Targeted isolation of cloned genomic regions by recombineering for haplotype phasing and isogenic targeting

Abstract

Studying genetic variations in the human genome is important for understanding phenotypes and complex traits, including rare personal variations and their associations with disease. The interpretation of polymorphisms requires reliable methods to isolate natural genetic variations, including combinations of variations, in a format suitable for downstream analysis. Here, we describe a strategy for targeted isolation of large regions (∼35 kb) from human genomes that is also applicable to any genome of interest. The method relies on recombineering to fish out target fosmid clones from pools and thereby circumvents the laborious need to plate and screen thousands of individual clones. To optimize the method, a new highly recombineering-efficient bacterial host, including inducible TrfA for fosmid copy number amplification, was developed. Various regions were isolated from human embryonic stem cell lines and a personal genome, including highly repetitive and duplicated ones. The maternal and paternal alleles at the MECP2/IRAK 1 loci were distinguished based on identification of novel allele-specific single-nucleotide polymorphisms in regulatory regions. Additionally, we applied further recombineering to construct isogenic targeting vectors for patient-specific applications. These methods will facilitate work to understand the linkage between personal variations and disease propensity, as well as possibilities for personal genome surgery.

Figure 1.

Fosmid library host optimization. (A) Recombineering assay with GB05RedTrfA + pSC101β. The strain carries in the genome the modified red operon (32) (gam, beta, exo, recA) (red) under the control of the rhamnose inducible Rha promoter and has the TrfA gene (blue), which promotes high copy fosmid replication under the control of the arabinose inducible BAD promoter. The BAD promoter also drives expression of the Redβ protein (red) located on the helper plasmid pSC101β. A random fosmid clone was chosen for the insertion of modified blasticidin (bsd) cassette via recombineering. The cassette is flanked with 50 bp homology arms, identical to the region of choice (green). After the selection step the temperature-sensitive plasmid pSC101β is lost at 37°C. OriV—bidirectional origin of replication, Fori—unidirectional origin of replication. (B) Strain history. All strains were derived from DH10B. The strains GB05, GB05Red and DY380 as well as the pSC101γβαA plasmid were described previously (32–34). The BADTrfA and RhaγβαA cassettes in GB05RedTrfA were inserted at ybcC locus as the BADγβαA cassette in GB05Red. (C) Comparison of the recombineering efficiencies of the host strains. To test recombineering efficiency, a modified blasticidin cassette was inserted in a randomly selected fosmid from the H7 library. The number of recombinants was normalized to the number of cells surviving electroporation. Higher recombineering frequencies were obtained using the pSC101β helper plasmid. High copy fosmid induction further promoted recombineering efficiency.

Figure 2.

Recombineering strategy for fishing out genomic regions. (A) Fosmid library preparation. High molecular weight DNA was isolated from hES cell line or patient tissue sample. The DNA was sheared to ∼40 kb fragments. The fragmented DNA was ligated to pCC2Fos (dark grey lines) as concatamers and packaged in λ phage particles. The DNA was transduced into the E. coli host strain GB05RedTrfA + pSC101β. (B) Screening of the library by recombineering. On average 3500 cfu were plated per petri dish and then collected as a pool to a single well of a 96-well plate, which were pre-screened in super pools of rows and columns by PCR. Positive wells were cultured to induce the fosmids to high copy and express the Red proteins for recombineering before electroporation with a bsd. Recombination into the fosmid of choice conveyed blasticidin resistance after plating.

Figure 3.

Genomic regions of interest isolated from the H7 hES cell line. Depicted are the fished out clones (red), the capturing cassettes (black triangles) and the characteristics of the genomic region showing exons (thick yellow lines), introns (thin yellow lines), repetitive elements (all repeats) and G+C content (%GC). (A) Clones containing the OCT4 locus. (B) The AK locus with the capturing cassette inserted in front of exon 5. (C) Methyl CpG binding protein 2 locus (MECP2) and two different probes used for fishing out regions of interest. (D) The 3′-end of the PAX6 locus. (E) Isolation of the NANOG locus from the highly similar NANOG P1 pseudogene (depicted in lilac). (F) Clone covering the MYCN gene and the probe used, which has 69% G+C content.

Figure 4.

Allele-specific SNPs at the MECP2/IRAK1 locus on the X chromosome in H7 hES cells. In the upper part of the diagram the distribution of uniquely mappable Illumina reads from fosmid clones H7-F and H7-C02 is shown as grey lines. Gaps indicate repetitive sequence and the average corresponds to 11 000 reads. The colored lines indicate the SNP positions in comparison with the human reference genome as follows: green-A; red-T; blue-C and orange-G. The lower part of the diagram shows a 14 kb region containing two allele-specific SNPs among seven other SNPs and one newly identified indel, which is present on both alleles. One of the alleles has A instead of G at position 153 290 956 and G instead of C at position 153 285 631. The SNPs are in DNaseI hypersensitive site (DHS) and in CpG island, respectively as indicated. The indel indicates an insertion of a G onto a poly G track at 153 287 325.

Figure 5.

Workflow for the generation of isogenic conditional targeting constructs. All recombineering steps after the first one were performed in GB05RedTrfA after rhamnose induction of the RedγβαA operon. All the genes conveying antibiotic resistance have prokaryotic promoters (data not shown). The antibiotics used for selection of the recombinants at each stage are indicated at the right of each step: cm, chloramphenicol; bsd, blasticidin; km, kanamycin; amp, ampicillin. (A) The fosmid clone was identified by insertion of the bsd (lilac) as described above using 50 bp homology arms to the genomic region of interest (orange) and thereby inserting the homology arms for the next recombineering step (blue). The insertion site was chosen to be nearby a frame-shifting exon (orange). Fori and oriV, fosmid replication origins; cat, chloramphenicol resistance gene. (B) The bsd gene is replaced by a lacZneo reporter/stop cassette that conveys kanamycin resistance by recombination through the homology arms (blue). The lacZneo cassette contains our standard design (27,28,33) including frt sites for FLP recombinase and a loxP site for Cre recombinase at the 3′-end. The cassette also contains splice acceptor (sA); ribosomal-skipping peptides (t2A) and the SV40 polyadenylation signal (SVpA). Neo is the kanamycin/G418 resistance gene and lacZ is the β-galactosidase gene. (C) A region containing the lacZneo cassette, the frame shifting exon and flanking ∼4.5 kb homology arms is subcloned by gap repair into a p15A plasmid vector. The plasmid vector also includes the HSVtk promoter (TK) driven diphtheria toxin A chain gene (DTA) for counter selection and the ampicillin resistance gene (bla). (D) A second loxP site is added on the 3′ side of the frameshifting exon using blasticidin selection. The PGK–BSD gene is flanked by rox sites for later removal by Dre recombinase.