 RNPs to enhance rates of site-specific genome editing by homology-directed repair.

3) The authors should establish the baseline nucleofection efficiencies for the different cell lines tested. This will help clarify whether nucleofection efficiency many be a contributing factor in the difference seen between HEK293T cells and H9 cells.

Although nucleofection efficiency is likely to affect observed differences in Cas9-mediated genome editing, we were not able to determine baseline RNP nucleofection efficiencies for these cells.

4) It is unclear whether the other cell cycle inhibitors besides nocodazole shown in Figure 1–figure supplement 1 were tested in ES cells. Minimally, this point should be clarified. If they were not tested, is there a reason why not?

We agree with the reviewer and have now clarified the manuscript by including a statement about cell cycle synchronization in hES cells. In preliminary experiments, we tested hES cells with the six cell cycle inhibitors. The results were disappointing, with only nocodazole showing enhancement in total editing, but no HDR was detected. Therefore, we adopted a method described by Pauklin and Vallier (2013) in which hES cells were treated sequentially first with nocodazole for 16h and then pulsed with aphidicolin for 3h, prior to nucleofection. With this modification, higher levels of total editing and detectable HDR were observed.

5) Given the interest in targeting efficiencies as a function of target loci, it would be useful to extend this study to more than the two loci tested here.

We now include new data showing the editing efficiency in the CXCR4 gene in HEK293T cells. Nocodazole synchronization led to markedly enhanced HDR efficiency. Similar to EMX1 and DYRK1, the most significant increase was observed for cells receiving a lower amount of Cas9 RNP. In this case, nocodazole synchronization yielded 27% HDR at 10 pmol of Cas9 RNP. A comparable level of HDR in the unsynchronized cells would require 100 pmol of RNP. Enhancement of HDR at three different loci demonstrates that this timed delivery of Cas9 RNP is a broadly applicable method in HEK293T cells.

6) In Figure 3B, the authors show that adding aphidicolin following release from a nocodazole block reduced HDR efficiency in HEK293T cells, suggesting that S-phase entry may be required for efficient HRD-mediated repair. They should show that this combined treatment actually did block S-phase entry in these experiments, especially given the odd cell cycle profiles shown in Figure 1–figure supplement 1. Also, how does this conclusion jibe with the increased efficiency of HDR in ES cells treated with this same combination when compared with nocodazole alone?

The experiments in Figure 3B third panel and hES cells involved two different conditions. We thank the reviewer for pointing out the confusion and we have now clarified this point in the manuscript.

In Figure 3B, third panel, HEK293T cells were synchronized with nocodazole prior to nucleofection. Immediately post nucleofection, one dose of aphidicolin was added to the growth media to prevent the transfected cells from proceeding into the S phase. The purpose was to reduce HDR efficiency, since the HDR pathway is thought to be most active during S phase. We labeled this one-time addition of aphidicolin “aphidicolin block” in Figure 3B, as opposed to the standard aphidicolin synchronization procedure used elsewhere in the manuscript. The standard aphidicolin synchronization procedure involves treating the cells with the drug for 17h, releasing the cells for 7-8h, and then treating the cells again for another 17h. Such sequential treatment does not fit our experimental scheme, because cells were harvested 24h after nucleofection for analysis. The goal of this experiment was to demonstrate that aphidicolin reduced the HDR efficiency, instead of attempting to completely abolish the HDR. Our results in Figure 3B second and third panels show that the HDR frequencies were indeed significantly reduced.

The hES cells in Figure 3B were treated differently. As described in the response above, we modified the standard one-drug synchronization procedure in the HEK293T experiments to incorporate two drugs for effective synchronization. We adopted a method described by Pauklin and Vallier (2013) in which hES cells were treated sequentially first with nocodazole for 16h and then pulsed with aphidicolin for 3h, prior to nucleofection. After nucleofection, hES cells were grown in inhibitor-free media.

7) The authors argue in the Discussion that their approach of nucleofection of Cas9 RNPs leads to higher cell viability than DNA transfection-based methods. However, no data is shown to support this claim.

We have now cited two published papers (Kim et al., 2014, and Zuris et al., 2014), both of which have investigated the cell viability between DNA- and RNP-based transfection methods.