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CRISPR/Cas9‐mediated genome editing of Schistosoma mansoni acetylcholinesterase
Author(s) -
You Hong,
Mayer Johannes U.,
Johnston Rebecca L.,
Sivakumaran Haran,
Ranasinghe Shiwanthi,
Rivera Vanessa,
Kondrashova Olga,
Koufariotis Lambros T.,
Du Xiaofeng,
Driguez Patrick,
French Juliet D.,
Waddell Nicola,
Duke Mary G.,
Ittiprasert Wannaporn,
Mann Victoria H.,
Brindley Paul J.,
Jones Malcolm K.,
McManus Donald P.
Publication year - 2021
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fj.202001745rr
Subject(s) - crispr , schistosoma mansoni , genome editing , biology , cas9 , genome , computational biology , acetylcholinesterase , genetics , gene , helminths , zoology , schistosomiasis , enzyme , biochemistry
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.