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Tobias Wimmer, Philipp Klekamp, Maria Weller, Birgit Lorenz, Knut Stieger; First steps to a genome editing approach correcting CLN3/Cln3deltaEx7/8. Invest. Ophthalmol. Vis. Sci. 2019;60(9):4935.
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Neuronal ceroid lipofuscinoses (NCL) are a heterogeneous group of autosomal-recessive neurodegenerative storage diseases. The clinical features include epilepsy, a progressive decline in motor skills, visual impairment and premature death. The juvenile form called CLN3 or Batten disease is characterized by a common homozygous 1.02 kbp deletion of exons 7 and 8 and an early degeneration of all retinal layers as the first presenting symptom. Since the description of CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR associated protein9) as a programmable tool generating genomic double strand breaks (DSBs), targeted genome editing strategies have become possible. With the use of homology directed repair (HDR) or microhomology mediated end joining (MMEJ) DNA repair pathways, the correction of this deletion seems to be possible by providing a wildtype (wt) DNA template after gRNA/Cas9 induced DSBs.
Eight guideRNA (gRNA) sequences were identified using ATUM (Newark, CA) within the human CLN3 and six gRNA sequences in murine Cln3 genomic region in intron 6 and intron 8. Target and gRNA oligonucleotides were hybridized and cloned into px459 (Addgene: #62988) and a bioluminescence biosensor (BRET) reporter vector. A HDR/MMEJ shuttle system with a fluorescent reporter was generated to study the HDR and MMEJ frequency. Additionally, wt-templates consisting of genomic DNA or mRNA derived sequences were synthesized and subcloned.
All hybridized DNA fragments were cloned successfully into the reporter systems and the gRNA/Cas9 vector. Three highly active gRNA/Cas9 complexes were identified and characterized using the BRET biosensor in CLN3/Cln3 intron 6 and intron 8. Furthermore, the fluorescent reporter system demonstrated successful HDR integration using the exon 6 and exon 9 homologous regions flanking the target site, with a combination of two selected gRNAs.
Methods to study and improve the therapeutic genome editing strategy in-vitro were generated in this study. With the identified gRNA sequences and the use of different wt-templates with homologous sequence sizes in-vitro HDR and MMEJ assays can be performed with a murine Cln3deltaEx7/8 cerebellar precursor cell line and a human CLN3deltaEx7/8 cell line derived from pluripotent stem cells.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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