SeLOX—a locus of recombination site search tool for the detection and directed evolution of site-specific recombination systems

Site-specific recombinases have become a resourceful tool for genome engineering, allowing sophisticated in vivo DNA modifications and rearrangements, including the precise removal of integrated retroviruses from host genomes. In a recent study, a mutant form of Cre recombinase has been used to excise the provirus of a specific HIV-1 strain from the human genome. To achieve provirus excision, the Cre recombinase had to be evolved to recombine an asymmetric locus of recombination (lox)-like sequence present in the long terminal repeat (LTR) regions of a HIV-1 strain. One pre-requisite for this type of work is the identification of degenerate lox-like sites in genomic sequences. Given their nature—two inverted repeats flanking a spacer of variable length—existing search tools like BLAST or RepeatMasker perform poorly. To address this lack of available algorithms, we have developed the web-server SeLOX, which can identify degenerate lox-like sites within genomic sequences. SeLOX calculates a position weight matrix based on lox-like sequences, which is used to search genomic sequences. For computational efficiency, we transform sequences into binary space, which allows us to use a bit-wise AND Boolean operator for comparisons. Next to finding lox-like sites for Cre type recombinases in HIV LTR sequences, we have used SeLOX to identify lox-like sites in HIV LTRs for six yeast recombinases. We finally demonstrate the general usefulness of SeLOX in identifying lox-like sequences in large genomes by searching Cre type recombination sites in the entire human genome. SeLOX is freely available at http://selox.mpi-cbg.de/cgi-bin/selox/index

Discovery of Nigri/nox and Panto/pox site-specific recombinase systems facilitates advanced genome engineering

Precise genome engineering is instrumental for biomedical research and holds great promise for future therapeutic applications. Site-specific recombinases (SSRs) are valuable tools for genome engineering due to their exceptional ability to mediate precise excision, integration and inversion of genomic DNA in living systems. The ever-increasing complexity of genome manipulations and the desire to understand the DNA-binding specificity of these enzymes are driving efforts to identify novel SSR systems with unique properties. Here, we describe two novel tyrosine site-specific recombination systems designated Nigri/nox and Panto/pox. Nigri originates from Vibrio nigripulchritudo (plasmid VIBNI_pA) and recombines its target site nox with high efficiency and high target-site selectivity, without recombining target sites of the well established SSRs Cre, Dre, Vika and VCre. Panto, derived from Pantoea sp. alpha B, is less specific and in addition to its native target site, pox also recombines the target site for Dre recombinase, called rox. This relaxed specificity allowed the identification of residues that are involved in target site selectivity, thereby advancing our understanding of how SSRs recognize their respective DNA targets

Engineering Cre Recombinase for Genome Engineering

Cre recombinase recombines its DNA target, loxP sites, without help of accessory proteins or DNA repair systems. The simplicity of Cre-lox system has been widely utilized for genome editing, especially in mouse genetics. The goal of this dissertation is to constructCrerecombinase variants that will operate uponrecombination target sites (RTs) present within the genome, instead of perturbing the genome by inserting wildtype RTs for subsequent genome engineering. In general, the desired RTs native to the genome are asymmetric. However, the loxP sequence is pseudo-palindromic, requiring a homotetrameric formation of Cre recombinase. As a first step, I broke the symmetry of Cre tetramer so that each Cre monomer could be arranged spatially to bind distinct RT halfsites. I designed an alternative protein-protein interface for Cre. Then, I separated the mutations into a pair of Cre monomers. I could then arrange the assembly of this pair of complementary Cre monomers to form a functional heterotetramer, even though neither monomer exhibits activity alone. When combined with other mutations that confer distinct DNA specificities, the monomers preferentially formed the desired complex and recombined asymmetric DNA sequences with greater fidelity. Ive successfully found a pair of Cre monomers that do not work in isolation, but do when combined together. This has been successfully demonstrated in vivo in E. coli, mouse ES cell cultures and mouse retinal explants. As the next step, I need to change the DNA specificity of Cre recombinase to recognize native genomic sites. Surprisingly, the DNA preferences of Cre recombinase have not been thoroughly characterized. The 34 bp RT site loxP contains two palindromic arm regions and an 8 bp spacer region. The arm region is recognized by Cre monomers while homology of the spacer region determines compatibility between RT sites. While the consensus sequence of loxP is known, I performed the first high-throughput studies to determine Cres sequence specificity in the arm region. I broke the 13 bp arm region into 3 overlapping 5-6 bp small windows and used in vitro recombination and high-throughput sequencing data to generate logos for each window. I found that non-specific recombination can interfere with the analysis and careful selection of NaCl concentration is important for observing in vitro specificity. I have not only determined Cres sequence preferences, but also used similar methods to determine CreC2#4 (a Cre mutant) and VCre (a Cre homolog). In contrast to zinc finger and TAL effector domains, no modular decomposition of DNA specificity exists for Cre recombinase homologs. As a result, the RT specificity of Cre has previously been modified using directed evolution, a laborious approach. To accelerate the process, I used sequence information from homologs of Cre. By searching across genomes of different bacteria species, I found hundreds of Cre homologs. Closely related homologs share similarity in both amino acid sequence and predicted RT DNA sequence. By comparing residues that differ between close homologs in the aligned regions where Cre contacts the switched base pairs, I found candidate possible mutations for a specificity switch. The change in specificity was validated by the high-throughput sequencing assay. This demonstrates the feasibility of leveraging sequence alignment data to alter the specificity of Cre recombinase, reducing the amount of effort needed to generate mutants with novel RT preferences

SeLOX--a locus of recombination site search tool for the detection and directed evolution of site-specific recombination systems.

Site-specific recombinases have become a resourceful tool for genome engineering, allowing sophisticated in vivo DNA modifications and rearrangements, including the precise removal of integrated retroviruses from host genomes. In a recent study, a mutant form of Cre recombinase has been used to excise the provirus of a specific HIV-1 strain from the human genome. To achieve provirus excision, the Cre recombinase had to be evolved to recombine an asymmetric locus of recombination (lox)-like sequence present in the long terminal repeat (LTR) regions of a HIV-1 strain. One pre-requisite for this type of work is the identification of degenerate lox-like sites in genomic sequences. Given their nature-two inverted repeats flanking a spacer of variable length-existing search tools like BLAST or RepeatMasker perform poorly. To address this lack of available algorithms, we have developed the web-server SeLOX, which can identify degenerate lox-like sites within genomic sequences. SeLOX calculates a position weight matrix based on lox-like sequences, which is used to search genomic sequences. For computational efficiency, we transform sequences into binary space, which allows us to use a bit-wise AND Boolean operator for comparisons. Next to finding lox-like sites for Cre type recombinases in HIV LTR sequences, we have used SeLOX to identify lox-like sites in HIV LTRs for six yeast recombinases. We finally demonstrate the general usefulness of SeLOX in identifying lox-like sequences in large genomes by searching Cre type recombination sites in the entire human genome. SeLOX is freely available at http://selox.mpi-cbg.de/cgi-bin/selox/index

Aplicación del sistema de recombinación específica de secuencia β-six al estudio de la regulación de la expresión génica en células eucariotas y modelos animales

Las recombinasas específicas de secuencia son herramientas muy valiosas en la generación de modificaciones génicas condicionales. Estos sistemas permiten controlar la recombinación de forma específica de tejido, temporalmente, o ambas, y sortean diversas limitaciones de los sistemas de knockout (KO) convencionales, como la letalidad embrionaria o la generación de mecanismos compensatorios. Actualmente los sistemas Cre/loxP y Flp/FRT son los más empleados tanto en modelos animales como vegetales. La necesidad de realizar modificaciones más complejas en un mismo organismo hace que sea primordial caracterizar otras recombinasas que complementen a las existentes. La b recombinasa (b-rec) es originaria del plásmido pSM19035 de Streptococcus pyogenes. A diferencia de Cre y Flp, que en ausencia de factores adicionales catalizan la integración en un nuevo sustrato, la b-rec necesita un sustrato superenrollado y un cofactor de la reacción, una proteína asociada a la cromatina (como la procariota Hbsu o la eucariota HMG1). Se ha demostrado que la b-rec cataliza de forma específicamente intramolecular (resolución o inversión) la recombinación en células eucariotas, tanto de sustratos episomales como integrados en la cromatina, lo que indica que el entorno eucariota es capaz de proveer del cofactor y del superenrollamiento necesarios para que la b-rec realice su función. En este trabajo hemos determinado que la tasa de recombinación mediada por la b-rec no se ve afectada en absoluto por la deficiencia en el cofactor HMG1, alcanzando el mismo valor de recombinación en MEF KO en HMG1 que en wt. Este y otros datos confirman que en el entorno eucariota hay otras proteínas accesorias que pueden actuar de cofactores y sugiere que estas reacciones pueden ocurrir en la mayor parte de tejidos y tipos celulares. Para estudiar detalladamente el potencial de la b-rec en eucariotas desarrollamos un sistema de RAGE (activación génica mediada por recombinación) dependiente de la actividad b-rec; este sistema ha resultado funcional tanto en sustratos episomales como en sustratos integrados en la cromatina. También hemos generado un vector retroviral que porta la proteína de fusión b-Egfp, permitiendo de forma rápida y eficiente la integración y expresión funcional de nuestra proteína..