Generation of LEPR K nockout Rabbits with CRISPR/CAS9 System

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In humans mutations in the LEPR gene, encoding leptin hormone receptor, are associated with the development of morbid obesity, lipid metabolism dysregulation, defects of fertility. In rodents spontaneous mutations in Lepr gene have been described. Lepr knock-out (KO) animals have been generated using classical KO technology or CRISPR/Cas9 system. In rodents lipid metabolism is significantly different from that in humans or rabbits, therefore rabbits are considered as the most relevant model of morbid obesity and lipid metabolism dysregulation in humans, but so far LEPR knock-out rabbits have not been reported. Here using CRISPR/Cas9 system we generated LEPR knock-out rabbit by introducing a deletion of the region around exon 10 of the LEPR gene. We showed that the body weight of the knock-out rabbit was significantly higher than an average body weight of the wild type rabbits. CRISPR/Cas9-mediated technology for the generation of the LEPR knock-out rabbits will allow for the development of the model of morbid obesity and endocrine defects in humans due to leptin receptor mutations.

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作者简介

Y. Silaeva

Institute of Gene Biology Russian academy of sciences

Email: mshepelev@mail.ru

Center for Precision Genome Editing and Genetic Technologies for Biomedicine

俄罗斯联邦, Moscow

P. Safonova

Institute of Gene Biology Russian academy of sciences

Email: mshepelev@mail.ru
俄罗斯联邦, Moscow

D. Popov

Afanas`ev Research Institute of Fur – Bearing Animal Breeding and Rabbit Breeding

Email: mshepelev@mail.ru
俄罗斯联邦, Rodniki, Moscow region

М. Filatov

Institute of Gene Biology Russian academy of sciences

Email: mshepelev@mail.ru

Center for Precision Genome Editing and Genetic Technologies for Biomedicine

俄罗斯联邦, Moscow

Y. Okulova

Institute of Gene Biology Russian academy of sciences

Email: mshepelev@mail.ru

Center for Precision Genome Editing and Genetic Technologies for Biomedicine

俄罗斯联邦, Moscow

R. Shafei

Moscow State University

Email: mshepelev@mail.ru
俄罗斯联邦, Moscow

О. Skobel

Afanas`ev Research Institute of Fur – Bearing Animal Breeding and Rabbit Breeding

Email: mshepelev@mail.ru
俄罗斯联邦, Rodniki, Moscow region

D. Vysotskii

Afanas`ev Research Institute of Fur – Bearing Animal Breeding and Rabbit Breeding

Email: mshepelev@mail.ru
俄罗斯联邦, Rodniki, Moscow region

Y. Gubarev

Belgorod National Research University

Email: mshepelev@mail.ru
俄罗斯联邦, Belgorod

V. Glazko

Afanas`ev Research Institute of Fur – Bearing Animal Breeding and Rabbit Breeding

Email: mshepelev@mail.ru
俄罗斯联邦, Rodniki, Moscow region

T. Glazko

Afanas`ev Research Institute of Fur – Bearing Animal Breeding and Rabbit Breeding

Email: mshepelev@mail.ru
俄罗斯联邦, Rodniki, Moscow region

P. Georgiev

Institute of Gene Biology Russian academy of sciences

Email: mshepelev@mail.ru

Academician of the RAS

俄罗斯联邦, Moscow

G. Kosovsky

Afanas`ev Research Institute of Fur – Bearing Animal Breeding and Rabbit Breeding

Email: mshepelev@mail.ru
俄罗斯联邦, Rodniki, Moscow region

М. Shepelev

Institute of Gene Biology Russian academy of sciences

编辑信件的主要联系方式.
Email: mshepelev@mail.ru

Center for Precision Genome Editing and Genetic Technologies for Biomedicine

俄罗斯联邦, Moscow

参考

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2. Fig. 1. Development of a CRISPR/Cas9-based LEPR gene knockout technology in rabbits. (a) Sanger sequencing results of the sgLepr-2 target site in the rabbit genome. SNP A/T at the 20th position of the protospacer is indicated by an arrow. The nucleotide sequence of the target site including the protospacer and PAM is shown below the chromatogram. (b) Results of an in vitro cleavage assay. A 642 bp PCR product was incubated with SpCas9 RNP containing either sgLepr-2 or a control guide RNA (sgScr) with no targets in the rabbit genome. Cleavage with expected fragments (340 and 302 bp) occurred only with sgLepr-2. “Mw” lane – DNA size marker: O’GeneRuler 50 bp DNA Ladder (Thermo Scientific, SM1133); some marker bands are indicated on the right. Images of rabbit embryos before (c) and 120 hours after (d) microinjection of genome editing reagents. Photographs taken in Petri dishes using a Nikon SMZ-800 microscope (Japan).

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3. Fig. 2. Efficient LEPR gene editing in rabbit embryos. (a) Heteroduplex analysis of PCR products from genomic DNA of six rabbit embryos (1–6) injected with SpCas9 mRNA and sgLepr-2. 5 µL of PCR product was separated in an 8% polyacrylamide gel. “Mw” lane – DNA size marker: O’GeneRuler 50 bp DNA Ladder; some marker bands are labeled on the right. (b) Sanger sequencing results of samples #2 and #3. The vertical dashed line and red triangle indicate the SpCas9 cleavage site. SNP genotype (A) is indicated above the chromatogram peak. (c) ICE analysis of sequencing chromatograms from samples #2 and #3. Nucleotide sequences show detected indels. Indel frequencies were 92% and 89% for samples #2 and #3, respectively.

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4. Fig. 3. Molecular genetic analysis of a rabbit with LEPR knockout. (a) PCR analysis of genomic DNA from wild-type (wt) and LEPR knockout (KO) rabbits using 8% acrylamide gel electrophoresis. A 642 bp band is amplified from wild-type DNA; a 356 bp band is observed only in the KO sample. DNA marker sizes (O’GeneRuler 50 bp DNA Ladder, ThermoFisher Scientific, USA) are indicated on the right. Arrows indicate the positions of the PCR products. (b) Schematic of the wild-type LEPR genomic locus (wt) showing exons 10 and 11 (291 and 118 bp). Primer positions (“F” and “R”) are indicated. The KO panel shows the genomic locus of a gene-edited rabbit. The Cas9-induced double-strand break guided by sgLepr-2 is marked by a red triangle. (c) Sanger sequencing result of the 356 bp PCR product. Below the nucleotide sequence is the translated open reading frame of transcript XM_051856399 from the knockout allele. Amino acids 1–450 encode wild-type LEPR (underlined in green), followed by a nonsense peptide FKVVPCS (due to a random 9 bp insertion, underlined in red), ending with a stop codon TGA (boxed).

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5. Fig. 4. Body weight dynamics of a LEPR knockout rabbit. (a) Photo of a gene-edited rabbit at approximately two weeks old. (b) Growth curve of a LEPR knockout rabbit (“LEPR KO”) compared to the average body weight of wild-type Soviet Chinchilla rabbits (“wt”).

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