Functional Genome Analysis  (B070)
Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 580
D-69120 Heidelberg, Germany.



  DNA / RNA Technologies



Genome-wide CRISPR-Cas9 knockout screen reveals genes with critical role in specifically metastatic PDAC cells

Early and aggressive metastasis is a crucial factor of the dismal prognosis of pancreatic ductaladenocarcinoma (PDAC). Toward identifying processes that are essential to PDAC metastasis, we performed a genome-wide gene knockout screen with a CRISPR-Cas9 library of 259,900 single guide RNA constructs on two sub-clones of a PDAC cell line, which exhibit very low or very high metastatic potential, respectively.

Data comparison identified several genes and pathways that were relevant for metastasising cells.
One knockout had a particularly strong effect and was analysed with respect to functional aspects. It was critical for the difference in metastatic potential. Also, a buffering interaction was found between this gene and a master regulator in cancer.

In vivo experiments, namely lung and liver colonisation assays after intravenous and intracardiac cell injections into the bloodstream as well as metastatic behaviour after resection of subcutaneously grown primary tumours, confirmed the very strong effect on metastasis.

Figure legend: Schematic outline of the genome-wide knockout experiment.

Process for an efficient lentiviral cell transduction

The combination of lentiviruses with techniques such as CRISPR-Cas9 has resulted in efficient and precise processes for targeted genome modification. An often-limiting aspect, however, is the efficiency of cell transduction. Low efficiencies with particular cell types and/or the high complexity of lentiviral libraries can cause insufficient representation.

We have developed a protocol that yielded substantial increases in transduction efficiency in various cell lines in comparison to several other procedures. By applying and combining relatively simple procedural modifications – (i) mixing lipofectamines 3000 and LTX for transfecting HEK-293T cells, (ii) virus concentration in spin-columns and (iii) gently pelleting cells prior to transfection – substantial improvements in transduction yields could be achieved.

Pirona et al. (2020) Biol. Method Protoc., bpaa005. pdf icon

Figure legend: Schematic overview of the various steps of the optimised transduction process. The steps in blue-labelled frames are different to standard protocols and jointly increase transduction efficiency substantially


CRISPR RNA-guided FokI nucleases repair a disease-causing variant in the PAH gene in a phenylketonuria model

In a proof-of-concept study, we demonstrated the potential of an improved CRISPR/Cas9 system the FokI-dCas9 system for precision medicine, in particular for targeting phenylketonuria (PKU) and other monogenic metabolic diseases. The FokI-dCas9 system can greatly improve the specificity of genome editing. In contrast to the standard system, it requires dimerization of the FokI-dCas9-sgRNA complex, meaning that monomeric FokI-dCas9-sgRNA is unable to cut the DNA strand, thus reducing substantially the chances of contaminating off-target effects.
PKU is the most common inherited disease in amino acid metabolism. It leads to severe neurological and neuropsychological symptoms if untreated or late diagnosed. Correction of the disease-causing variants in the phenylalanine hydroxylase (PAH) gene could rescue residual activity and restore normal function. The CRISPR/Cas9 system is a recently developed genome editing technique. We applied a modification, which employs the fusion of inactive Cas9 (dCas9) and the FokI endonuclease (FokI-dCas9) to correct the most common variant (allele frequency 21.4%) in the PAH gene - c.1222C>T (p.Arg408Trp) - as an approach toward curing PKU. Co-expression of a single guide RNA plasmid, a FokI-dCas9-zsGreen1 plasmid, and the presence of a single-stranded oligodeoxynucleotide in PAH_c.1222C>T COS-7 cells – an in vitro model of PKU – corrected the PAH variant and restored PAH activity.

Pan et al. (2016) Sci. Rep. 6, 35794.  pdf icon

Scheme of the process: one dimer of the FokI-dCas9 complex binds to two “half-sites” on the genome with a certain spacer length and generate double-strand breaks in the DNA. The double-strand breaks are then repaired by homology directed repair, introducing the non-mutated sequence provided as an oligonucleotide.

Scheme of shRNA knockdown process.

Scheme of shRNA knockdown experiments. An shRNA-construct is brought into a cell by means of a lentivirus system. The construct integrates into the genome and the respective shRNA is constitutively expressed at high levels, acting as an inhibitory RNA intracellularly. Using a common primer pair (P1, P2), a barcode sequence that is unique for each shRNA construct can be amplified from each cell.  Therefore, also complex mixtures of cells transduced with different constructs can be studied simultaneously.

Functional screens by means of lentiviral shRNA libraries

RNA interference (RNAi) has become a popular and important tool for the analysis of gene function, although being partly superseeded by CRISPR-Cas already. Loss-of-function studies have greatly facilitated functional analyses of the human transcriptome.
Limitations of early siRNA experiments, most importantly the transient inhibition of gene expression as well as the inefficient transfection into non-dividing cells, were generally overcome by short hairpin RNA (shRNA) expression vectors, which stably integrate into a target cell's genome via retro- or lentiviral gene transfer. Intracellular processing of shRNAs results in short duplex RNAs with siRNA-like properties. Viral integration ensures not only a broad range of infectable target cell types but also the stable expression of specific shRNAs, resulting in the permanent reduction of the targeted gene product. Complex shRNA expression libraries allow the targeted knockdown of thousands of different genes in a single experiment.
Using such lentiviral vector shRNA libraries and initially barcode arrays and meanwhile next-generation sequencing analysis for decoding of the pooled RNAi screens, we are able to quantify the abundance of individual shRNAs and thus determine in a complex pool the number of cells infected with an individual shRNA construct.
We used the technique to predict anti-proliferative effects of individual shRNAs from pooled negative selection screens. By such screens, we identified synthetic-lethal activities toward combination therapies, defined genes which are required for a stem-cell like phenotype and found tumour suppressor genes by in vivo studies. Further studies are under way, both for the elucidation of basic regulative processes associated to cancer and for the identification of pathways that are affected by particular drugs or compounds. In particular, we use the technique for obtaining more detailed information on the functional effects of particularly potentially druggable gene products.

Wolf et al. (2014) Oncogene 33, 4273-4278. pdf icon
Fredebohm et al. (2013) J. Cell Sci. 126, 3380-3389. pdf icon

Böttcher et al. (2014) BMC Genomics 15, 158. pdf icon
Böttcher et al. (2010) BMC Genomics 11, 7. pdf icon

Wolf et al. (2013) Breast Cancer Res. 15, R109. pdf icon
Böttcher & Hoheisel (2010) Curr. Genom. 11, 162-167.

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