CRISPR/Cas9-mediated genome editing of Schistosoma mansoni acetylcholinesterase.
FASEB J
; 35(1): e21205, 2021 01.
Article
in En
| MEDLINE
| ID: mdl-33337558
ABSTRACT
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.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Acetylcholinesterase
/
Schistosoma mansoni
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Schistosomiasis mansoni
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Helminth Proteins
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CRISPR-Cas Systems
/
Gene Editing
Limits:
Animals
Language:
En
Journal:
FASEB J
Journal subject:
BIOLOGIA
/
FISIOLOGIA
Year:
2021
Type:
Article
Affiliation country:
Australia