Method to Assemble Genomic DNA Fragments or Genes on Human Artificial Chromosome with Regulated Kinetochore Using a Multi-Integrase System

Abstract

The production of cells capable of carrying multiple transgenes to Mb-size genomic loci has multiple applications in biomedicine and biotechnology. In order to achieve this goal, three key steps are required: (i) cloning of large genomic segments; (ii) insertion of multiple DNA blocks at a precise location and (iii) the capability to eliminate the assembled region from cells. In this study, we designed the iterative integration system (IIS) that utilizes recombinases Cre, ΦC31 and ΦBT1, and combined it with a human artificial chromosome (HAC) possessing a regulated kinetochore (alphoid^tetO-HAC). We have demonstrated that the IIS-alphoid^tetO-HAC system is a valuable genetic tool by reassembling a functional gene from multiple segments on the HAC. IIS-alphoid^tetO-HAC has several notable advantages over other artificial chromosome-based systems. This includes the potential to assemble an unlimited number of genomic DNA segments; a DNA assembly process that leaves only a small insertion (<60 bp) scar between adjacent DNA, allowing genes reassembled from segments to be spliced correctly; a marker exchange system that also changes cell color, and counter-selection markers at each DNA insertion step, simplifying selection of correct clones; and presence of an error proofing mechanism to remove cells with misincorporated DNA segments, which improves the integrity of assembly. In addition, the IIS-alphoid^tetO-HAC carrying a locus of interest is removable, offering the unique possibility to revert the cell line to its pretransformed state and compare the phenotypes of human cells with and without a functional copy of a gene(s). Thus, IIS-alphoid^tetO-HAC allows investigation of complex biomedical pathways, gene(s) regulation, and has the potential to engineer synthetic chromosomes with a predetermined set of genes.

Keywords: DNA assembly; HAC; IIS; human artificial chromosome; iterative integration system; synthetic biology.

Figure 1

Scheme of plasmids used in the IIS-alphoid^tetO-HAC system. (a) The platform cassette A037. (b) Type I A167 and Type I-ARS A168 carrier plasmids to deliver a genomic DNA fragment. (c) Type II A169 and Type II-ARS A170 carrier plasmids to deliver a genomic fragment. (d) A135-JH plasmid expressing ΦBT1 integrase and Cre recombinase. (e) A139 plasmids expressing ΦC31 integrase and Cre recombinase. Restriction sites used in this work are marked in blue.

Figure 2

Insertion of the platform cassette into alphoid^tetO-HAC in hamster CHO cells. (a) The XhoI-linearized platform cassette A037 was inserted into the Ch13 genomic segment present within alphoid^tetO-HAC by homologous recombination in DT40 cells. Then the alphoid^tetO-HAC carrying the platform cassette was MMCT transferred from chicken DT40 cells to hamster CHO cells. (b) Diagnostic PCRs to verify correct targeting of the platform cassette A037 into the Ch13 region present within the HAC. Two pairs of diagnostic primes, B128/B124 and B129/B126, amplified the expected products of 4.4 kb and 3.9 kb in size, respectively, confirming correct recombination between the M2A hook sequence of A037 and a homologous sequence of the Ch13 segment. Two pairs of diagnostic primes, B034/B126 and B034/B127, amplified the expected products of 4.6 kb and 4.4 kb in size, respectively. This confirmed the correct recombination between the M2B hook sequence of A037 and a homologous sequence of the Ch13 segment. M, a GeneRuler DNA ladder mix (Fermentas). Lane 1, DT40 cells carrying alphoid^tetO-HAC without A037 insertion (a negative control); Lane 2, targeted DT40:BH3:A037 clone #48; Lane 3, targeted DT40:BH3:A037 clone #49. (c) FISH analysis of the alphoid^tetO-HAC carrying the platform cassette in CHO cells. Arrows indicate to the HAC visualized with the BAC specific probe (in red).

Figure 3

Scheme of DNA segment integration by the iterative integration system (IIS). (a) The starting platform cassette. Cells express the GHT marker, i.e., a green fluorescence protein (GFP). Also, they are hygromycin resistant (hph) and Ganciclovir sensitive (TK). (b) Recombination between a Type I carrier vector and a platform cassette by Cre recombinase and ΦC31 integrase. The GHT marker is replaced by the PCF marker and the first DNA segment of interest is integrated into the platform cassette (DNA1). The integration event is selected using Puromycin and Ganciclovir. (c) A structure of the platform cassette after the 1st round of integration. The PCF marker is expressed. Therefore, the cells have red fluorescence (mCherry), Puromycin resistance (Pac) and 5-Fluorocytosin sensitivity (FcyFur). (d) Recombination between a Type II carrier vector and a platform cassette by Cre recombinase and ΦBT1 integrase. The PCF marker is replaced by the GHT marker and the second DNA segment of interest is integrated into the platform cassette (DNA2). The integration event is selected using Hygromycin and 5-Fluorocytosine. (e) A structure of the platform cassette after the second round of recombination. The cells express the GHT marker, i.e., a green florescence protein (GFP). They become again Hygromycin resistant (hph) and Ganciclovir sensitive (TK). This structure is identical to the stating cassette aside from the integration of DNA segments of interest, DNA1 and DNA2.

Figure 4

Error proofing design of the IIS-alphoid^tetO-HAC. (a,b) Products of incomplete recombination between Type I carrier plasmid with an active GHT cassette. (c,d) Products of incomplete recombination between Type II carrier plasmid with an active PCF cassette. The selection agents to remove each misassembled product are listed.

Figure 5

VHL gene reconstruction using the IIS-alphoid^tetO-HAC system. (a) Construction of Type I A168 and Type II A170 carrier plasmids containing fragments with exon1 or exon 2 or exon 3 of the VHL gene. Positions of the VHL fragments in the human genome (GRCH38/hg38) are indicated. (b) Three rounds of insertion of the VHL fragments into alphoid^tetO-HAC carrying the platform cassette are shown. Representative images of changing cell color after each round are shown. (c) Diagnostic PCRs for insertion of the VHL fragments after each round of recombination. Round 1: Lanes 1 to 5, diagnostic PCR for the fragment 3 with the expected size 1.7 kb. Lane 1, Clone #1; Lane 2, Clone #11; Lane 3, Clone #12; Lane 4, Clone #14; Lane 5, genomic DNA from CHO cells as a negative control. Round 2: Lanes 6–10, diagnostic PCR for junction between fragments 2 and 3. The expected size is 688 bp. The expected size for a negative control is 608 bp. Lane 6, Clone #14–4; Lane 7, Clone #14–12; Lane 8, alphoid^tetO-HAC carrying the full-length human VHL gene in CHO cells; Lane 9, a TAR/BAC clone containing the VHL gene; Lane 10, human genomic DNA as a positive control. Round 3: Lanes 11–14, diagnostic PCR for junction between fragments 1 and 2. The expected size is 2788 bp. The expected size for a negative control is 2716 bp. Lane 11, Clone #14–12–3; Lane 12, alphoid^tetO-HAC carrying the full-length VHL gene in CHO cells; Lane 13, a TAR/BAC clone containing the VHL gene; Lane 14, human genomic DNA as a positive control. (d) RT-PCR for mRNA of VHL. The expected product size is 642 bp. Lane 15, Clone #14–12–3; Lane 16, genomic DNA from CHO cells as a negative control; Lane 17, human genomic DNA from HeLa cell line as a positive control; Lane 18, human genomic DNA from HT1080 cell line as a positive control.