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.

Authors	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
Journal	Faseb Journal : Official Publication Of The Federation Of American Societies For Experimental Biology
Pages	e21205
Volume	35
Date	1/01/2021
Grant ID
Funding Body
URL	http://www.ncbi.nlm.nih.gov/pubmed/?term=10.1096/fj.202001745RR