Generation of isogenic human iPSC line from a DCM patient carrying the Finnish founder mutation (p. S143P) in LMNA gene using the CRISPR/Cas9 system
Auri, Heini (2019)
Auri, Heini
2019
Bioteknologian tutkinto-ohjelma
Lääketieteen ja terveysteknologian tiedekunta - Faculty of Medicine and Health Technology
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Hyväksymispäivämäärä
2019-12-11
Julkaisun pysyvä osoite on
https://urn.fi/URN:NBN:fi:tuni-201911155974
https://urn.fi/URN:NBN:fi:tuni-201911155974
Tiivistelmä
Background and aims: CRISPR (clustered, regularly interspaced, short, palindromic repeats) system is an adaptive immune system against foreign genetic elements that has been found in various bacterial and archaeal species. CRISPR system has been used efficiently and precisely to edit diverse genomes of eukaryotes. It has provided an ability to repair mutated alleles in patient lines that enable the analysis of isogenic cell pairs that differ in a single genetic change, allowing a detailed study of the molecular and cellular phenotypes that result from this abnormality. So far, CRISPR/Cas genome editing experiments have been primarily performed using human immortalized cell lines (e.g. HEK293T cells). These cell lines are easy to manipulate and transfect. While only little information is available considering gene correction in human iPS cells. The purpose of this study was to precisely correct the Finnish founder mutation (p. S143P) in the LMNA gene that causes Dilated cardiomyopathy (DCM) using CRISPR/Cas9 genome editing technique.
Methods: Isogenic gene-corrected cell line was obtained using the CRISPRs/Cas9 system that comprises a Cas9 and a guide RNA plasmid in combination with a donor plasmid serving as a homologous template covering the site of the mutation. T7 Endonuclease assay was used to detect the cleavage activity of three selected sgRNAs in HEK293 cells. For that, HEK293 cells were transfected using Fugene HD Transfection reagent with Cas9 plasmid and sgRNA plasmid. Agarose gel electrophoresis was used to observe the efficiency of Cas9-mediated cleavage. Further, quick change mutagenesis analysis was performed to insert an exogenous restriction enzyme site in the donor plasmid template for enabling the screening of positive CRISPR iPSC clones. Nucleofection of the patient iPSC line, UTA.12619.LMNA carrying S143P mutation in LMNA gene, was performed using the P1 primary cell 4D-Nucleofector X kit. Antibiotic was used to select the resistant cell clones. The transfected iPSC clones were mechanically picked into the wells of 96-well plate. Successful gene engineering was confirmed by DNA isolation, PCR and Sanger sequencing. Finally, iPSCs were differentiated into cardiomyocytes using small molecule differentiation method.
Results: Based on the T7 endonuclease assay results, the best sgRNA with the highest cleavage activity and the PAM sequence located close to the point mutation site was selected for nucleofection. The restriction site EcoR1 was successfully introduced into the L2 donor plasmid for screening the corrected iPSC clones. The efficiency of the iPSC nucleofection was confirmed by EGFP expression by Cas9 plasmid. The analysis of each resistant clone by sequencing using a primer close to the corrected locus validated the generation of isogenic cell line.
Conclusions: The correction of a point mutation to obtain isogenic iPSC line utilizing CRISPR/Cas9 system was successful. However, the correction efficiency remained low, so the efficient optimization of the CRISPR method and off-targets effects minimization would be the main goals of future research work before utilizing CRISPR system in therapeutic applications.
Methods: Isogenic gene-corrected cell line was obtained using the CRISPRs/Cas9 system that comprises a Cas9 and a guide RNA plasmid in combination with a donor plasmid serving as a homologous template covering the site of the mutation. T7 Endonuclease assay was used to detect the cleavage activity of three selected sgRNAs in HEK293 cells. For that, HEK293 cells were transfected using Fugene HD Transfection reagent with Cas9 plasmid and sgRNA plasmid. Agarose gel electrophoresis was used to observe the efficiency of Cas9-mediated cleavage. Further, quick change mutagenesis analysis was performed to insert an exogenous restriction enzyme site in the donor plasmid template for enabling the screening of positive CRISPR iPSC clones. Nucleofection of the patient iPSC line, UTA.12619.LMNA carrying S143P mutation in LMNA gene, was performed using the P1 primary cell 4D-Nucleofector X kit. Antibiotic was used to select the resistant cell clones. The transfected iPSC clones were mechanically picked into the wells of 96-well plate. Successful gene engineering was confirmed by DNA isolation, PCR and Sanger sequencing. Finally, iPSCs were differentiated into cardiomyocytes using small molecule differentiation method.
Results: Based on the T7 endonuclease assay results, the best sgRNA with the highest cleavage activity and the PAM sequence located close to the point mutation site was selected for nucleofection. The restriction site EcoR1 was successfully introduced into the L2 donor plasmid for screening the corrected iPSC clones. The efficiency of the iPSC nucleofection was confirmed by EGFP expression by Cas9 plasmid. The analysis of each resistant clone by sequencing using a primer close to the corrected locus validated the generation of isogenic cell line.
Conclusions: The correction of a point mutation to obtain isogenic iPSC line utilizing CRISPR/Cas9 system was successful. However, the correction efficiency remained low, so the efficient optimization of the CRISPR method and off-targets effects minimization would be the main goals of future research work before utilizing CRISPR system in therapeutic applications.