Lentiviral Tet-On system for inducible expression of fluorescent chimeras of dCas9 orthologs and its application

Abstract

ABSTRACT
The aim of the study is to achieve inducible expression of genetically encoded chimeric sensor molecules
based on catalytically inactive mutants of the Cas9 endonuclease family (dCas9) and fluorescent proteins (FP,
including EGFP, mCherry as well as a photoswitchable protein SAASOTI). Inducible expression of chimeric
proteins is important for performing in vitro and in vivo studies of spatial organization of the genome under the
conditions of reduced toxicity due to protein overxpression. Very important that the chimeric expression products
should not be accumulated in cells at a high concentration, which may result in cytotoxicity and phototoxicity. A
lentiviral vector of the 3rd generation (FU-tet-o-hOct4) was chosen for obtaining constructs with regulated
expression of the target construct upon the addition of doxycycline and rtTA (Tet-On system). The length of
selected dCas9 ortholog sequences was limited to 4 KB due to lentiviral cassette capacity. We selected two nuclear
localization sequences (NLS) - fused dCas9 orthologs from Streptococcus pyogenes (Sp) and Neisseria meningitides
(Nm). The ratio of dCas9: fluorescent protein (FP) was chosen as 1:1 for directing the obtained dCas9-FPs to
nuclear DNA loci by NLS targeting to achieve the assembly of DNA-bound and closely spaced pairs of
fluorophores that may potentially engage in Forster resonance energy transfer (FRET). EGFP and mCherry were
selected as FP capable of a FRET pair formation. Results: 1) a FU-tet-o-linker constructs carriing a polylinker with
unique restriction sites for dCas9 and FP orthologs were made in various combinations; 2) constructs FU-Tet-o-
spdCas9-EGFP and FU-Tet-o-nmdCas9-mCherry were engineered in order to achieve doxycycline-inducible in
which dCas9-FP expression; 3) telomere-specific sequences were selected by using two dCas9 ortholog-specific
PAMs and genetically engineered constructs for guide RNA expression pLH-spsgRNA-T, pLH-nmsgRNA-T for
telomere targeting were obtained. The obtained transfer vectors were used to assemble lentiviral particles. The
resultant lentiviral particles were used to transduce HEK293T and A549 cells. The transduction efficiency in the
case of NmdCas9-mCherry expression was 70%, and for the construct encoding SpdCas9-EGFP the expression was
observed in approximately 50% of cells. Stable cell lines were obtained by using puromycin selection. Fluorescence
microscopy of HEK293 and A549 cells was performed 48 h after induction of expression of spdCas9-EGFP and
NmdCas9-mCherry protein chimeras by doxycycline (1 μg/ml). In FACS-sorted (Department of Cell Biology and
Histology, Faculty of Biology, Moscow State University). A549 cells the expression of dCas9-FP pair was observed
with predominant localization of fluorescence in the nuclei. Cells expressing Sp dCas9-EGFP provided
homogeneous phenotype (i.e. morphology, localization of the construct with fluorescent protein, division rate). Cell
lines expressing Nm dCas9-mCherry ortholog chimeric protein showed varying degrees of fluorescence in the
nucleus as well as in the cytoplasm. The expression of dCas9-EGFP in human cells was then tested in the presence
and the absence of the doxycycline, that demonstrated a complete absence of fluorescence before the induction and
the development of fluorescence 24 hours after induction with a clear tendency of a decrease of nuclear transport of
fluorescent chimeras over time, and with the accumulation of fluorescent products in the cytoplasm in the case of
NmdCas9-mCherry. There was a stable growth of fluorescent chimeric proteins in the nuclei over time in the case of
SpdCas9-EGFP. Conclusions: Tet-On system for inducible expression of dCas9 was constructed and successfully
tested by using lentiviral transduction of human cell lines. The efficacy of nuclear transport as well as the
cytoplasmic retention was found to be ortholog and NLS-dependent. We plan to improve nuclear transport of FP
targeting to the nucleus using dCas9 optimization.

Speaker

Gerel Abushinova
Federal Research Centre “Fundamentals of Biotechnology” of the Russian Academy of Sciences
Russia

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