Genome Engineering

A series of game-changing developments have revolutionized the field of genome engineering within in the past years. With the advent of designer nucleases such as zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs) or the CRISPR/Cas9 system it has now become possible to specifically alter the genomic architecture of a wide range of different organisms. As such, redirecting the nuclease activity of a synthetically engineered endonuclease allows the efficient and specific introduction of double-strand breaks (DSBs) at a user-defined genetic locus. This in turn triggers cell-inherent repair mechanisms that can be exploited to delete, insert or replace a DNA sequence of interest at almost any genomic region.

DESIGNER NUCLEASES

Given their high on-target activity and ease of use designer nucleases based on the TALE scaffold and the CRISPR/Cas9 technology have established themselves as the method of choice for a wide array of genome engineering applications. TALE proteins derived from Xanthomonas bacteria provide a fully modular DNA binding architecture as these proteins consist of an array of 15-30 protein domains, each of which is 34-amino acids in length and determines binding to one DNA base through its repeat variable di-residue (RVD). Every repeat unit binds it target base in a largely context-independent manner, thus allowing the re-arrangement of the individual repeat units to obtain a TALE protein with user-defined sequence specificity. Tagging TALE-proteins with a nuclease domain (FokI) allows the specific and efficient cutting of genomes at user-defined loci. On the other hand, clustered regularly interspaced short palindromic repeats (CRISPR) in conjunction with CRISPR-associated proteins (Cas) provide an adaptive immune system to bacteria and archaea targeting foreign genetic material. Cas9, which is a member of the type II CRISPR-Cas system, requires two RNA molecules to be directed against its target sequence to operate as a sequence-specific endonuclease. The specificity of the guiding RNA molecule(s) can be changed and as such re-directed to target virtually any genomic sequence, subsequently introducing double-strand breaks (DSB) at high efficiency.

GENERATING KNOCKOUT AND KNOCK-IN CELL LINES

Following DSB, either a templated repair mechanism (homologous recombination from the sister chromatid, HR) or non-templated repair mechanisms (non-homologous end joining, NHEJ) are employed in order to rescue the cell's ability to replicate. Both repair mechanisms can be exploited to specifically manipulate the target genome. For example, NHEJ-mediated repair often results in insertions or deletions (indels), which can be exploited to generate knockout cell lines. At the same time, by providing a template with homology arms, heterologous genetic material can be introduced into the genetic locus of choice (knock-in).

For many projects we are primarily interested in the generation of knockout cell lines. To this end, we target a critical exon of the gene of interest and aim at the disruption of the reading frame by introducing indel mutations. This can lead to the introduction of premature stop codons and as such truncated proteins or even degradation of the cognate mRNA of interest due to nonsense mediated mRNA decay. In order to generate a knockout cell line, all alleles of a gene of interest have to be targeted for frame shift causing indel mutations. To facilitate the identification of knockout out cell clones by genotype, we make use of targeted deep sequencing coupled to an analysis tool that identifies knockout cell clones harboring all-allelic frame shift mutations (Opens external link in new windowOutKnocker). Using this workflow, we have thus far generated more than 180 individual knockouts in various cell lines, including human monocytic THP1 cells, HEK 293T cells, A549 cells, murine immortalized fibroblasts and murine immortalized macrophages. For more details on our knockout technology please visit the website of our genome engineering platform.

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