The Targeted Integration of a Reporter System for CRISPR-Cas9 Gene Editing into dbDNA-derived AAVS1 Safe Harbor
10.3870/j.issn.1672-0741.24.08.019
- VernacularTitle:CRISPR-Cas9基因编辑报道系统于dbDNA来源的iPSC AAVS1安全港的定点整合研究
- Author:
Jiajia HE
1
;
Yao CHEN
;
Chun CAO
Author Information
1. 江苏大学医学院基础医学系,镇江 212013
- Publication Type:Journal Article
- Keywords:
induced pluripotent stem cell;
doggybone DNA;
CRISPR-Cas9;
adeno-associated virus site 1
- From:
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
2025;54(2):151-158
- CountryChina
- Language:Chinese
-
Abstract:
Objective To establish a simple technical procedure for preparing doggybone DNA(dbDNA),and to discuss the distinctions in gene expression and immune response between dbDNA and conventional plasmid DNA.Induced pluripotent stem cell(iPSC)was generated from human peripheral blood mononuclear cells(PBMCs)by using dbDNA.To investigate a protocol for inserting ultra-long DNA fragments into the iPSC adeno-associated virus site 1(AAVS1),concurrently with the integration of the Cas9 expression frame point into the dbDNA-derived iPSC AAVS1 safe harbor,so as to establish a stable cell line that can achieve iPSC gene editing exclusively through the introduction of the single guide RNA(sgRNA)for the targeted gene.Methods A simple dbDNA preparation technique was established through utilization of Phi29 DNA polymerase multiple displacement amplification,protelomerase TelN digestion and covalent ligation,as well as the subsequent removal of the back-bone loop through the action of a restriction enzyme and exonuclease.The efficiency of green fluorescent protein(GFP)was eval-uated following electroporation of the dbDNA-GFP vector and the pMax-dbDNA-GFP traditional plasmid vector into PB-MCs.The mRNA expression of interferon-γ(IFN-γ)in the two groups was quantified by qPCR.The dbDNA reprogramming vectors dbDNA-MOS,dbDNA-KLF4,dbDNA-MYC and dbDNA-BCL were prepared and subsequently transferred into PBMCs to induce reprogramming.The dbDNA-iPSC cell line was identified through morphological analysis,alkaline phosphatase stai-ning,RT-PCR,and detection of pluripotent stem cell markers.The ZFN and TALEN gene editing techniques were employed to insert an 11.5 kb ultra-long DNA fragment containing the Cas9 expression cassette,EGFP reading frame with stop codons in the sequence,a eukaryotic cell screening marker and other elements at the iPSC AAVS1.The identification of the iPSC lines with Cas9 integrated into AAVS1 was conducted through the utilization of red fluorescence detection,genomic PCR reaction and Western blot.The EGFP single-strand homologous repair template and EGFP sgRNA were co-transfected into positive iPSC cell lines to repair the erroneous stop codons in the EGFP gene and to express green fluorescent protein correctly.This approach was undertaken in order to verify the gene editing ability of the Cas9 protein.Results The dbDNA vector was successfully pre-pared and employed for gene expression.The GFP gene expression efficiency of dbDNA was similar to that of traditional plas-mid DNA.The expression level of IFN-γ mRNA in the dbDNA group was lower than that of traditional plasmid DNA group.The iPSC cell lines resembling human embryonic stem cells were successfully obtained,iPSC cell lines with positive alka-line phosphatase staining,pluripotency related gene expression and stem cell pluripotency markers.The ultra-long fragment CRISPR-Cas9 gene editing reporting system was successfully inserted into the safe harbor AAVS1 site of the cell line,and the integrated fragment was successfully inserted into the genome by genomic PCR.Red fluorescent protein was expressed.Cas9 protein expression and EGFP gene recovery was confirmed by Western blot.Green fluorescence was observed.Conclusion A simple technical procedure for the preparation of dbDNA is successfully established.The gene expression efficiency of the dbD-NA vector is comparable to that of traditional plasmid DNA,with less immune response.The dbDNA-derived iPSCs are success-fully obtained,which is confirmed by morphological analysis,alkaline phosphatase staining,RT-PCR,and stem cell pluripotency markers.An 11.5 kb ultra-long DNA fragment is successfully inserted into the iPSC AAVS1 safe harbor to establish a stable iPSC cell line with Cas9-mediated gene editing.
