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Hyperhomocysteinemia and Dyslipidemia in point mutation G307S of cystathionine β-synthase-deficient rabbit generated using CRISPR/Cas9
Yong Cheng
Yangzhou University
Corresponding Author
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This work is licensed under a CC BY 4.0 License. Read Full License
View the published version at Lipids in Health and Disease.
Background: Congenital hyper-homocysteinemia (HHcy) is caused by a defective cystathionine β-synthase (CBS) gene, and is frequently associated with dyslipdemia. The aim of this study was to further elucidate the effect of mutated CBS gene on circulating lipids using a rabbit model harboring a homozygous G307S point mutation in CBS.
Methods: CRISPR/Cas9 system was used to edit the CBS gene in rabbit embryos. The founder rabbits were sequenced, and their plasma homocysteine (Hcy) and lipid profile were analyzed.
Results: Six CBS-knockout (CBS-KO) founder lines with biallelic modifications were obtained. Mutation in CBS caused significant growth retardation and high mortality rates within 6 weeks after birth. In addition, the 6-week old CBS-KO rabbits showed higher plasma levels of Hcy, triglycerides (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) compared to the age-matched wild-type (WT) controls. Histological analysis of the mutants showed accumulation of micro-vesicular cytoplasmic lipid droplets in the hepatocytes. However, gastric infusion of vitamin B and betaine complex significantly decreased the plasma levels of TG, TC and LDL-C in the CBS-KO rabbits, and alleviated hepatic steatosis compared to the untreated animals.
Conclusion: A CBSG307S rabbit model was generated that exhibited severe dyslipidemia when fed on a normal diet, indicating that G307S mutation in the CBS gene is a causative factor for dyslipidemia.
Keywords
Cystathionine β-synthase, hyperhomocysteinemia, dyslipidemia, rabbits, CRISPR/Cas9, G307S mutation
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Table S1. The sequences of potential off-target sites. Table S2. The sequences of potential off-target loci PCR primers.
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Reviewer #4 agreed
On 14 Sep, 2020
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Received 14 Sep, 2020
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Editorial decision: Major revision
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Review #5 received
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Reviewer #5 agreed
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Review #3 received
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Review #1 received
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Reviewers invited
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Reviewer #1 agreed
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Reviewer #2 agreed
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Reviewer #3 agreed
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Reviewer #4 agreed
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This preprint is under consideration at Lipids in Health and Disease. A preprint is a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Learn more about In Review
Research
Hyperhomocysteinemia and Dyslipidemia in point mutation G307S of cystathionine β-synthase-deficient rabbit generated using CRISPR/Cas9
Yong Cheng
Yangzhou University
Corresponding Author
Badges
Prescreen
This manuscript passed our Prescreen assessment
Complete author information
Appropriate declaration statements
Potential risks to human health
Prescreen
Peer Review Timeline
CURRENT STATUS: Published
Version 3
Posted 22 Sep, 2020
Published
On 14 Oct, 2020
No community comments so far
Review #1 received
Received 23 Sep, 2020
Editorial decision: Accept
On 23 Sep, 2020
Reviewers invited
Invitations sent on 22 Sep, 2020
Reviewer #1 agreed
On 22 Sep, 2020
Editor assigned
On 21 Sep, 2020
Submission checks complete
On 20 Sep, 2020
Editor invited
On 20 Sep, 2020
No comments provided
Review #5 received
Received 19 Sep, 2020
Editorial decision: Major revision
On 19 Sep, 2020
Reviewer #5 agreed
On 18 Sep, 2020
Review #2 received
Received 17 Sep, 2020
Review #1 received
Received 15 Sep, 2020
Review #4 received
Received 14 Sep, 2020
Editor assigned
On 14 Sep, 2020
Reviewers invited
Invitations sent on 14 Sep, 2020
Reviewer #1 agreed
On 14 Sep, 2020
Reviewer #2 agreed
On 14 Sep, 2020
Reviewer #3 agreed
On 14 Sep, 2020
Reviewer #4 agreed
On 14 Sep, 2020
Review #3 received
Received 14 Sep, 2020
Submission checks complete
On 13 Sep, 2020
Editor invited
On 13 Sep, 2020
No comments provided
Editorial decision: Major revision
On 18 Aug, 2020
Review #5 received
Received 13 Aug, 2020
Reviewer #5 agreed
On 11 Aug, 2020
Review #3 received
Received 08 Aug, 2020
Review #2 received
Received 08 Aug, 2020
Review #1 received
Received 08 Aug, 2020
Review #4 received
Received 08 Aug, 2020
Reviewers invited
Invitations sent on 02 Aug, 2020
Reviewer #1 agreed
On 02 Aug, 2020
Reviewer #2 agreed
On 02 Aug, 2020
Reviewer #3 agreed
On 02 Aug, 2020
Reviewer #4 agreed
On 02 Aug, 2020
Editor assigned
On 31 Jul, 2020
First submitted
On 30 Jul, 2020
Submission checks complete
On 30 Jul, 2020
Editor invited
On 30 Jul, 2020
Metrics
Comments: 0
PDF Downloads: ...
HTML Views: ...
License:
This work is licensed under a CC BY 4.0 License. Read Full License
View the published version at Lipids in Health and Disease.
Background: Congenital hyper-homocysteinemia (HHcy) is caused by a defective cystathionine β-synthase (CBS) gene, and is frequently associated with dyslipdemia. The aim of this study was to further elucidate the effect of mutated CBS gene on circulating lipids using a rabbit model harboring a homozygous G307S point mutation in CBS.
Methods: CRISPR/Cas9 system was used to edit the CBS gene in rabbit embryos. The founder rabbits were sequenced, and their plasma homocysteine (Hcy) and lipid profile were analyzed.
Results: Six CBS-knockout (CBS-KO) founder lines with biallelic modifications were obtained. Mutation in CBS caused significant growth retardation and high mortality rates within 6 weeks after birth. In addition, the 6-week old CBS-KO rabbits showed higher plasma levels of Hcy, triglycerides (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) compared to the age-matched wild-type (WT) controls. Histological analysis of the mutants showed accumulation of micro-vesicular cytoplasmic lipid droplets in the hepatocytes. However, gastric infusion of vitamin B and betaine complex significantly decreased the plasma levels of TG, TC and LDL-C in the CBS-KO rabbits, and alleviated hepatic steatosis compared to the untreated animals.
Conclusion: A CBSG307S rabbit model was generated that exhibited severe dyslipidemia when fed on a normal diet, indicating that G307S mutation in the CBS gene is a causative factor for dyslipidemia.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5