Abstract
BackgroundTargeted 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.ResultsWe 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.ConclusionWe 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.
Highlights
Targeted DNA integration is widely used in basic research and commercial applications because it eliminates positional effects on transgene expression
We tested whether targeted integration of multiple copies of a reporter expression cassette can be achieved in human cells through single crossover recombination upon the introduction of double-strand breaks by ZFN or CRISPR/Cas9
It is possible that when the chromosome and the donor plasmid are cut by ZFN or CRISPR/Cas9, the entire plasmid can be integrated into the target site in either forward or reverse orientation through the non-homologous end joining (NHEJ) repair pathway, as previously reported [11] (Fig. 2a and b)
Summary
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 This strategy is generally less efficient at introducing larger DNA fragments. 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]. Single crossovers allow the integration of multiple copies of expression vectors in yeast [8], and have the advantage of being able to integrate larger DNA fragments into specific genomic loci compared to double crossover [4]
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