Engineering spacer specificity of the Cre/loxP system

Translational research on the Cre/loxP recombination system focuses on enhancing its specificity by modifying Cre/DNA interactions. Despite extensive efforts, the exact mechanisms governing how Cre distinguishes between substrates remains elusive. Cre recognizes 13 bp inverted repeats, initiating recombination in the 8 bp spacer region. While literature suggests that efficient recombination proceeds between lox sites with non-loxP spacer sequences when both lox sites have matching spacers, experimental validation for this assumption is lacking. To fill this gap, we investigated target site variations of identical pairs of the loxP 8 bp spacer region, screening 6,000 unique loxP-like sequences. Approximately 84% of these sites exhibited efficient recombination, affirming the flexibility of spacer sequences for catalysis. However, certain spacers negatively impacted recombination, emphasizing sequence dependence. Directed evolution of Cre on inefficiently recombined spacers not only yielded recombinases with enhanced activity but also mutants with reprogrammed selective activity. Mutations altering spacer specificity were identified, and molecular modelling and dynamics simulations elucidated the mechanism behind this specificity switch. These findings highlight the potential to fine-tune site-specific recombinases for spacer sequence specificity, offering a novel concept to enhance the applied properties of designer-recombinases for genome engineering applications. Characterizing the activity of Cre and an evolved Cre variant, RecS3. In order to profile the activity, an oligo library of 6000 different target sites are assembled to an E. coli inducible expression vector. The vector carries the recombinase of interest, either Cre (Exp6) or RecS3 (Exp13). Once library plasmids are assembled, they are transformed to E. Coli and expression of the recombinase is induced over night. The subsequent vectors are then purified from the sample. In order to determine how many times each target was either edited (recombined) or not edited (non-recombined) we run a PCR over the target sites then send the amplicon for illumination deep sequencing. Each experiment is done in triplicates at different induction concentrations.

针对Cre/loxP重组系统的转化研究，核心在于通过改造Cre蛋白与DNA的相互作用以提升其重组特异性。尽管已开展大量研究，但Cre区分不同底物的确切机制仍未阐明。Cre可识别13 bp的反向重复序列，并在8 bp间隔区（spacer region）启动重组反应。尽管已有文献报道，当两个lox位点的间隔区序列匹配时，即便间隔区并非野生型loxP序列，二者之间仍可发生高效重组，但该推论尚未得到实验验证。为填补这一研究空白，本研究针对loxP位点8 bp间隔区的同源配对靶位点变异展开探究，共筛选得到6000条独特的类loxP序列。其中约84%的位点可发生高效重组，证实了间隔区序列在催化重组过程中的灵活性；但部分间隔区会对重组反应产生抑制作用，凸显了序列依赖性的影响。针对重组效率低下的间隔区开展Cre的定向进化实验，不仅获得了活性提升的重组酶，还得到了重组选择性被重编程的突变体。研究人员鉴定出可改变间隔区识别特异性的突变位点，并通过分子建模与动力学模拟阐明了该特异性转换背后的分子机制。本研究结果证实，可通过精准调控位点特异性重组酶的间隔区序列识别特异性，为优化定制重组酶在基因组工程领域的应用性能提供了全新思路。为表征Cre及其进化突变体RecS3的重组活性，本研究构建了包含6000种不同靶位点的寡核苷酸文库，并将其克隆至大肠杆菌（E. coli）诱导型表达载体中。该载体携带目标重组酶，分别为Cre（实验6组，Exp6）与RecS3（实验13组，Exp13）。待文库质粒构建完成后，将其转化至大肠杆菌中，并于过夜培养期间诱导重组酶的表达。随后从菌体样本中纯化得到载体质粒。为统计每个靶位点发生编辑（重组）与未发生编辑（非重组）的次数，我们针对靶位点开展PCR扩增，并将扩增产物送至测序平台进行Illumina深度测序。所有实验均设置三次生物学重复，并在不同的诱导浓度下开展。