Design and selection of guides for CRISPR/Cas9-mediated knockout of the Kcnv2 gene in mouse cells

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Abstract

Mutations in the human KCNV2 gene cause a rare hereditary disease — cone dystrophy with supernormal rod response (CDSRR), characterized by progressive vision loss and impaired color discrimination. The KCNV2 gene encodes the Kv8.2 subunit of a potassium channel that is critical for the normal function of retinal photoreceptors. Gene therapy offers a promising treatment approach for this condition. To test the efficacy of gene therapy, an appropriate experimental disease model, such as a knockout mouse model, is required. This study focused on selecting optimal guide RNAs for knocking out the Kcnv2 gene using the CRISPR/Cas9 system and testing their efficiency in a mouse cell line. The selected guide RNAs can be utilized to generate a Kcnv2-/- mouse model.

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About the authors

E. N. Antonova

Moscow Institute of Physics and Technology (National Research University)

Author for correspondence.
Email: antonova.en@genlab.llc
Russian Federation, Dolgoprudny, Moscow Oblast, 141701

A. B. Soroka

Moscow Institute of Physics and Technology (National Research University)

Email: antonova.en@genlab.llc
Russian Federation, Dolgoprudny, Moscow Oblast, 141701

O. N. Mityaeva

Moscow Institute of Physics and Technology (National Research University); Lomonosov Moscow State University; Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies

Email: antonova.en@genlab.llc
Russian Federation, Dolgoprudny, Moscow Oblast, 141701; Moscow, 119991; Moscow, 125315

P. Yu. Volchkov

Moscow Institute of Physics and Technology (National Research University); Lomonosov Moscow State University; Federal Research Center for Innovator and Emerging Biomedical and Pharmaceutical Technologies

Email: antonova.en@genlab.llc
Russian Federation, Dolgoprudny, Moscow Oblast, 141701; Moscow, 119991; Moscow, 125315

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Supplementary files

Supplementary Files
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1. JATS XML
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2. Fig. 1. Diagram of the Kcnv2 gene with designated sites of recognition by guides in the SnapGene program. The purple color shows the names and locations of the primers for amplification of the region covering the large and small deletions.

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3. Fig. 2. Electrophorogram based on the results of PCR screening of B16-F10 cells. a – Large deletions, b – small deletion. (K–) – PCR product from untransfected cells (negative control), M – DNA length marker. Electrophoresis in 1.5% (a) and 3% (b) agarose gel.

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4. Fig. 3. The efficiency of deletion induction, obtained from the results of electrophoregram analysis using densitometry in the ImageJ program.

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5. Fig. 4. Indel frequencies in the range of 10 nucleotides from the predicted cutting site. a – for the gx/2 guide, b – for the g4 guide.

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6. Fig. 5. Confirmation of a small deletion by Sanger sequencing. Chromatogram corresponding to deletion by g3/1–gx/2 guides (top) and control chromatogram obtained from cells without deletion (bottom).

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7. Fig. 6. Confirmation of large deletions by Sanger sequencing. The upper chromatograms were obtained from a DNA fragment with a deletion, and the lower ones from a DNA fragment without a deletion: a – gx/2–g4, b – g3/1–g6, c – gx/2–g6, d – g3/1–g4.

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8. Fig. DM1. Plasmid (# 52961; Addgene, USA). a is a diagram showing the correspondence of the sticky ends of the vector and the sticky ends of paired oligonucleotides; the BsmBI restrictase recognition sites are highlighted in purple, and the unique part of the guide is shown in green using the g1 guide as an example. b is a plasmid map with the BsmBI restriction sites and the cut–out product. b is the restriction product on the electrophoregram.

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9. Fig. DM2. Electrophoregram based on the results of PCR screening of genomic DNA with small deletions isolated from B16-F10 cells. The electrophorogram shows the pairs of guides used to create the small deletion: g3/1 and g2/1, g2/1 and gx/2, g3/1 and gx/2, g4/1 and gx/2. M is a marker of DNA lengths, K is a negative control.

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10. Fig. DM3. Gel electrophoresis based on the results of PCR screening of genomic DNA with large deletions isolated from B16-F10 cells. The electrophorogram shows the pairs of guides used to create a large deletion: g1–gx/24; g1–g3/1; g3–gx/2; g-g2/1; g3/1–g6; g1–g6; g3–g6; g3/1–g4; g1–g4; gx/2–g4; gx/2–g6; g3/1–g6. M is a marker of DNA lengths, K is a negative control.

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11. Fig. DM4. Secondary guide structures obtained in RNAfold Web Server. a are the secondary structures of the guides used; b is the secondary structure with the designations RAR-hairpin (repeat and anti-repeat region), hairpins (stem loop) 1, 2 and 3, 20–nucleotide sequence (Bruegmann et al., 2019).

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