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. 2022 Jan 20;13:422. doi: 10.1038/s41467-022-28080-7

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 1 — a Overview of the target site identification in the inverted repeats (int1h) surrounding the F8 gene. For each 8 bp sequence in int1h-1 the surrounding 13 bp were compared. Every 13 bp sequence pair with less than 8 mismatch positions was analyzed for similarity to previous target sites targeted by evolved Cre-type recombinases. After off-target analysis the loxF8 target site was nominated. Underlined nucleotides display asymmetric positions. The nucleotide sequence of the left (orange) and the right (blue) half-sites are indicated. b Overview of substrate-linked directed evolution (SLiDE). The evolution starts by cloning recombinase libraries into the pEVO expression vector (I.). Single recombinase genes are displayed in different gray scales. Two lox-like sites for the recombinases are indicated as small triangles (excision orientation). After expression (II.) of the recombinases, plasmids are analyzed (III.). A library activity assay is performed for each cycle (IVa.). A schematic representation of the different recombination products on an agarose gel is shown. Marker and sizes are indicated. Active recombination variants that are further evolved (IVb.). Upon recombination a unique restriction site (scissors) is excised. Applying a restriction digest will linearize the non-recombined plasmid, recombined plasmid remains circular. A PCR (error-prone) using the indicated primers (arrows) will only generate a product from recombined plasmids. This amplified and mutated active recombinase variants are then subjected to the next evolution round. c Strategy to evolve a heterodimer of recombinases to target the loxF8 site. The monomers of the heterodimer recognize either the left (loxF8-L, orange) or the right (loxF8-R, blue) half-site of the loxF8 target site. Recombinases are shown as homo- or heterotetramers binding to two target sites in the recombination synapses. d Plasmid-based activity assay of the recombinase heterodimer library evolved for targeting loxF8. The agarose gel picture of the final heterodimer library recombining the loxF8 site is shown at two different expression levels (0 and 10 μg/ml L-arabinose). The upper band represents the unrecombined plasmid (illustrated by a line with two triangles), with the lower band showing the recombined plasmid (a line with one triangle). Marker (M) and sizes are indicated. Source data are provided as a Source data file.