Figure 2.

Applications using ssDNA of various lengths. (A) Genome editing in human cells using CRISPR/Cas9. (left) A genomically integrated GFP coding sequence is disrupted by the insertion of a stop codon and a 68-bp genomic fragment from the AAVS1 locus. Restoration of the GFP sequence by HDR with a ssDNA donor sequence results in GFP+ cells that can be quantified by FACS. (right) Bar graph depicting HDR efficiencies induced by MeRPy-PCR derived ssDNAs of different lengths versus a 200mer chemically synthesized strand from IDT. Data are shown as mean ± STD (N = 3). (B) ssDNA scaffold was generated via MeRPy-PCR from the phage genome, p7308 and used in the folding of a 30 nm DNA origami barrel and a 20 nm rectangle. Agarose gel electrophoresis shows the 1 kb Ladder (L), purified scaffold strand (S) alongside the folded barrel and 20 nm rectangle structures (F). TEM depicts the folded DNA origami barrel (left) and a 20 nm rectangle (right). Scale bars denote 100 and 50 nm for small insert. (C) A library comprising 42 000 probe sequences designed to tile along an 8.4 Mbp region of Human Chromosome 8 was amplified from a small amount of template using MeRPy-PCR with a Cy3-labeled reverse primer and subsequent recovery of fluor-tagged strand 1 library. The generated fluor-labeled ssDNA library was validated insitu on fixed human metaphase spreads and interphase cells. Scale bars denote 20 μm (zoom of metaphase spread scale bar denotes 5 μm).