Software-based screening for efficient sgRNAs in Lactococcus lactis.
CRISPR/Cas9
Lactococcus lactis
genomic engineering
sgRNA screening
Journal
Journal of the science of food and agriculture
ISSN: 1097-0010
Titre abrégé: J Sci Food Agric
Pays: England
ID NLM: 0376334
Informations de publication
Date de publication:
30 Jan 2024
30 Jan 2024
Historique:
revised:
23
08
2023
received:
26
06
2023
accepted:
30
08
2023
pubmed:
30
8
2023
medline:
30
8
2023
entrez:
30
8
2023
Statut:
ppublish
Résumé
The two essential editing elements in the clustered regularly interspaced short palindromic repeats (CRISPR) editing system are promoter and single-guide RNA (sgRNA), the latter of which determines whether Cas protein can precisely target a specific location to edit the targeted gene. Therefore, the selection of sgRNA is crucial to the efficiency of the CRISPR editing system. Various online prediction tools for sgRNA are currently available. These tools can predict all possible sgRNAs of the targeted gene and rank sgRNAs according to certain scoring criteria according to the demands of the user. We designed sgRNAs for Lactococcus lactis NZ9000 LLNZ_RS02020 (ldh) and LLNZ_RS10925 (upp) individually using online prediction software - CRISPOR - and successfully constructed a series of knockout strains to allow comparison of the knockout efficiency of each sgRNA and analyze the differences between software predictions and actual experimental results. Our experimental results showed that the actual editing efficiency of the screened sgRNAs did not match the predicted results - a phenomenon that suggests that established findings from eukaryotic studies are not universally applicable to prokaryotes. Software prediction can still be used as a tool for the initial screening of sgRNAs before further selection of suitable sgRNAs through experimental experience. © 2023 Society of Chemical Industry.
Sections du résumé
BACKGROUND
BACKGROUND
The two essential editing elements in the clustered regularly interspaced short palindromic repeats (CRISPR) editing system are promoter and single-guide RNA (sgRNA), the latter of which determines whether Cas protein can precisely target a specific location to edit the targeted gene. Therefore, the selection of sgRNA is crucial to the efficiency of the CRISPR editing system. Various online prediction tools for sgRNA are currently available. These tools can predict all possible sgRNAs of the targeted gene and rank sgRNAs according to certain scoring criteria according to the demands of the user.
RESULTS
RESULTS
We designed sgRNAs for Lactococcus lactis NZ9000 LLNZ_RS02020 (ldh) and LLNZ_RS10925 (upp) individually using online prediction software - CRISPOR - and successfully constructed a series of knockout strains to allow comparison of the knockout efficiency of each sgRNA and analyze the differences between software predictions and actual experimental results.
CONCLUSION
CONCLUSIONS
Our experimental results showed that the actual editing efficiency of the screened sgRNAs did not match the predicted results - a phenomenon that suggests that established findings from eukaryotic studies are not universally applicable to prokaryotes. Software prediction can still be used as a tool for the initial screening of sgRNAs before further selection of suitable sgRNAs through experimental experience. © 2023 Society of Chemical Industry.
Types de publication
Journal Article
Langues
eng
Sous-ensembles de citation
IM
Pagination
1200-1206Subventions
Organisme : National Science Foundation for Distinguished Young Scholars
ID : 32025029
Organisme : CIFST-Yili Foundation of Health Science
ID : 2021-Y06
Organisme : Shanghai Education committee scientific research innovation projects
ID : 2101070007800120
Organisme : National Natural Science Foundation of China
ID : 32101928
Informations de copyright
© 2023 Society of Chemical Industry.
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