Targeted insertion and reporter transgene activity at a gene safe harbor of the human blood fluke,Schistosoma mansoni

Abstract

We identified genomic safe harbor sites (GSH) in the human blood fluke, Schistosoma mansoni and developed a CRISPR-focused protocol for insertion of a reporter transgene into a representative GSH. The protocol employed ribonuclear protein complexes of Cas9 nuclease, three overlapping guide RNAs, and phosphorothioate-modified, double stranded donor DNAs encoding green fluorescent protein driven by a strong endogenous promoter. Gene-editing efficiencies of >20% and reporter transgene fluorescence of >50% of gene-edited eggs were obtained by five days after CRISPR transfection. These methods and results advance functional genomics for multicellular parasites, and represent a tractable path towards transgenic schistosomes using homology directed repair catalyzed transgene insertion. Identification and characterization of GSH is expected to facilitate consistent transgene activity with neutral influence on the host cell genome and, concurrently, provide a privileged locus for transgene activity. This approach should be adaptable to platyhelminths generally.

ENHANCED EFFICIENCY OF RNA-GUIDED CAS12a VERSUS CAS9 TRANSGENE KNOCK-IN AND ACTIVITY AT ASCHISTOSOMA MANSONIGENOME SAFE HARBOR

“…Cas12a is better suited than Cas9 for editing AT-rich regions. Furthermore, we showed previously that overlapping of three gRNAs combined with Cas9 cleaved the target DNA with resultant staggered (overhanging rather than blunt ended) strands (3, 21, 26). This outcome in turn led to marked increase of ∼70% of donor transgene integration and chromosomal repair by HDR in the schistosome egg.…”

“…CLSM was employed to evaluate reporter gene expression in eggs/miracidia following programmed KI at days 5 and 10 after transfection. Emission spectra were analyzed as described (21), which revealed EGFP fluorescence in cells of eggs that contained a miracidium. Fluorescence intensity of eggs emitted at 509 nm was quantified, which revealed significantly more EGFP-positive eggs in the Cas12a group than the Cas9 group at days 5 and 10 after electroporation (Figure 4A).…”

“…To study GOI, knock-out (KO) models are common for various model organisms, but not yet established for trematodes or other helminths. Towards this end, a recent breakthrough achieved CRISPR/Cas9 transgene KI into a schistosome genome safe harbor site (21).…”

“…An ideal GSH has been defined as a region that does not overlap functional genomic elements and that lacks heterochromatic marks that could impede transcription. We have predicted several GSHs in S. mansoni using criteria that included a location in euchromatin to avoid transgene silencing, a unique genome-target sequence to minimize off-target events, avoidance of long noncoding RNA-encoding genes, and the presence of histone marks for open chromatin structure and, conversely, the absence of epigenetic marks indicating heterochromatin (21). We reported the Cas9-mediated integration of an exogenous DNA sequence into GSH1 (genome safe harbor number 1) of S. mansoni .…”

“…We reported the Cas9-mediated integration of an exogenous DNA sequence into GSH1 (genome safe harbor number 1) of S. mansoni . Programmed cleavage improved when three overlapping guide RNAs complexed with the Cas9 nuclease from Streptococcus pyogenes (21) were used in unison. As biological targets, we used liver-stage S. mansoni eggs (LE), which represent a heterogenous population at different stages of development.…”