- Research article
- Open Access
Targeted integration in human cells through single crossover mediated by ZFN or CRISPR/Cas9
© The Author(s). 2018
- Received: 26 September 2017
- Accepted: 28 September 2018
- Published: 19 October 2018
Targeted DNA integration is widely used in basic research and commercial applications because it eliminates positional effects on transgene expression. Targeted integration in mammalian cells is generally achieved through a double crossover event between the genome and a linear donor containing two homology arms flanking the gene of interest. However, this strategy is generally less efficient at introducing larger DNA fragments. Using the homology-independent NHEJ mechanism has recently been shown to improve efficiency of integrating larger DNA fragments at targeted sites, but integration through this mechanism is direction-independent. Therefore, developing new methods for direction-dependent integration with improved efficiency is desired.
We generated site-specific double-strand breaks using ZFNs or CRISPR/Cas9 in the human CCR5 gene and a donor plasmid containing a 1.6-kb fragment homologous to the CCR5 gene in the genome. These DSBs efficiently drove the direction-dependent integration of 6.4-kb plasmids into the genomes of two human cell lines through single-crossover recombination. The integration was direction-dependent and resulted in the duplication of the homology region in the genome, allowing the integration of another copy of the donor plasmid. The CRISPR/Cas9 system tended to disrupt the sgRNA-binding site within the duplicated homology region, preventing the integration of another plasmid donor. In contrast, ZFNs were less likely to completely disrupt their binding sites, allowing the successive integration of additional plasmid donor copies. This could be useful in promoting multi-copy integration for high-level expression of recombinant proteins. Targeted integration through single crossover recombination was highly efficient (frequency: 33%) as revealed by Southern blot analysis of clonal cells. This is more efficient than a previously described NHEJ-based method (0.17–0.45%) that was used to knock in an approximately 5-kb long DNA fragment.
We developed a method for the direction-dependent integration of large DNA fragments through single crossover recombination. We compared and contrasted our method to a previously reported technique for the direction-independent integration of DNA cassettes into the genomes of cultured cells via NHEJ. Our method, due to its directionality and ability to efficiently integrate large fragments, is an attractive strategy for both basic research and industrial application.
- Single crossover
Genome editing techniques such as ZFNs (zinc-finger nucleases), TALENs (transcription activator-like effector nucleases), and the CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9) system, have greatly improved the efficiency and accuracy of site-specific modifications in cell lines and organisms. ZFNs use protein-DNA interactions for targeting, while the CRISPR/Cas9 system uses simple RNA-DNA base-pairing rules for targeting . Both ZFNs and CRISPR/Cas9 have been used to efficiently create knock-out alleles in mammalian cells by inducing DNA double-strand breaks (DSBs) which are repaired through the error-prone non-homologous end joining (NHEJ) mechanism. However, knock-in of DNA cassettes at defined loci using ZFNs or CRISPR/Cas9 and homology-directed repair (HDR) is generally less efficient , since NHEJ predominates over HDR for DSB repair [2–4]. Knock-in of DNA cassettes into the genomes of mammalian cells is generally achieved through double crossover with a linear donor containing two flanking homology arms and is aided by the presence of genome editors. This method has a relatively lower frequency of integration (10− 6–10− 5) [5, 6]. In contrast to gene knock-in in mammalian cells, integrating DNA cassettes can be achieved more easily in microorganisms through single “Campbell-like” crossover with a circular plasmid DNA containing a region of homology to a genomic target-locus . Single crossovers allow the integration of multiple copies of expression vectors in yeast , and have the advantage of being able to integrate larger DNA fragments into specific genomic loci compared to double crossover .
The targeted insertion of a gene of interest into the genome of mammalian cells is a strategy commonly used to overcome positional effects encountered in traditional transgene expression. This can be achieved through the generation of a double-strand break at the target site by engineered nucleases and subsequent HDR via double crossover with a provided plasmid containing a DNA sequence with substantial identity to the region flanking the desired site of integration [3, 10]. However, HDR in human cells is much slower and less efficient than NHEJ . One study showed that in human pluripotent stem cells, the efficiency of knock-in of 3-kb cassettes via CRISPR/Cas9-induced HDR was estimated to be around 10− 6–10− 5 . Moreover, the efficiency of knock-in through double crossover generally decreases as the size of the insert increases . Therefore, alternative strategies are required to improve the efficiency of targeted insertion of larger DNA fragments into the genome of mammalian cells.
NHEJ-mediated homology-independent targeted integration of in vivo linearized transgene donors has recently been shown to improve targeting efficiency, as demonstrated in one study where site-specific DSBs in the genome and the donor plasmid generated using CRISPR/Cas9 were used to efficiently target ~ 5-kb plasmids into mammalian genomes via NHEJ. They were able to achieve efficiencies of up to 0.17% in HEK293T cells and 0.45% in CHO cells . Our results show that through homology-based single crossover induced by either ZFN or CRISPR/Cas9, the efficiency of the targeted integration of a 6.4-kb plasmid can reach 10% in HeLa cells, which is more efficient than homology-independent targeting via NHEJ. Homology-independent targeted integration via NHEJ generally results in the integration of plasmids in both forward and reverse orientations . In contrast, we showed that targeted integration via homology-based single crossover generally results in the integration of plasmids in the forward orientation, which would be more usefully in applications requiring the uniformity of the orientation of integration. In addition, integration by homology-based single crossover can result in a duplication of the homologous region in the genome, which provides the opportunity to successively integrate multiple copies of the plasmid. This generally increases the expression level dramatically, meeting the requirement for large-scale protein production . Interestingly, we found that CRISPR/Cas9 tended to disrupt the sgRNA-binding sequence in the duplicated homology region, possibly preventing the sgRNA-Cas9 complex from binding the site again for a second round of cutting. Thus, the successive integration of another plasmid donor is inhibited (Fig. 3c and Additional file 3: Figure S2C). In contrast, ZFNs tended to induce small indel mutations in the spacer sequence, but kept the binding sites intact in the duplicated homology region. This would possibly allow ZFNs to bind the site again for another round of cutting, and thus allow the successive integration of additional copies of plasmid donors (Fig. 3b & e; Additional file 3: Figure S2B). This property confers ZFNs an advantage over CRISPR/Cas9 in generating cell lines with stably integrated multiple copies of plasmids. This advantage implies that ZFN should not be totally replaced by CRISPR/Cas9 due to its advantages in specific applications. It is interesting that both the 5′ and 3′ junction sequences of ZFN-driven knock-ins contained insertions of an additional spacer sequence, when co-transfected with only the EGFP donor plasmid (Fig. 2d), while both the 5′ and 3′ junction sequences remained intact in the EGFP-positive/DsRed-negative cells, when co-transfected with both EGFP and DsRed donor plasmids (Fig. 3c). We speculate that the doubled concentration of the donor plasmids used for ZFN-driven knock-in (Fig. 3c), in relative to the concentration of only EGFP donor plasmid used for ZFN-driven knock-in (Fig. 2d), may have contributed to the differences observed in the 5′ and 3′ junctions. As donor plasmids harbor the binding sites of ZFN monomers, a higher number of donor plasmids in the cells would provide more opportunities for ZFN monomer binding. This reduces the chances of the ZFN monomers binding to the genome with the EGFP donor plasmid already integrated for re-cutting, therefore resulting in reduced chances of observing indels at the 5′ and 3′ junction sequences.
It is noticeable that the analysis of multi-copy integration induced by ZFN by junction PCR showed that the internal junction DNA segment could be amplified from 65% of clones, while 5′ and 3′ junction DNA segments can be successfully amplified from only 10% of clones (Fig. 3e). The amplification of the internal junction DNA segment indicates the occurrence of multi-copy integration (Fig. 3a); however, the failure to amplify the 5′ or 3′ junction DNA segments from these clones may imply the occurrence of complex DNA rearrangements (deletions, duplications, insertions, and inversions) at the junctions, possibly induced by illegitimate recombination. Illegitimate recombination is an important competing pathway of homologous recombination for DSB repair in mammalian cells. One study has shown that illegitimate recombination can be stimulated 1,000-fold, as compared to 100-fold for homology recombination at site-specific DSBs . Further characterization of a small portion of clones of ZFN-driven knock-in by Southern blot supports our speculation. Incomplete integration of donor plasmid at target site happened in 33.3% (#1 and #6) clones, as they showed only one putatively perfect junction. Therefore, imperfect recombination could happen at the junctions possibly induced by illegitimate, making junction PCR prone to under estimate the true frequency of targeted integrations through single crossover mediated by ZFN. In addition, we found 50% (#3, #4 and #5) clones presented multi-copy integration, which is comparable to that detected by junction PCR analysis, indicating that junction PCR is able to detect most multi-copy integration events, however, it is hard to discriminate targeted and random integration, as in the three clones presenting multi-copy integration, one (#4 clone) seems to contain random integration.
ZFNs have certain limitations as compared with TALENs. For example, when different integration sites need to be targeted, it is relatively easier to assemble TALEN pairs than ZFN pairs. As TALENs share similar editing patterns with ZFNs, it would be interesting for us to test in the future whether TALENs would induce only small indels in the spacer sequence and keep the binding sites intact after integration of one copy of the donor plasmid into the genome, and subsequently mediate multiple targeted integration through single crossover.
Compared with direction-independent integration of DNA cassettes into the genomes of cultured cells via the NHEJ repair pathway, the direction-dependent integration of large DNA fragments through single crossover in this study is highly efficient and is capable of mediating multi-copy integration, making it an attractive strategy for both basic research and industrial applications.
ZFN pairs targeting the human CCR5 sequence were assembled based on previous studies [9, 10]. Briefly, the left and right halves of the ZFN coding sequences were synthesized (Generay Biotech, China) and cloned into a ZFN expression vector purchased from Sigma-Aldrich. An sgRNA was designed to target the human CCR5 using the online CRISPR Design Tool (http://crispr.mit.edu/v2). The sequences of the target sites of the designed ZFNs and CRISPR/Cas9 are summarized in Additional file 1: Table S1. For the co-expression of sgRNA and Cas9 in human cells, the synthesized sgRNA oligos were cloned into the pX330 plasmid (Addgene #42230) at the Bbs I sites as previously described . To construct the donor plasmid pEGFP-CCR5-Donor, a 1.6-kb fragment homologous to the CCR5 locus containing a ZFN or CRISPR/Cas9 target site in the middle was synthesized (Generay Biotech, China) and cloned into the pEGFP-N1 vector (Clontech, USA) at the Ase I restriction site (Fig. 2a). The donor plasmid pDsRed-CCR5-Donor was generated by replacing the EGFP coding region with the synthesized DsRed-coding DNA segment (Generay Biotech, China).
Cell culture and transfection
HeLa cells or HEK293T cells were seeded at 1.0 × 105 cells/well in a 24-well plate, cultured with 10% fetal bovine serum-DMEM, and incubated at 37 °C with 5% CO2. Upon 70–90% confluence, the cells were transfected with 150 ng each of the plasmids coding for the ZFN pair or 150 ng of the Cas9/sgRNA co-expression vector pX330 using Lipofectamine 3000 (Thermo Fisher Scientific). At day 3 post-transfection, genomic DNA was isolated from cells using the DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer’s protocol and was subsequently analyzed through PCR and the T7E1 assay. For targeted integration using donor plasmids, 150 ng each of the plasmids encoding the ZFN pair, 100 ng of pEGFP-CCR5-Donor, and 100 ng of pDsRed-CCR5-Donor were transfected into the cells. For CRISPR/Cas9-mediated knock-in, 150 ng of the Cas9/sgRNA co-expression vector pX330, 100 ng of pEGFP-CCR5-Donor, and 100 ng of pDsRed-CCR5-Donor were transfected into the cells.
The T7E1 assay was performed as previously described . Briefly, primers (Additional file 1: Table S2) were designed to amplify a 750-bp fragment across the target sites of the designed ZFNs or sgRNA from genomic DNA of cells transfected with ZFN or CRISPR/Cas9. The PCR products were denatured and annealed to form heteroduplex DNA, which could be cut by treatment with 0.5 μL T7E1 (NEB, USA) for 30 min at 37 °C. The digestion products were run on a 10% polyacrylamide Tris-borate-EDTA (TBE) gel. After staining the gel with SYBR Gold, mutation frequencies were calculated based on relative band intensities determined using the software Image J .
The CCR5 gene fragment containing the ZFN and sgRNA target sites were amplified using LA Taq DNA polymerase (Takara, Japan) and the designed primers (Additional file 1: Table S2). PCR products were analyzed by agarose gel electrophoresis, purified using a gel extraction kit (OMEGA, USA), and then cloned into the vector pMD18-T (Takara, Japan). Cloned plasmids were sequenced using M13 primers. Similarly, junction PCR products (Additional file 1: Table S2) were cloned into pMD18-T vector for Sanger sequencing.
Clonal cell culture
For clonal expansion of single cells stably expressing EGFP or co-expressing EGFP and DsRed, a single cell from the cell population edited by engineered nucleases and donor plasmids was seeded into each well in a 96-well plate after fluorescence-activated cell sorting (FACS). The cells were cultured in condition medium (medium from log-phase HeLa cells filtered through a 0.45 μm pore size filter, and supplemented with 10% fetal bovine serum (FBS)) for 9 days to form a compact clonal population of cells. The clonal cells were then cultured for an additional 11 days and harvested for junction PCR analysis to detect targeted integration events.
Southern blot assay
Genomic DNAs were isolated from single cell clones of ZFN-driven knock-in of only the EGFP donor plasmid for Southern blot analysis. 8 μg of isolated DNA was digested with Bam HI for 5′ junction analysis, or Hpa I for 3′ junction analysis. Digested samples were separated on a 0.7% agarose gels at 25 V overnight. DNA in 2 × standard saline citrate (SSC) was transferred to a charged nylon membrane (Roche). Probes for detecting donor plasmids integration were prepared as followings: probes were PCR-amplified from donor plasmid using primers, CCR5-5F and CCR5-5R for 5′ junction analysis and CCR5-3F and CCR5-3R for 3′ junction analysis (Additional file 1: Table S4). DNA probes were labelled by PCR amplification in the presence of digoxigenin-11-dUTP according to the instruction of PCR DIG Probe Synthesis Kit (Roche). For Southern blot hybridizations, nylon membranes were prehybridized for 2 h at 37 °C in hybridization solution without labelled probe and then hybridized separately at 37 °C with specific DNA probes overnight. The DNA was washed twice in 2 × SSC and 0.1% SDS (15 min each time) at room temperature, and once in 1× SSC and 0.1% SDS for 15 min at 65 °C, and was blocked in blocking buffer for 30 min. DNA hybridizations and detection were conducted by using the DIG labelling and CSPD substrate according to the instruction of DIG High Prime DNA Labling and Detection Starter II (Roche), and were exposed on an X-ray film.
This work was jointly supported by National Transgenic Major Program (2016ZX08006003–006) and the Natural Science Foundation of Guangdong Province (2016A030313310).
Availability of data and materials
The datasets used and/or analyzed during the current study available from the corresponding author on reasonable request.
XFL, MW, YFQ, XS, ZYH performed and analysed experiments, ZYH, YSC, PQC, XFL designed the project and wrote the paper. All authors read and approved the final manuscript.
Ethics approval and consent to participate
Human cell line HeLa and HEK293T were purchased from ATCC.
Consent for publication
The authors declare that they have no competing interests.
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