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Updated: Oct 21, 2025
CRISPR-Cas9 Mediated Gene Deletion in Human Pluripotent Stem Cells Cultured Under Feeder-Free Conditions
Published on: November 1, 2024
Generation of a homozygous LRPAP1 knockout human embryonic stem cell line (FDCHDPe009-B) by CRISPR/Cas9 system
Jie You1, Hairui Xi2, Shuangping Ma3
1Department of Ophthalmology & Vision Science, Eye & ENT Hospital, Shanghai Medical School, Fudan University, Shanghai, China; Key NHC Key Laboratory of Myopia (Fudan University), Laboratory of Myopia, Chinese Academy of Medical Sciences, China; Shanghai Key Laboratory of Visual Impairment and Restoration, Eye & ENT Hospital, Shanghai Medical School, Fudan University, Shanghai, China.
Abstract:
The homozygous autosomal recessive truncating mutations of LDL receptor related protein associated protein 1 (LRPAP1) is a possible reason for Nonsyndromic Extreme Myopia, patients with which show typical chorioretinal degeneration. We generated an LRPAP1 knockout FDCHDPe009-B embryonic stem cell line to study mechanisms of retinal degeneration underlying LRPAP1 deficiency with the help of the CRISPR/Cas9 system. Two distinct biallelic deletions in the cell line have been confirmed, which causing a frameshift and premature stop codons thus influence the translation of LRPAP1. FDCHDPe009-B has maintained normal stem cell morphology, pluripotent gene expression, parental karyotype, and ability to differentiate into three germ layers.
Insights
Nonsyndromic extreme myopia may stem from mutations in LDL receptor related protein associated protein 1 (LRPAP1). Researchers created an LRPAP1 knockout stem cell line to investigate retinal degeneration mechanisms, confirming gene deletions that disrupt LRPAP1 function.
Area of Science:
- Ophthalmology
- Genetics
- Cell Biology
Background:
- Nonsyndromic extreme myopia is associated with chorioretinal degeneration.
- Homozygous autosomal recessive truncating mutations in LDL receptor related protein associated protein 1 (LRPAP1) are implicated.
- Understanding LRPAP1's role in retinal health is crucial.
Purpose of the Study:
- To investigate the mechanisms of retinal degeneration caused by LRPAP1 deficiency.
- To generate and characterize a cellular model for studying LRPAP1-related eye conditions.
- To utilize CRISPR/Cas9 gene editing for precise genetic modification.
Main Methods:
- Generation of an LRPAP1 knockout embryonic stem cell line (FDCHDPe009-B) using CRISPR/Cas9.
- Confirmation of biallelic deletions in LRPAP1, leading to frameshift and premature stop codons.
- Assessment of stem cell characteristics, including morphology, pluripotency, karyotype, and differentiation potential.
Main Results:
- Successful generation of an LRPAP1 knockout stem cell line.
- Confirmation of genetic modifications that disrupt LRPAP1 translation.
- The modified stem cell line retained normal stem cell properties and differentiation capacity.
Conclusions:
- The generated LRPAP1 knockout cell line serves as a valuable model for studying retinal degeneration.
- This model will aid in elucidating the pathogenic mechanisms underlying LRPAP1 deficiency in myopia.
- Further research can explore therapeutic strategies targeting LRPAP1-related retinal diseases.
