CRISPR/Cas9-mediated genome editing of Schistosoma mansoni acetylcholinesterase.
Schistosoma mansoni
CRISPR/Cas9
acetylcholinesterase
eggs
genome editing
Journal
FASEB journal : official publication of the Federation of American Societies for Experimental Biology
ISSN: 1530-6860
Titre abrégé: FASEB J
Pays: United States
ID NLM: 8804484
Informations de publication
Date de publication:
01 2021
01 2021
Historique:
received:
19
07
2020
revised:
16
10
2020
accepted:
03
11
2020
entrez:
18
12
2020
pubmed:
19
12
2020
medline:
16
6
2021
Statut:
ppublish
Résumé
CRISPR/Cas9-mediated genome editing shows cogent potential for the genetic modification of helminth parasites. We report successful gene knock-in (KI) into the genome of the egg of Schistosoma mansoni by combining CRISPR/Cas9 with single-stranded oligodeoxynucleotides (ssODNs). We edited the acetylcholinesterase (AChE) gene of S. mansoni targeting two guide RNAs (gRNAs), X5 and X7, located on exon 5 and exon 7 of Smp_154600, respectively. Eggs recovered from livers of experimentally infected mice were transfected by electroporation with a CRISPR/Cas9-vector encoding gRNA X5 or X7 combining with/ without a ssODN donor. Next generation sequencing analysis of reads of amplicon libraries spanning targeted regions revealed that the major modifications induced by CRISPR/Cas9 in the eggs were generated by homology directed repair (HDR). Furthermore, soluble egg antigen from AChE-edited eggs exhibited markedly reduced AChE activity, indicative that programed Cas9 cleavage mutated the AChE gene. Following injection of AChE-edited schistosome eggs into the tail veins of mice, an significantly enhanced Th2 response involving IL-4, -5, -10, and-13 was detected in lung cells and splenocytes in mice injected with X5-KI eggs in comparison to control mice injected with unmutated eggs. A Th2-predominant response, with increased levels of IL-4, -13, and GATA3, also was induced by X5 KI eggs in small intestine-draining mesenteric lymph node cells when the gene-edited eggs were introduced into the subserosa of the ileum of the mice. These findings confirmed the potential and the utility of CRISPR/Cas9-mediated genome editing for functional genomics in schistosomes.
Identifiants
pubmed: 33337558
doi: 10.1096/fj.202001745RR
doi:
Substances chimiques
Helminth Proteins
0
Acetylcholinesterase
EC 3.1.1.7
Types de publication
Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e21205Subventions
Organisme : Wellcome Trust
ID : 107475/Z/15/Z
Pays : United Kingdom
Informations de copyright
© 2020 Federation of American Societies for Experimental Biology.
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