Genome Analysis (B070)
Im Neuenheimer Feld 580
DNA / RNA Technologies
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
meaning that monomeric FokI-dCas9-sgRNA is unable to cut the
thus reducing substantially the chances of contaminating off-target
is the most common inherited disease in amino acid metabolism. It leads
severe neurological and neuropsychological symptoms if untreated or
diagnosed. Correction of the disease-causing variants in the
hydroxylase (PAH) gene could rescue residual activity and
function. The CRISPR/Cas9 system is a recently developed genome editing
technique. We applied a modification, which employs the fusion of
(dCas9) and the FokI endonuclease (FokI-dCas9) to
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,
presence of a single-stranded oligodeoxynucleotide in PAH_c.1222C>T
COS-7 cells – an in vitro model of PKU – corrected the PAH
and restored PAH activity.
Pan et al. (2016) Sci. Rep. 6, 35794.
Scheme of the process: one dimer of the FokI-dCas9
complex binds to two “half-sites” on the genome with a certain spacer
and generate double-strand breaks in the DNA. The double-strand breaks
repaired by homology directed repair, introducing the non-mutated
sequence provided as an oligonucleotide.
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.
screens by means of lentiviral shRNA libraries
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
Limitations of early siRNA experiments, most
importantly the transient inhibition of gene expression as well
inefficient transfection into non-dividing cells, were generally
overcome by short hairpin RNA (shRNA)
stably integrate into a target cell's genome via
or lentiviral gene transfer. Intracellular processing of shRNAs results
short duplex RNAs with siRNA-like properties. Viral integration ensures
only a broad range of infectable target cell types but also the stable
expression of specific shRNAs, resulting in the permanent reduction of
targeted gene product. Complex shRNA expression libraries
allow the targeted knockdown of
of different genes in a single experiment.
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
individual shRNAs and thus determine in a complex pool the number of
cells infected with an individual shRNA construct.
We used the
predict anti-proliferative effects of individual shRNAs from pooled
selection screens. By such screens, we identified synthetic-lethal
activities toward combination therapies, defined genes which are
for a stem-cell
like phenotype and found tumour
suppressor genes by in vivo
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
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