- Open Access
Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells
- Yuanming Wang†1, 2,
- Kaiwen Ivy Liu†2,
- Norfala-Aliah Binte Sutrisnoh2, 3,
- Harini Srinivasan1, 2,
- Junyi Zhang1, 2,
- Jia Li1, 2,
- Fan Zhang2,
- Charles Richard John Lalith2,
- Heyun Xing1,
- Raghuvaran Shanmugam1, 2,
- Jia Nee Foo2, 4,
- Hwee Ting Yeo2, 3,
- Kean Hean Ooi1, 3,
- Tore Bleckwehl1,
- Yi Yun Rachel Par2, 5,
- Shi Mun Lee1, 2,
- Nur Nadiah Binte Ismail2, 6,
- Nur Aidah Binti Sanwari2, 6,
- Si Ting Vanessa Lee2, 6,
- Jan Lew2, 6 and
- Meng How Tan1, 2Email authorView ORCID ID profile
© The Author(s). 2018
- Received: 17 June 2017
- Accepted: 7 May 2018
- Published: 29 May 2018
While CRISPR-Cas systems hold tremendous potential for engineering the human genome, it is unclear how well each system performs against one another in both non-homologous end joining (NHEJ)-mediated and homology-directed repair (HDR)-mediated genome editing.
We systematically compare five different CRISPR-Cas systems in human cells by targeting 90 sites in genes with varying expression levels. For a fair comparison, we select sites that are either perfectly matched or have overlapping seed regions for Cas9 and Cpf1. Besides observing a trade-off between cleavage efficiency and target specificity for these natural endonucleases, we find that the editing activities of the smaller Cas9 enzymes from Staphylococcus aureus (SaCas9) and Neisseria meningitidis (NmCas9) are less affected by gene expression than the other larger Cas proteins. Notably, the Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) are able to perform precise gene targeting efficiently across multiple genomic loci using single-stranded oligodeoxynucleotide (ssODN) donor templates with homology arms as short as 17 nucleotides. Strikingly, the two Cpf1 nucleases exhibit a preference for ssODNs of the non-target strand sequence, while the popular Cas9 enzyme from Streptococcus pyogenes (SpCas9) exhibits a preference for ssODNs of the target strand sequence instead. Additionally, we find that the HDR efficiencies of Cpf1 and SpCas9 can be further improved by using asymmetric donors with longer arms 5′ of the desired DNA changes.
Our work delineates design parameters for each CRISPR-Cas system and will serve as a useful reference for future genome engineering studies.
- Genome editing
- Non-homologous end joining (NHEJ)
- Homology-directed repair (HDR)
- Cas9 nucleases
- Cpf1 nucleases
The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas system is a versatile tool that has been successfully used to modify the genome of myriad organisms [1–10]. In this system, an endonuclease (typically Cas9 or Cpf1) is recruited to a specific genomic locus by a chimeric single guide RNA (sgRNA), which comprises both a crRNA spacer that recognizes the target site by reverse complementary base pairing and a stem loop-containing scaffold for the nuclease [11–13]. Additionally, the target locus must carry a short sequence signature, known as the protospacer adjacent motif (PAM), before DNA cleavage can occur. Hence, the PAM places a constraint on which parts of a genome may be cut by a particular Cas nuclease. Nevertheless, Cas enzymes from different bacteria species generally recognize different PAMs. Therefore, by incorporating various CRISPR-Cas systems into our engineering toolbox, we can expand the range of sites to target in a genome.
Distinct endogenous DNA repair pathways are harnessed to achieve desired genome engineering outcomes [12, 13]. Typically, after the Cas endonuclease cleaves the DNA at the target site, the double-stranded break is repaired by either the non-homologous end joining (NHEJ) pathway or the homology-directed repair (HDR) pathway. The former is activated in the absence of a repair template. Being error-prone, it frequently introduces insertions or deletions (indels) during the repair process, which may result in frameshift mutations and gene knockouts. On the other hand, the latter is activated when a homologous repair template is supplied. Precise DNA changes are specified in the template and are hence incorporated into the target locus with high fidelity by the HDR pathway. However, NHEJ is the dominant repair pathway in higher eukaryotes. Consequently, precise genome editing via homologous recombination (HR) usually occurs at a very low frequency.
Several CRISPR-Cas systems from different bacterial species have been deployed for genome engineering in human cells [1, 2, 4, 14–17]. So far, the vast majority of studies utilize SpCas9 for both NHEJ-mediated and HDR-mediated genome editing largely because it is the first Cas endonuclease to be successfully used in human cells [1, 2, 4] and is also the best characterized enzyme to date. Additionally, the nuclease’s relatively simple NGG PAM requirement contributes to its popularity for genome engineering. However, a major disadvantage of SpCas9 is its relatively large size (1368 amino acids), which hinders certain in vivo therapeutic applications. Some Cas9 homologs, including SaCas9 (1053 amino acids) and NmCas9 (1082 amino acids), help alleviate the size issue, but they typically require more complex PAMs, such as NNGRRT for SaCas9 and NNNNGATT for NmCas9 .
Given the widespread occurrence of CRISPR-Cas in bacteria, users are currently uncertain about the relative performance of each system in engineering mammalian genomes. Despite the abundant literature available, it is difficult to directly compare the studies that employ different Cas nucleases due to inconsistencies in cellular context, target sites, protein expression levels, and other experimental conditions. Two recent reports attempted to assess SpCas9 with Cpf1 nucleases, but they were limited in scope and focused mainly on the specificities of Cpf1 [19, 20]. Here, we rigorously characterize three Cas9 nucleases (SpCas9, SaCas9, and NmCas9) and two Cpf1 nucleases (also known as Cas12a) from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) across a plethora of target sites. Our data provide much-needed guidance to others who are keen to leverage the CRISPR-Cas technology to perform genome editing in human cells and potentially also in other organisms.
Framework for fair evaluation of different CRISPR-Cas systems
We sought to establish an evaluation framework that allowed an unbiased assessment of the five Cas endonucleases (SpCas9, SaCas9, NmCas9, AsCpf1, and LbCpf1). Every protein was fused to two nuclear localization signals (NLS) and an identical V5 epitope tag. Additionally, we expressed each enzyme and its cognate sgRNA from the same plasmid backbone. The CAG or the EF1α promoter was used to drive the expression of the Cas nuclease, while the same U6 promoter was used to drive the expression of the sgRNA. After cloning, we verified the activity of each construct using target sites that were known to be edited robustly by the respective nucleases (Additional file 1: Figure S1 and Tables S1 and S2).
The various Cas enzymes should ideally be targeting identical genomic loci in order for the results to be comparable. As each endonuclease requires a different PAM for efficient cleavage and the PAMs for SaCas9 and NmCas9 are incompatible, we initially selected 61 matched target sites that are flanked by TTTN (PAM for AsCpf1 and LbCpf1) and either NGGRRT (combined PAM for SpCas9 and SaCas9) or NGGNGATT (combined PAM for SpCas9 and NmCas9) (Additional file 1: Table S3). The sites ranged in length from 17 to 23 nucleotides (nt). Additionally, since each Cas nuclease may be differentially affected by chromatin accessibility, we targeted genes with varying expression levels in HEK293T cells because gene transcription is largely controlled by the underlying chromatin architecture . Based on our RNA-seq data, the chosen genes showed more than 4000-fold difference in expression (Additional file 1: Figure S2a). Consistently, we also observed higher levels of H3K27ac at the promoters of more actively transcribed genes (Additional file 1: Figure S2b).
We asked whether our evaluation studies may be influenced by the choice of promoter used to express the Cas enzymes. We checked the expression of each endonuclease from either the CAG or the EF1α promoter by quantitative real-time PCR (qRT-PCR) and found that transcript levels were approximately 1.5-fold higher under the latter promoter (Additional file 1: Figure S2c). However, the cleavage efficiencies of Cas nucleases expressed under the CAG promoter were highly correlated with the cleavage efficiencies of the enzymes expressed under the EF1α promoter, regardless of whether T7 endonuclease I (T7E1) mismatch cleavage assays or Illumina deep sequencing experiments were used to measure the rate of indel formation (Pearson R2 = 0.75 or 0.96, respectively) (Additional file 1: Figure S2d). The data obtained from the CAG promoter was also not significantly different from the data obtained from the EF1α promoter (P > 0.5, Wilcoxon rank sum test; Additional file 1: Figure S2e). This may be because both promoters were strong enough to produce sufficient amounts of Cas proteins, so that enzyme concentration in the cells was no longer a limiting factor. Hence, we pooled the data obtained using the CAG promoter with the data obtained using the EF1α promoter to perform a combined analysis.
Performance of CRISPR-Cas in NHEJ-mediated genome editing
Notably, NmCas9 performed poorly at most of the target sites irrespective of spacer lengths, with editing frequencies considerably lower than the other nucleases (Fig. 1b and Additional file 1: Figure S5b). We also observed that with our chimeric sgRNAs, NmCas9 did not show a preference for longer spacer lengths, consistent with a recent study on the usage of NmCas9 in mammalian genome editing . Nevertheless, since the length of naturally occurring crRNA spacers in N. meningitides was found to be 24 nt , we selected nine new 24 nt- or 25 nt-long target sites that are flanked by the PAMs for Cpf1 and NmCas9 (Additional file 1: Figure S7a, b and Table S5). Moreover, these sites are in highly expressed genes to ensure accessibility of the chromatin. When we quantified editing efficiencies at these new genomic loci by T7E1 cleavage assays (Additional file 1: Figure S7c, d) and Illumina deep sequencing experiments (Fig. 1e, f) in HEK293T cells, we again found that the editing activity of NmCas9 was lower than those of both AsCpf1 and LbCpf1 at all nine matched target sites. We further verified the poorer performance of NmCas9 in other cell lines (Additional file 1: Figure S8). Collectively, our results suggest that NmCas9 might not be an ideal Cas nuclease for many genome editing applications, such as multiplex gene targeting.
While AsCpf1 performed generally well in our NHEJ-mediated genome editing experiments, it was usually surpassed by some other enzyme at most target sites, regardless of whether they are located in lowly expressed or highly expressed genes. When we carried out a four-way comparison of the different Cas nucleases using spacers that were either perfectly matched or contained matched seed regions, we found that AsCpf1 was the best performing enzyme at only a minority of the sites, even for optimal spacer lengths (Additional file 1: Figure S12). When we carried out a pairwise comparison of AsCpf1 with either SpCas9 or LbCpf1 alone, focusing only on the sgRNAs of optimal lengths for both enzymes under consideration, we also found that AsCpf1 exhibited significantly lower cleavage efficiencies than the other two nucleases (P < 0.05, Wilcoxon rank sum test; Fig. 2c and Additional file 1: Figure S11c). Nevertheless, despite its overall weaker editing activity, AsCpf1 showed the lowest tolerance to single mismatches between the sgRNA and the target DNA (Fig. 2d, e and Additional file 1: Figure S11d, e). Hence, our results suggest that there is a compromise between cleavage efficiency and specificity of naturally occurring Cas endonucleases.
Performance of CRISPR-Cas in HDR-mediated genome editing with single-stranded oligodeoxynucleotide donor
We wondered whether the results from our HDR-mediated editing experiments might be due to differences in cleavage efficiencies. After co-transfecting ssODNs with our CRISPR plasmids, we performed T7E1 assays and RFLP analysis on the same genomic DNA samples. Overall, we observed that SpCas9 generated indels as efficiently as AsCpf1 and LbCpf1 in the T7E1 assays, but yet it produced weaker cleavage bands than the Cpf1 nucleases after restriction digest with XbaI or HindIII (Figs. 3e, f and 4d). Additionally, we sequenced the targeted genomic loci and examined the sequencing reads. Strikingly, SpCas9 produced random indels at least as efficiently as AsCpf1 and LbCpf1 at all the tested loci (Additional file 1: Figure S20), but clearly fewer sequencing reads had the desired restriction site correctly incorporated (Additional file 1: Figure S21). Hence, the lower efficiency of precise genome editing exhibited by SpCas9 compared to the Cpf1 nucleases when ssODNs of non-target strand sequences were used was not simply due to a poorer ability to cut the target sites.
Optimization of ssODN donor templates
The design of the ssODN donor template can influence HDR efficiency [29–32]. So far, all our experiments had relied on symmetric ssODNs of the non-target strand sequence. Hence, we first sought to explore the extent to which the editing activity of each CRISPR-Cas system may be influenced by the orientation of the donor template. To this end, we targeted the CACNA1D and PPP1R12C loci as well as the A3, A11, B8, and B18 loci using ssODNs that were complementary to either the target or the non-target strand. All the ssODNs contained 27-nt homology arms. We also tested ssODNs with 17-nt arms for the B8 locus. Surprisingly, we did not detect a consistent strand bias for each Cas nuclease by deep sequencing experiments (Additional file 1: Figure S22) or by RFLP analysis (Additional file 1: Figure S23). Instead, at five out of the six targeted sites, we observed a trend for the editing activity of all the enzymes to change in the same direction when we altered the orientation of the donor template, thereby suggesting that each genomic locus may have an inherent ssODN strand preference. For example, at the PPP1R12C locus, the HDR frequencies of all the enzymes showed an increase when we switched from the original ssODN template that was of the non-target strand sequence (NT) to a new donor that was of the target strand sequence (T), although this increase was much larger for SpCas9 (Additional file 1: Figures S22b and S23b). Conversely, at the A11 locus, the HDR frequencies of SpCas9, AsCpf1, and LbCpf1 all decreased when we used T ssODNs in place of the original NT ssODNs, although this reduction was more significant for the Cpf1 nucleases (Additional file 1: Figures S22d and S23d). Furthermore, the changes in HDR frequencies were not simply due to differences in cleavage rates as every nuclease yielded similar amounts of indels in the presence of either the NT or the T ssODNs (Additional file 1: Figure S24).
Subsequently, we sought to determine whether the structure of the ssODN could further impact on the editing efficiency of the Cas enzymes. A previous study demonstrated that homology-directed editing by SpCas9 could be enhanced by using asymmetric donor templates . Here, to create such asymmetric donors, we extended either the PAM-proximal or the PAM-distal side of each ssODN from 37 to 77 nt (Fig. 5a). Again, we tested donor DNA that was complementary to either the target or the non-target strand of the CACNA1D or PPP1R12C locus. Consistent with the published report , we found that for SpCas9, extending the homology arm at the PAM-distal side of the T ssODN could improve HDR efficiency, while extending the homology arm at the PAM-proximal side was either neutral or detrimental to the performance of the enzyme (Fig. 5b, c and Additional file 1: Figure S25). In contrast, we discovered that for the Cpf1 nucleases, extending the homology arm at the PAM-proximal side of the NT ssODN instead led to an increase in HDR frequency, while extending the homology arm at the PAM-distal side decreased the rate of HDR. Overall, LbCpf1 still exhibited a higher HDR efficiency than SpCas9 at the CACNA1D locus when all possible types of donor DNA had been considered, but at the PPP1R12C locus, the HDR rate exhibited by SpCas9 with its optimal ssODN template was significantly higher than that exhibited by LbCpf1 with its optimal donor template (P < 0.05, Student’s t-test). Taken together, our results indicate that both SpCas9 and LbCpf1 may be used for ssODN-mediated editing, but strand preferences of the genomic locus and the enzyme as well as the structure of the donor template need to be carefully considered.
Enhancement of error-prone repair with long single-stranded DNA
Performance of CRISPR-Cas in HDR-mediated genome editing with plasmid donor
The ideal CRISPR-Cas system is one with both high cleavage efficiency and high target specificity. Two recent studies reported that AsCpf1 and LbCpf1 appeared to satisfy both these criteria [19, 20], suggesting that they may be the model Cas endonucleases to pursue in future applications. However, our analysis indicates that there may be a compromise between editing activity and target specificity in naturally occurring Cas enzymes (Fig. 2c–e and Additional file 1: Figure S11c–e). Specifically, both SpCas9 and LbCpf1 showed more robust editing activity than AsCpf1, but they also exhibited higher tolerance for mismatches between their sgRNA and the target DNA. It may be possible that the genome-wide methods used in the two recent studies [19, 20] have some limitations that preclude comprehensive detection of all off-target sites cleaved by the Cpf1 nucleases. Digenome-seq [34, 35] requires very high sequencing depth to capture cleavage sites, while GUIDE-seq  requires the incorporation of blunt double-stranded oligodeoxynucleotides (dsODNs) into DNA breakage sites, which may inherently be biased against the staggered cuts generated by Cpf1. Indeed, the efficiency of tag integration for AsCpf1 and LbCpf1 was found to be lower than that for SpCas9 . Hence, additional work is needed to fully investigate the relationship between editing activity and target specificity of all promising natural CRISPR-Cas systems. Newer and more sensitive methods of detecting off-target effects, such as CIRCLE-seq  and SITE-Seq , may help to resolve the issue. We further note that one may also evolve natural Cas enzymes into variants that achieve both high editing efficiency and targeting specificity, as demonstrated recently for SpCas9 .
HDR-mediated precise genome editing typically occurs at low frequencies. This roadblock needs to be overcome before CRISPR-Cas can realize its full potential in gene therapy, whereby accurate correction of disease-causing mutations can lead to a permanent cure. As a result, there have been numerous efforts over the past few years to improve its efficiency [29, 31, 40–46]. Our data indicate that AsCpf1 and LbCpf1 are able to introduce precise genome edits in human cells efficiently when ssODNs are used as donor templates (Figs. 3, 4 and 5 and Additional file 1: Figures S13, S17, S18, S22, S23, S25). We found that the Cpf1 nucleases prefer single-stranded DNA donors that are complementary to the target strand, in contrast to SpCas9, which prefers donor templates that are complementary to the non-target strand instead. In addition, we observed that asymmetric donors with a longer PAM-proximal homology arm could further improve Cpf1-mediated editing. From these results, we propose that Cpf1 may asymmetrically release the 3′ end of the cleaved target strand, thereby allowing the shorter arm of the optimized donor template to anneal and consequently enabling the longer arm of the template to invade and displace the base-paired non-target DNA strand at the other side of the break. We further note that our data, which were obtained in human cells, are in agreement with another recent study performed in zebrafish embryos .
It is tempting to speculate that the Cpf1 nucleases should perform precise genome editing more efficiently than SpCas9. First, Cpf1 generates a staggered cut that may facilitate HDR, while Cas9 generates a blunt cut. Second, Cpf1 cleaves outside the critical seed region and hence repeated targeting may occur, while Cas9 cleaves within the seed region and hence re-targeting is less likely to happen because indel mutations will prevent any subsequent recognition by the enzyme . Indeed, our results showed that even when we used ssODNs of the optimal strand sequence, both AsCpf1 and LbCpf1 still yielded higher HDR frequencies than SpCas9 at five out of the six target sites tested (Additional file 1: Figures S22 and S23). However, we note that there might be other confounding factors. For example, our data indicate the presence of an inherent strand bias at each target genomic locus, possibly due to native chromatin context. This localized bias can play in favor of SpCas9’s preference for a ssODN template that is complementary to the non-target strand, which is what we observed at the PPP1R12C locus. Furthermore, we found that SpCas9 and Cpf1 performed comparably when linearized plasmids were used as donor templates (Fig. 7), suggesting that it is not the nature of the cut per se that affects HR efficiency. While this may be explained by the fact that ssODN-mediated editing and plasmid-mediated editing are resolved through different DNA repair pathways, further work is needed to carefully dissect the underlying mechanisms.
We systematically assessed the ability of different CRISPR-Cas systems to perform NHEJ- and HDR-mediated genome editing in human cells. We targeted numerous genomic loci with matched spacers or matched seeds to obtain a clearer and fairer picture of how the various Cas enzymes compared against one another. Our extensive survey enabled us to formulate a set of rules and design parameters that others may follow to carry out their genome editing experiments with CRISPR-Cas (Fig. 8). We anticipate that the guidelines will evolve with time as the technology matures and more Cas nucleases are discovered and characterized in the future.
Cell culture and transfection
All cell lines were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS, 2 mM L-glutamine, and 1% penicillin/streptomycin. Cells were incubated at 37 °C in a humidified 5% CO2 air incubator. The cell lines were routinely checked by PCR for mycoplasma contamination using the following primers: forward, GGG AGC AAA CAG GAT TAG ATA CCC T; reverse, TGC ACC ATC TGT CAC TCT GTT AAC CTC.
Transfections were performed using either Turbofect (Thermo Scientific), jetPRIME (Polyplus), or Lipofectamine 2000 (Life Technologies), according to the manufacturers’ instructions. We seeded 350,000 or 120,000 cells in each well of a 12-well plate or a 24-well plate, respectively, one day prior to transfection, so that the cells would be approximately 60% confluent the next day. For NHEJ-mediated editing experiments, HEK293T cells were transfected with 500 ng CRISPR plasmids in 12-well plates and sorted 24 h post-transfection for fluorescent cells. For HDR-mediated editing experiments with ssODNs, HEK293FT cells were co-transfected with 300 ng CRISPR plasmids and 300 ng ssODNs, which were purchased from Integrated DNA Technologies, in 24-well plates and sorted 3 days post-transfection for fluorescent cells. Sequences of the donor ssODNs are provided in Additional file 1: Appendix S1. For HDR-mediated editing experiments with donor plasmids, HEK293FT cells were co-transfected with 300 ng CRISPR plasmids and 300 ng donor templates, which were linearized with either KpnI or SalI, in 24-well plates and analyzed by flow cytometry for GFP-positive cells 9 days post-transfection.
T7E1 assay and RFLP analysis
Genomic DNA was isolated using QuickExtract (Epicentre) according to the manufacturer’s instructions. The loci-of-interest were then amplified using Q5 High-Fidelity DNA Polymerase (New England Biolabs) and the following PCR parameters: 98 °C for 30s, 98 °C for 10s, 63–65 °C for 30s, 72 °C for 20s (repeat from step 2 for 34 more cycles), and 72 °C for 2 min. Sequences of the primers used are provided in Additional file 1: Table S7. Subsequently, the PCR products were purified using the GeneJET Gel Extraction Kit (Thermo Scientific).
For the T7E1 assay, 200 ng PCR products was incubated at 95 °C for 5 min in 1× NEBuffer 2 and then slowly cooled at a rate of − 0.1 °C/second. After annealing, 5 U T7 endonuclease I (New England Biolabs) was added to each sample and the reactions were incubated at 37 °C for 50 min. The T7E1-digested products were separated on a 2.5% agarose gel stained with GelRed (Biotium) and the gel bands were quantified using ImageJ. For the RFLP analysis, 200 ng PCR products were digested overnight with either XbaI or HindIII-HF (New England Biolabs) in CutSmart buffer. The reactions were separated on a 2% agarose gel stained with GelRed (Biotium) and the gel bands were quantified using ImageJ.
Flow cytometry was performed on FACSAriaIII (Becton Dickinson). FSC and SSC were used to separate singlets from cell aggregates. Subsequently, RFP- and GFP-positive cells were gated relative to untransfected control cells. Data analysis was performed using FACSDiva (Becton Dickinson) and FlowJo.
Illumina deep sequencing analysis
Sequencing libraries were constructed as previously described . Sequences of the PCR primers used to amplify the loci-of-interest are provided in Additional file 1: Table S8. To process the data, we first built a local reference library comprising the amplicon sequences of the targeted genes. The sequenced reads were then mapped against this reference library with BWA-MEM (mismatch penalty = 2 and clipping penalty = 8). The uniquely mapped reads with mapping quality ≥ 20 were sorted and assigned group information using Picard. GATK toolkit was used to perform local realignment and recalibration. The ‘CIGAR’ string of the BAM file was used to classify the reads as ‘Insertion’, ‘Deletion’, and ‘Match’. We used only the portions of the reads within 40 bp upstream and downstream of the spacer for the calculation of indel percentages. Randomly selected reads were also aligned with the relevant reference sequences using the Needleman-Wunsch algorithm. These alignments were then manually compared with our script outputs to ensure the accuracy of our analysis.
For the characterization of HDR-mediated genome editing, we built another local reference library consisting of the modified genes. The procedures described above were repeated, but the mapping was done to the newly built reference library (consisting of the modified genes). The reads were then classified into three categories for calculating the HDR incorporation percentages: ‘Correct incorporation’, when the read is tagged as a match and the restriction site is present; ‘Wrong incorporation’, when the read is tagged as an insertion or deletion and the restriction site is present; ‘No incorporation’, when the restriction site is not present.
Quantitative real-time PCR
First, RNA was isolated using Direct-zol RNA Miniprep kit (Zymo Research) according to manufacturer’s instructions. cDNA was then synthesized using qScript cDNA Supermix (QuantaBio). Finally, PCR was performed using Perfecta SYBR Fastmix (QuantaBio) according to manufacturer’s instructions and the following primers: OFP set 1 forward, TGA GCA AAA ACG TGA GCG TG; OFP set 1 reverse, ACC ATA CTG AAA TGC CGT GGT; OFP set 2 forward, AAA CGG GGT TCT TGT TGG CT; OFP set 2 reverse, TCA GTC TGC TCA ACC GTC TT.
Statistical tests, including Student’s t-test, Wilcoxon rank sum test, and Kolmogorov-Smirnov test, were performed as described in the figure captions. All P values were calculated with either the R software package or Microsoft Excel.
The authors thank Shyam Prabhakar and Talal Bin Amin for sharing the H3K27ac ChIP-seq data and Yue Wan for critical reading of the manuscript.
M.H.T. is supported by an Agency for Science Technology and Research’s Joint Council Office grant (1431AFG103), a National Medical Research Council grant (OFIRG/0017/2016), National Research Foundation grants (NRF2013-THE001-046 and NRF2013-THE001-093), a Ministry of Education Tier 1 grant (RG50/17 (S)), a startup grant from Nanyang Technological University, and funds for the International Genetically Engineering Machine (iGEM) competition from Nanyang Technological University. T.B. is supported by a student grant from Studierendenwerk Tübingen-Hohenheim.
Availability of data and materials
The deep sequencing data from this study have been deposited in the National Center for Biotechnology Information (NCBI) Sequence Read Archive (SRA) under accession code SRP144449 .
MHT conceived the project and supervised the research. YW, KIL, JL, TB, and MHT designed the experiments. YW, KIL, NABS, JZ, JL, CRJL, HX, RS, HTY, KHO, TB, YYRP, SML, NNBI, NABS, STVL, JL, and MHT performed the experiments. HS, FZ, JNF, and MHT analyzed the Illumina sequencing data. MHT wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that they have no competing interests.
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- Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339:819–23.View ArticlePubMedPubMed CentralGoogle Scholar
- Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. RNA-guided human genome engineering via Cas9. Science. 2013;339:823–6.View ArticlePubMedPubMed CentralGoogle Scholar
- Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol. 2013;31:227–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Jinek M, East A, Cheng A, Lin S, Ma E, Doudna J. RNA-programmed genome editing in human cells. elife. 2013;2:e00471.View ArticlePubMedPubMed CentralGoogle Scholar
- Li D, Qiu Z, Shao Y, Chen Y, Guan Y, Liu M, Li Y, Gao N, Wang L, Lu X, et al. Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat Biotechnol. 2013;31:681–3.View ArticlePubMedGoogle Scholar
- Friedland AE, Tzur YB, Esvelt KM, Colaiacovo MP, Church GM, Calarco JA. Heritable genome editing in C. elegans via a CRISPR-Cas9 system. Nat Methods. 2013;10:741–3.View ArticlePubMedPubMed CentralGoogle Scholar
- Bassett AR, Tibbit C, Ponting CP, Liu JL. Highly efficient targeted mutagenesis of Drosophila with the CRISPR/Cas9 system. Cell Rep. 2013;4:220–8.View ArticlePubMedPubMed CentralGoogle Scholar
- Li JF, Norville JE, Aach J, McCormack M, Zhang D, Bush J, Church GM, Sheen J. Multiplex and homologous recombination-mediated genome editing in Arabidopsis and Nicotiana benthamiana using guide RNA and Cas9. Nat Biotechnol. 2013;31:688–91.View ArticlePubMedPubMed CentralGoogle Scholar
- Blitz IL, Biesinger J, Xie X, Cho KW. Biallelic genome modification in F(0) Xenopus tropicalis embryos using the CRISPR/Cas system. Genesis. 2013;51:827–34.View ArticlePubMedPubMed CentralGoogle Scholar
- Harel I, Benayoun BA, Machado B, Singh PP, Hu CK, Pech MF, Valenzano DR, Zhang E, Sharp SC, Artandi SE, Brunet A. A platform for rapid exploration of aging and diseases in a naturally short-lived vertebrate. Cell. 2015;160:1013–26.View ArticlePubMedPubMed CentralGoogle Scholar
- Mali P, Esvelt KM, Church GM. Cas9 as a versatile tool for engineering biology. Nat Methods. 2013;10:957–63.View ArticlePubMedPubMed CentralGoogle Scholar
- Doudna JA, Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9. Science. 2014;346:1258096.View ArticlePubMedGoogle Scholar
- Hsu PD, Lander ES, Zhang F. Development and applications of CRISPR-Cas9 for genome engineering. Cell. 2014;157:1262–78.View ArticlePubMedPubMed CentralGoogle Scholar
- Zetsche B, Gootenberg JS, Abudayyeh OO, Slaymaker IM, Makarova KS, Essletzbichler P, Volz SE, Joung J, van der Oost J, Regev A, et al. Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell. 2015;163:759–71.View ArticlePubMedPubMed CentralGoogle Scholar
- Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature. 2015;520:186–91.View ArticlePubMedPubMed CentralGoogle Scholar
- Hou Z, Zhang Y, Propson NE, Howden SE, Chu LF, Sontheimer EJ, Thomson JA. Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis. Proc Natl Acad Sci U S A. 2013;110:15644–9.View ArticlePubMedPubMed CentralGoogle Scholar
- Kim E, Koo T, Park SW, Kim D, Kim K, Cho HY, Song DW, Lee KJ, Jung MH, Kim S, et al. In vivo genome editing with a small Cas9 orthologue derived from Campylobacter jejuni. Nat Commun. 2017;8:14500.View ArticlePubMedPubMed CentralGoogle Scholar
- Komor AC, Badran AH, Liu DR. CRISPR-based technologies for the manipulation of eukaryotic genomes. Cell. 2017;168:20–36.View ArticlePubMedGoogle Scholar
- Kleinstiver BP, Tsai SQ, Prew MS, Nguyen NT, Welch MM, Lopez JM, McCaw ZR, Aryee MJ, Joung JK. Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells. Nat Biotechnol. 2016;34:869–74.View ArticlePubMedPubMed CentralGoogle Scholar
- Kim D, Kim J, Hur JK, Been KW, Yoon SH, Kim JS. Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol. 2016;34:863–8.View ArticlePubMedGoogle Scholar
- Clapier CR, Cairns BR. The biology of chromatin remodeling complexes. Annu Rev Biochem. 2009;78:273–304.View ArticlePubMedGoogle Scholar
- Lee CM, Cradick TJ, Bao G. The Neisseria meningitidis CRISPR-Cas9 system enables specific genome editing in mammalian cells. Mol Ther. 2016;24:645–54.View ArticlePubMedPubMed CentralGoogle Scholar
- Zhang Y, Heidrich N, Ampattu BJ, Gunderson CW, Seifert HS, Schoen C, Vogel J, Sontheimer EJ. Processing-independent CRISPR RNAs limit natural transformation in Neisseria meningitidis. Mol Cell. 2013;50:488–503.View ArticlePubMedPubMed CentralGoogle Scholar
- Hinz JM, Laughery MF, Wyrick JJ. Nucleosomes Inhibit Cas9 Endonuclease Activity in Vitro. Biochemistry. 2015;54:7063–6.View ArticlePubMedGoogle Scholar
- Horlbeck MA, Witkowsky LB, Guglielmi B, Replogle JM, Gilbert LA, Villalta JE, Torigoe SE, Tjian R, Weissman JS. Nucleosomes impede Cas9 access to DNA in vivo and in vitro. eLife. 2016;5:e12677.Google Scholar
- Isaac RS, Jiang F, Doudna JA, Lim WA, Narlikar GJ, Almeida R. Nucleosome breathing and remodeling constrain CRISPR-Cas9 function. eLife. 2016;5:e13450.Google Scholar
- Daer RM, Cutts JP, Brafman DA, Haynes KA. The impact of chromatin dynamics on Cas9-mediated genome editing in human cells. ACS Synth Biol. 2017;6:428–38.View ArticlePubMedGoogle Scholar
- Chen F, Pruett-Miller SM, Huang Y, Gjoka M, Duda K, Taunton J, Collingwood TN, Frodin M, Davis GD. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases. Nat Methods. 2011;8:753–5.View ArticlePubMedPubMed CentralGoogle Scholar
- Lin S, Staahl BT, Alla RK, Doudna JA. Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery. elife. 2014;3:e04766.PubMedPubMed CentralGoogle Scholar
- Yang L, Guell M, Byrne S, Yang JL, De Los AA, Mali P, Aach J, Kim-Kiselak C, Briggs AW, Rios X, et al. Optimization of scarless human stem cell genome editing. Nucleic Acids Res. 2013;41:9049–61.View ArticlePubMedPubMed CentralGoogle Scholar
- Richardson CD, Ray GJ, DeWitt MA, Curie GL, Corn JE. Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA. Nat Biotechnol. 2016;34:339–44.View ArticlePubMedGoogle Scholar
- Moreno-Mateos MA, Fernandez JP, Rouet R, Vejnar CE, Lane MA, Mis E, Khokha MK, Doudna JA, Giraldez AJ. CRISPR-Cpf1 mediates efficient homology-directed repair and temperature-controlled genome editing. Nat Commun. 2017;8:2024.View ArticlePubMedPubMed CentralGoogle Scholar
- Richardson CD, Ray GJ, Bray NL, Corn JE. Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes. Nat Commun. 2016;7:12463.View ArticlePubMedPubMed CentralGoogle Scholar
- Kim D, Bae S, Park J, Kim E, Kim S, Yu HR, Hwang J, Kim JI, Kim JS. Digenome-seq: genome-wide profiling of CRISPR-Cas9 off-target effects in human cells. Nat Methods. 2015;12:237–43. 231 p following 243View ArticlePubMedGoogle Scholar
- Kim D, Kim S, Kim S, Park J, Kim JS. Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq. Genome Res. 2016;26:406–15.View ArticlePubMedPubMed CentralGoogle Scholar
- Tsai SQ, Zheng Z, Nguyen NT, Liebers M, Topkar VV, Thapar V, Wyvekens N, Khayter C, Iafrate AJ, Le LP, et al. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 2015;33:187–97.View ArticlePubMedGoogle Scholar
- Tsai SQ, Nguyen NT, Malagon-Lopez J, Topkar VV, Aryee MJ, Joung JK. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets. Nat Methods. 2017;14(6):607–614.View ArticlePubMedPubMed CentralGoogle Scholar
- Cameron P, Fuller CK, Donohoue PD, Jones BN, Thompson MS, Carter MM, Gradia S, Vidal B, Garner E, Slorach EM, et al. Mapping the genomic landscape of CRISPR-Cas9 cleavage. Nat Methods. 2017;14(6):600–606.View ArticlePubMedGoogle Scholar
- Hu JH, Miller SM, Geurts MH, Tang W, Chen L, Sun N, Zeina CM, Gao X, Rees HA, Lin Z, Liu DR. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity. Nature. 2018;556(7699):57–63.View ArticlePubMedGoogle Scholar
- Chu VT, Weber T, Wefers B, Wurst W, Sander S, Rajewsky K, Kuhn R. Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells. Nat Biotechnol. 2015;33:543–8.View ArticlePubMedGoogle Scholar
- Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotechnol. 2015;33:538–42.View ArticlePubMedPubMed CentralGoogle Scholar
- Yu C, Liu Y, Ma T, Liu K, Xu S, Zhang Y, Liu H, La Russa M, Xie M, Ding S, Qi LS. Small molecules enhance CRISPR genome editing in pluripotent stem cells. Cell Stem Cell. 2015;16:142–7.View ArticlePubMedPubMed CentralGoogle Scholar
- Pinder J, Salsman J, Dellaire G. Nuclear domain ‘knock-in’ screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing. Nucleic Acids Res. 2015;43:9379–92.View ArticlePubMedPubMed CentralGoogle Scholar
- Song J, Yang D, Xu J, Zhu T, Chen YE, Zhang J. RS-1 enhances CRISPR/Cas9- and TALEN-mediated knock-in efficiency. Nat Commun. 2016;7:10548.View ArticlePubMedPubMed CentralGoogle Scholar
- Gutschner T, Haemmerle M, Genovese G, Draetta GF, Chin L. Post-translational regulation of Cas9 during G1 enhances homology-directed repair. Cell Rep. 2016;14:1555–66.View ArticlePubMedGoogle Scholar
- Zhang JP, Li XL, Li GH, Chen W, Arakaki C, Botimer GD, Baylink D, Zhang L, Wen W, Fu YW, et al. Efficient precise knockin with a double cut HDR donor after CRISPR/Cas9-mediated double-stranded DNA cleavage. Genome Biol. 2017;18:35.View ArticlePubMedPubMed CentralGoogle Scholar
- Liu KI, Ramli MN, Woo CW, Wang Y, Zhao T, Zhang X, Yim GR, Chong BY, Gowher A, Chua MZ, et al. A chemical-inducible CRISPR-Cas9 system for rapid control of genome editing. Nat Chem Biol. 2016;12(11):980–987.View ArticlePubMedGoogle Scholar
- Wang Y, Liu KI, Sutrisnoh NAB, Srinivasan H, Zhang J, Li J, Zhang F, Lalith CRJ, Xing H, Shanmugam R, et al. Systematic evaluation of CRISPR-Cas systems reveals design principles for genome editing in human cells. https://www.ncbi.nlm.nih.gov/sra/?term=SRP144449.