Here, we report a rapid CRISPR–Cas9-mediated gene knock-in strategy that uses Cas9 ribonucleoprotein and 5′-modified double-stranded DNA donors with 50-base-pair homology arms and achieved unprecedented 65/40% knock-in rates for 0.7/2.5 kilobase inserts, respectively, in human embryonic kidney 293T cells. The identified 5′-end modification led to up to a fivefold increase in gene knock-in rates at various genomic loci in human cancer and stem cells.
CRISPR–Cas9-induced double-strand breaks enable various genome editing applications including gene knockout, gene knock-in (KI) and introduction of point mutations1,2,3. Gene KI has been explored for a wide variety of purposes ranging from functional genomics4,5 to gene therapies6. Despite its enormous potential in the biotechnology and pharmaceutical industry, gene KI is very inefficient in human cells, especially in stem cells (typical gene KI rate in human stem cells is lower than 0.1% without selection)7. To realize the ultimate potential of gene KI in biotechnology and for therapeutic development, we set out to improve the efficiency of CRISPR–Cas9-mediated gene KI in human cells.
Initially, we decided to investigate gene KI strategies that use linear double-stranded DNA as donor templates (herein referred to as ‘dsDNA donors’). This decision was based on a lack of correlation between gene KI efficiency and Cas9-induced insertion and deletion (indel) rate (Supplementary Fig. 1a–c). On the other hand, gene KI efficiency was shown to be sensitive to variations in donor concentration, homology arm (HA) length and types of donor template (Supplementary Fig. 2a–c). Moreover, end modifications of single-stranded DNA donors by replacing the phosphate linkages with the more stable phosphorothioate linkages was shown to increase gene editing efficiency8. As a result, we sought to identify additional end modifications of dsDNA donors that improve gene KI efficiency.
We selected 13 modifications and tested their effect on dsDNA donor-mediated gene KI, including 5′-end modifications, 3′-end modifications and two phosphorothioate linkages (Fig. 1a,b). Primers with 5′ modifications including an amine group with a C6 linker (AmC6) or C12 linker (AmC12) were synthesized (Fig. 1b). Other secondary modifications were added to AmC6/AmC12 linkers by conjugating the amine group with their N-hydroxysuccinimide (NHS) esters (Supplementary Fig. 3). These 5′ modifications were easily incorporated into dsDNA donors by PCR reaction. Enzymatic installation of 3′-end modifications was limited by the scale, so we focused on testing 5′ modifications.
We first tested the effect of these end modifications on gene KI using a published gene KI report system at the GAPDH locus in HCT116 cells9. A (1.8 ± 0.36)-fold increase in KI rate was observed for dsDNA donors with phosphorothioate bonds (Fig. 1c). The 5′ modifications led to more substantial enhancements in KI rates (Fig. 1c), ranging from 2.3- to 5.1-fold at 2.2 pmol donor concentration (Fig. 1c). AmC6/C12 linkers brought a (4.0 ± 0.18)/(3.8 ± 0.47)-fold increase in KI rate, and no C6/C12 linked modifications showed decreased KI rates (Fig. 1c). C6-azidoacetic acid (AAA) and C6-polyethylene glycol (PEG10) provided an additional stimulating effect compared to AmC6, while C6-Alexa647 and C6-acetylthioglycolic acid (AGA) showed an opposite effect (Fig. 1c). Modifications at 3′ ends such as Am-ddU and ddU-PEG10 also caused (2.6 ± 0.21)-fold and (1.9 ± 0.06)-fold increases in gene KI rate. A slight additive effect ((0.13 ± 0.05)-fold increase) was observed when 3′ ddU-PEG10 was added to 5′ C6-PEG10 modified dsDNA donors (Fig. 1c). By incorporating end modifications, the need for long HAs to maximize gene KI rate is eliminated at the GAPDH locus (Fig. 1c and Supplementary Fig. 2b). A slightly higher KI rate was observed for 5′ C6-PEG10 modified dsDNA donors with 50-base-pair (bp) HAs compared to unmodified dsDNA donor with 500-bp HAs (Fig. 1c). Moreover, the background signal from the templates was greatly reduced when short HAs were used (Supplementary Fig. 4a). The stimulating effect on KI rate was retained for all 5′ modifications tested at a saturating concentration of dsDNA donors (Supplementary Fig. 4b). No significant increase was observed when 5′ C6-PEG10 modified dsDNA donor with 500-bp HAs was used (Supplementary Fig. 4c). Due to much better scalability and high KI efficiency, 5′ C6-PEG10 modified dsDNA donors with 50-bp HAs were tested in the following studies.
Next, we compared the efficiency of our strategy to previous studies using donors with short HAs. For GAPDH locus, dsDNA donors with 5′ C6-PEG10 modifications resulted in a 6.6 ± 0.45% KI rate in hESC H1 (Fig. 2a), whereas a 1.7% KI rate was reported for double-cut nonhomologous end joining (NHEJ) donors using the same report system9. A recent study demonstrated that by using PCR fragments with 33-bp HAs as donor templates, a 15% KI rate for a 0.7-kilobase (kb) enhanced green fluorescent protein (EGFP) sequence at lamin A/C locus was achieved in human embryonic kidney 293T (HEK293T) cells10. Although 13-fold less amount of Cas9 protein was used in this study, a 30.4 ± 0.96% gene KI rate was observed using the conditions described (Fig. 2b). Simply by adding 5′ C6-PEG10 modifications to this dsDNA donor, the KI rate was increased to 65.4 ± 0.44% (Fig. 2b), which is a high preselection gene KI rate for insertions of longer than 500 bp, despite the use of short HAs (Supplementary Table 3). It is worth mentioning that other factors besides donor modification might contribute to the increase in gene KI rate, such as Cas9 ribonucleoprotein (RNP) delivery optimization.
To demonstrate its generality, we tested this strategy in diverse applications. For GAPDH locus, the KI rate after applying 5′ C6-PEG10 modifications was increased from 35.7 ± 2.5% to 59.4 ± 0.50% in HEK293T, 8.0 ± 0.98% to 19.8 ± 1.8% in human induced pluripotent stem cell (hiPSC) WTC G3 and 2.9 ± 0.55% to 6.6 ± 0.45% in human embryonic stem cell (hESC) H1 (Fig. 2a). Gene KIs at safe harbor sites such as AAVS1 and CCR5 were evaluated because these sites are preferred for predictable gene integration6. The 5′ C6-PEG10 modifications of the dsDNA donor improved its KI rate from 3.3 ± 0.19% to 5.0 ± 0.24% at the AAVS1 site and 2.0 ± 0.28% to 2.9 ± 0.26% at the CCR5 site in hiPSC WTC G3 (Fig. 2c). Gene KI at heterochromatin regions can be applied in the study of chromatin structure, function and dynamics11. With 5′ C6-PEG10 end modification, we achieved a 0.5- to 1.6-fold increase in gene KI rate at four predicted heterochromatin loci11, with a remarkable 19.7 ± 0.72% KI rate at region 2 (Fig. 2d). In addition, we were able to achieve a 40.0 ± 1.0% gene KI rate by using a 2.5-kb 5′ PEG10 modified dsDNA donor at AAVS1 locus in a HEK293T cell (Fig. 2e), which showed that our strategy could be used in many other applications besides reporter gene KI. Moreover, a (0.64 ± 0.15)-fold increase was observed at the GAPDH locus in Cpf1-mediated gene KI rate with 5′ C6-PEG10 modification (Fig. 2f). To examine the potential of multiplexing, dual gene KI of GAPDH and lamin A/C was tested. An increase in double positive population from 5.7 ± 0.29% to 17.2 ± 0.42% was observed for dsDNA donors with 5′ C6-PEG10 modification (Fig. 2g). Allele-specific gene editing techniques are potentially useful in allelic gene functional studies. Using 5′ C6-PEG10 modified donors containing an EGFP or mCherry, the EGFP/mCherry double positive population was promoted from 9.4 ± 2.3% to 28.0 ± 3.2% at the lamin A/C locus (Fig. 2h). Taken together, these results demonstrated the versatility of this strategy in challenging situations.
To determine the editing accuracy of this strategy, we examined the indels in KI junction regions. Decreases of 7.3 ± 0.77% to 4.2 ± 0.36% and 5.9 ± 0.40% to 3.4 ± 0.59% in indel rates were measured by TIDE analysis12 at the 5′ and 3′ junctions, respectively, with 5′ C6-PEG10 modification (Supplementary Fig. 5a,b). The indels in the KI junctions are usually caused by NHEJ-mediated KI, while homology directed recombination (HDR)-mediated KI generally results in precise editing10. NHEJ-KI rate was evaluated using dsDNA donors with no HAs, and no increase in NHEJ-KI rate was observed as donor concentration increased (Supplementary Fig. 5c). Together, these results indicated that the increase in gene KI rate by end modification is not caused by promoting error-prone NHEJ-mediated repair. Gene integration copy number was determined for individual KI clones to investigate whether there was significant concatemer formation at the KI site. We examined a total of 66 clones from KI experiments performed with dsDNA donors with 5′ C6-PEG10 modification targeting six different sites. Out of these clones, 61 showed one copy gene integration at one allele or both alleles of the target genes (Supplementary Fig. 6a–c). Only three clones showed concatemer formation at the target sites, and two clones showed off-target integration (either Cas9-guided or random integration, Supplementary Fig. 6a–c). These results are consistent with the recent study that used 5′ biotin modified dsDNA donors to reduce NHEJ-based KI and concatemer formation13. They also indicate that random integration rate is at a low level (≤2 out of 66) in gene KI experiments performed with end-modified donors.
In summary, we described a simple and effective strategy for gene KI combining Cas9 RNP with 5′-modified dsDNA donors with short HAs. The present method exhibited the advantages of enhanced KI efficiency, shorter workflow and high accuracy. We screened 13 modifications with diverse structures and identified 5′ C6-PEG10 as the most beneficial one. The 5′ C6-PEG10 modified dsDNA resulted in an up to fivefold increase in gene KI efficiency, which resulted in, to the best of our knowledge, the highest reported gene KI rate (65.4 ± 0.44%) in human cells. In addition, lower indel rates in the KI junctions were observed with end-modified dsDNA donors. Therefore, end-modified dsDNA donors with short HAs could be used as an appealing alternative to traditional plasmid donor templates. We tested 13 modifications in this study, and further screening may reveal more potent chemical moieties. Moreover, it might be interesting to study the mechanism underlying this phenomenon. Finally, as the stimulating effect of 5′ C6-PEG10 modification was verified at diverse genomic loci including safe harbor sites and heterochromosome regions, and also in different cell lines including human cancer and stem cell lines, we envision our strategy may benefit a wide range of studies such as gene functional studies, allele-specific gene expression, chromatin structure studies and genetic disease treatment.
Generation of plasmids, dsDNA, Cas9 RNP and Cpf1 RNP
Plasmids used as templates for dsDNA donor PCR were assembled by Gibson assembly using pCRIS-PITChv2-FBL (Addgene, plasmid no. 63672) as the backbone. The insertion sequences were between BamHI and NheI sites and are listed in Supplementary Table 4. dsDNA donors were obtained by PCR amplification using Q5 High-Fidelity DNA polymerase (New England BioLabs). The 0- to 50-bp HA sequences were incorporated in the primers listed in Supplementary Table 5. For KI at GAPDH locus, donors with longer HAs were amplified from a plasmid donor with 500-bp HAs (IRES-GFP donor plasmid, GAPDH, 500-bp HAs). PCR products were gel-purified or PCR-purified depending on their purities using QIAquick gel extraction kit or QlAquick PCR purification kit (Qiagen). The typical yield from a 500-μl reaction was around 15–30 μg. Guide RNAs and CRISPR RNA used for transfections were obtained using the GeneArt Precision gRNA Synthesis Kit (Thermo Fisher Scientific) using primers listed in Supplementary Table 5. gRNAs/crRNAs were eluted in TE buffer and annealed using a PCR thermocycler using the following program: 95 °C for 5 min and then cooling to 25 °C at 0.1 °C s−1. All primers were ordered from IDT (Integrated DNA Technologies). Cas9 protein was purchased from IDT or purified as described14. Cpf1 protein was purified as described15. Cas9/Cpf1 protein was expressed in Escherichia coli Rosetta 2 (DE3) cells using Cas9 expression plasmid (Addgene, plasmid no. 47327) or Cpf1 expression plasmid (Addgene, plasmid no. 79007).
For 5′ modification and phosphorothioate bond modification, primers with 2PS bonds, Acrydite, C6-Alex 647, amine C6 and amine C12 were ordered from IDT. Bis-PEG10-NHS ester was purchased from Broadpharm (BP-22588). Biotin NHS ester (H1759), azidoacetic acid NHS ester (900919), s-acetylthioglycolic acid NHS ester (A9043) and propargyl NHS ester (764221) were purchased from Sigma-Aldrich. NHS esters were incubated with 10 μM primers with 5′ amine C6 or C12 group at 1 mM concentration in ×1 borate buffer (Thermo Fisher, 28341) overnight at room temperature. Primers were then desalted using Bio-Spin 30 columns (Bio-Rad). The labeling efficiencies were measured by high-performance liquid chromatography and matrix-assisted laser desorption/ionization–time of flight mass spectrometry (Supplementary Fig. 3). For 3′ Am-ddU modification, an aliquot of 10 μg of dsDNA donors, 20 μM amino-11-ddUTP (Lumiprobe), 50 μM CoCl2, and 1 U TdT polymerase (New England Biolabs) and 1×TdT reaction buffer were incubated in a 50-μl reaction at 37 °C for 4 h. Amino-11-ddUTP modified dsDNA was purified using QlAquick PCR purification kit (Qiagen) after stopping the reaction with 10 μl of 0.2 M EDTA (pH = 8.0). The resulting product was then used to generate 3′ ddU-PEG10 modified dsDNA by incubation with Bis-PEG10-NHS ester at the conditions described above.
Cell culture and transfection
HCT116, HEK293T, H1 and WTC G3 cells were cultured as described on the ATCC website. Cell transfection was performed using the Amaxa Nucleofector II device (Lonza): Cell Line Nucleofector kit V and Program D-032 were used for HCT116 and HEK293T, and Human Stem Cell Nucleofector Kit 2 and Program B-016 were used for H1 and WTC G3. The cells were expanded to 70–90% confluence, and then dissociated using TypLE (Thermo Fisher Scientific) or Accutase (StemCell Technologies), and washed twice with phosphate buffered saline. Cas9 and gRNA were incubated in 10 μl of electroporation solution for 15 min, and then added to 0.5 million cancer cells or 1.5 million stem cells resuspended in 90 μl of electroporation solution. The donor templates were added into the tubes before the electroporation. The components were added in the amount specified for each experiment.
Flow cytometry analysis, cell sorting and KI clone isolation
For each experiment, 5,000–50,000 cells were analyzed using a BD LSR II or a BD LSR Fortessa flow cytometer (BD Biosciences). For cell sorting, cells inside the defined gates were sorted using BD fluorescence activated cell sorting ARIA II (BD Biosciences). KI clone isolation was performed either by direct sorting of EGFP positive single cells onto a 96-well plate or use of micropipette tips to pick single colonies from a 10-cm plate seeded with 1,000 EGFP positive cells16.
Genomic PCR and DNA sequencing
Genomic DNA from cell pellets was extracted using QuickExtract DNA solution 1.0 (Epicenter). Genomic PCR was performed using Q5 High-Fidelity DNA polymerase (New England BioLabs) with the primers listed in Supplementary Table 5. The PCR products were subjected to direct DNA sequencing service (ACGT).
Genomic DNA from cell pellets was extracted using QuickExtract DNA Extraction Solution 1.0 (Epicentre). Amplification of the junction regions surrounding gene KI sites in edited cells was carried out by PCR using Q5 High-Fidelity DNA polymerase (New England BioLabs) and 100 ng of genomic DNA as template in a 50-μl reaction. PCR products were purified using QlAquick PCR purification kit (Qiagen) and then subjected to direct DNA sequencing service (ACGT). The indel rates were analyzed by the online software12 (http://tide.nki.nl) using the sequence without indels as a reference.
Droplet digital PCR (ddPCR)
ddPCR17 was used to determine the copy number of target gene integration using ACTB gene as a reference. Primers specific for the reference ACTB gene and the EGFP sequence in the insert were used, and primer sequences are listed in Supplementary Table 5. Isolated KI clones were expanded in 24-well plates and cells were gathered at 90% confluency. Genomic DNA was purified by Quick-DNA microprep kit (Zymo Research), and then digested overnight with EcoRI (New England Biolabs). The ddPCR mixture was prepared in PCR tubes by adding 10.5 μl of 2× EvaGreen Supermix (Bio-Rad), 1.05 μl of 4 μM forward and reverse primers, 1 μl of digested genomic DNA (12.5 ng μl−1) and 7.4 μl of ddH2O. For each ddPCR reaction, around 15,000-nl-sized water emulsion droplets were generated using a QX200 Automated Droplet Generator with DG8 Cartridges (186-4007, Bio-Rad) according to the manufacturer’s instructions. The resulting droplet mixture was transferred to a 96-well PCR plate and gene amplification was performed using the following program: 95 °C for 5 min, 40 cycles of 95 °C for 30 s and 59.1 °C for 60 s, 4 °C for 5 min, 90 °C for 5 min and then cooling to 4 °C. The amplified products were analyzed using the QX200 droplet reader (Bio-Rad) and the concentration of target genes was analyzed using QuantaSoft software (Bio-Rad, v.1.7.4.019). The copy number of the gene integration was calculated based on the ratio of the EGFP copy number to the ACTB copy number.
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
The data that support the findings of this study are available from the corresponding author upon request.
Ran, F. A. et al. Genome engineering using the CRISPR–Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).
Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).
Doudna, J. A. et al. The new frontier of genome engineering with CRISPR–Cas9. Science 346, 1258096 (2014).
Leonetti, M. D. et al. A scalable strategy for high-throughput GFP tagging of endogenous human proteins. Proc. Natl Acad. Sci. USA 113, 3501–3508 (2016).
Diao, Y. R. et al. A tiling-deletion-based genetic screen for cis-regulatory element identification in mammalian cells. Nat. Methods 14, 629–633 (2017).
Stephens, C. J. et al. Targeted in vivo knock-in of human alpha-1-antitrypsin cDNA using adenoviral delivery of CRISPR/Cas9. Gene Ther. 25, 139–156 (2018).
Merkle, F. T. et al. Efficient CRISPR-Cas9-mediated generation of knockin human pluripotent stem cells lacking undesired mutations at the targeted locus. Cell Rep. 11, 875–883 (2015).
Renaud, J. B. et al. Improved genome editing efficiency and flexibility using modified oligonucleotides with TALEN and CRISPR-Cas9 nucleases. Cell Rep. 14, 2263–2272 (2016).
He, X. et al. Knock-in of large reporter genes in human cells via CRISPR/Cas9-induced homology-dependent and independent DNA repair. Nucleic Acids Res. 44, e85 (2016).
Paix, A. et al. Precision genome editing using synthesis-dependent repair of Cas9-induced DNA breaks. Proc. Natl Acad. Sci. USA 114, 10745–10754 (2017).
Tasan, I. et al. CRISPR/Cas9-mediated knock-in of an optimized TetO repeat for live cell imaging of endogenous loci. Nucleic Acids Res. 46, e100 (2018).
Brinkman, E. K. et al. Easy quantitative assessment of genome editing by sequence trace decomposition. Nucleic Acids Res. 42, e168 (2014).
Gutierrez-Triana, J. A. et al. Efficient single-copy HDR by 5′ modified long dsDNA donors. eLife 7, e39468 (2018).
DeWitt, M. A. et al. Genome editing via delivery of Cas9 ribonucleoprotein. Methods 121-122, 9–15 (2017).
Hur, J. K. et al. Targeted mutagenesis in mice by electroporation of Cpf1 ribonucleoproteins. Nat. Biotechnol. 34, 807–808 (2016).
Strukov Y. G. et al. Development of mammalian cell lines with lac operator-tagged chromosomes. Cold Spring Harbor Protoc. https://doi.org/10.1101/pdb.prot4903 (2008).
Hindson, B. J. et al. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Anal. Chem. 83, 8604–8610 (2011).
This work was supported by the US National Institutes of Health (grant nos. 1UM1HG009402 and 1U54DK107965) (H.Z.). We thank B. Pilas and B. Balhan (Flow Cytometry Facility, Biotechnology Center, University of Illinois at Urbana Champaign) for cell sorting experiments. We thank S. Long and K. Sobanska for help with ddPCR experiments. We thank W. Tang, T. Si, J. Lian, C. Field and S. Tsai for helpful suggestions for manuscript writing and experimental designs.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
About this article
Cite this article
Yu, Y., Guo, Y., Tian, Q. et al. An efficient gene knock-in strategy using 5′-modified double-stranded DNA donors with short homology arms. Nat Chem Biol 16, 387–390 (2020). https://doi.org/10.1038/s41589-019-0432-1
Efficient construction of a stable linear gene based on a TNA loop modified primer pair for gene delivery
Chemical Communications (2020)
Suppression of SHROOM1 Improves In Vitro and In Vivo Gene Integration by Promoting Homology-Directed Repair
International Journal of Molecular Sciences (2020)
Genome editing enables reverse genetics of multicellular development in the choanoflagellate Salpingoeca rosetta
Recent advances in chemical modifications of guide RNA, mRNA and donor template for CRISPR-mediated genome editing
Advanced Drug Delivery Reviews (2020)