Correction of a CD55 mutation to quantify the efficiency of targeted knock-in via flow cytometry.
CD55
CRISPR/Cas9
Genome editing
Genome engineering
Targeted knock-in
Journal
Molecular biology reports
ISSN: 1573-4978
Titre abrégé: Mol Biol Rep
Pays: Netherlands
ID NLM: 0403234
Informations de publication
Date de publication:
Jul 2022
Jul 2022
Historique:
received:
06
09
2021
accepted:
24
03
2022
pubmed:
15
4
2022
medline:
15
4
2022
entrez:
14
4
2022
Statut:
ppublish
Résumé
Targeted knock-in assisted by the CRISPR/Cas9 system is an advanced technology with promising applications in various research fields including medical and agricultural sciences. However, improvements in the efficiency, precision, and specificity of targeted knock-in are prerequisites to facilitate the practical application of this technology. To improve the efficiency of targeted knock-in, it is necessary to have a molecular system that allows sensitive monitoring of targeted knock-in events with simple procedures. We developed an assay, named CD55 correction assay, with which to monitor CD55 gene correction accomplished by targeted knock-in. To create the reporter clones used in this assay, we initially introduced a 7.7-kb heterozygous deletion covering CD55 exons 2-5, and then incorporated a truncating mutation within exon 4 of the remaining CD55 allele in human cell lines. The resultant reporter clones that lost the CD55 protein on the cell membrane were next transfected with Cas9 constructs along with a donor plasmid carrying wild-type CD55 exon 4. The cells were subsequently stained with fluorescence-labeled CD55 antibody and analyzed by flow cytometry to detect CD55-positive cells. These procedures allow high-throughput, quantitative detection of targeted gene correction events occurring in an endogenous human gene. The current study demonstrated the utility of the CD55 correction assay to sensitively quantify the efficiency of targeted knock-in. When used with the PIGA correction assay, the CD55 correction assay will help accurately determine the efficiency of targeted knock-in, precluding possible experimental biases caused by cell line-specific and locus-specific factors.
Sections du résumé
BACKGROUND
BACKGROUND
Targeted knock-in assisted by the CRISPR/Cas9 system is an advanced technology with promising applications in various research fields including medical and agricultural sciences. However, improvements in the efficiency, precision, and specificity of targeted knock-in are prerequisites to facilitate the practical application of this technology. To improve the efficiency of targeted knock-in, it is necessary to have a molecular system that allows sensitive monitoring of targeted knock-in events with simple procedures.
METHODS AND RESULTS
RESULTS
We developed an assay, named CD55 correction assay, with which to monitor CD55 gene correction accomplished by targeted knock-in. To create the reporter clones used in this assay, we initially introduced a 7.7-kb heterozygous deletion covering CD55 exons 2-5, and then incorporated a truncating mutation within exon 4 of the remaining CD55 allele in human cell lines. The resultant reporter clones that lost the CD55 protein on the cell membrane were next transfected with Cas9 constructs along with a donor plasmid carrying wild-type CD55 exon 4. The cells were subsequently stained with fluorescence-labeled CD55 antibody and analyzed by flow cytometry to detect CD55-positive cells. These procedures allow high-throughput, quantitative detection of targeted gene correction events occurring in an endogenous human gene.
CONCLUSIONS
CONCLUSIONS
The current study demonstrated the utility of the CD55 correction assay to sensitively quantify the efficiency of targeted knock-in. When used with the PIGA correction assay, the CD55 correction assay will help accurately determine the efficiency of targeted knock-in, precluding possible experimental biases caused by cell line-specific and locus-specific factors.
Identifiants
pubmed: 35420385
doi: 10.1007/s11033-022-07422-0
pii: 10.1007/s11033-022-07422-0
doi:
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
6241-6248Subventions
Organisme : Japan Society for the Promotion of Science
ID : 18K14703
Organisme : Japan Society for the Promotion of Science
ID : 18K08342
Organisme : Japan Society for the Promotion of Science
ID : 21K08426
Organisme : Japan Society for the Promotion of Science
ID : 18H02645
Organisme : Japan Society for the Promotion of Science
ID : 19K08668
Organisme : Japan Society for the Promotion of Science
ID : 17K07263
Organisme : Japan Society for the Promotion of Science
ID : 20K06613
Organisme : Japan Society for the Promotion of Science London
ID : 19K09292
Informations de copyright
© 2022. The Author(s), under exclusive licence to Springer Nature B.V.
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