CRISPR-mediated Transfection of Brugia malayi

Abstract

The application of reverse genetics in the human filarial parasites has lagged due to the difficult biology of these organisms. Recently, we developed a co-culture system that permitted the infective larval stage of Brugia malayi to be transfected and efficiently develop to fecund adults. This was exploited to develop a piggyBac transposon-based toolkit that can be used to produce parasites with transgene sequences stably integrated into the parasite genome. However, the piggyBac system has generally been supplanted by Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) based technology, which allows precise editing of a genome. Here we report adapting the piggyBac mediated transfection system of B. malayi for CRISPR mediated knock-in insertion into the parasite genome. Suitable CRISPR insertion sites were identified in intergenic regions of the B. malayi genome. A dual reporter piggybac vector was modified, replacing the piggyBac inverted terminal repeat regions with sequences flanking the insertion site. B. malayi molting L3 were transfected with a synthetic guide RNA, the modified plasmid and the CAS9 nuclease. The transfected parasites were implanted into gerbils and allowed to develop into adults. Progeny microfilariae were recovered and screened for expression of a secreted luciferase reporter encoded in the plasmid. Approximately 3% of the microfilariae were found to secrete luciferase; all contained the transgenic sequences inserted at the expected location in the parasite genome. Using an adaptor mediated PCR assay, transgenic microfilariae were examined for the presence of off target insertions; no off-target insertions were found. These data demonstrate that CRISPR can be used to modify the genome of B. malayi, opening the way to precisely edit the genome of this important human filarial parasite.

Fig 1. Map of pCHR1-BmGLuc-GFP: Reporter open reading frames and direction of transcription are indicated by the colored arrows.

Fig 2

In vitro digestion of the putative insertion site: An amplicon corresponding to Chr1scaffold 001:6484000–6485999 was produced and digested with the corresponding sgRNA and CAS9 nuclease, as described in Materials and Methods. Lane A = undigested amplicon. Lane A+R+C = amplicon incubated with the sgRNA and CAS9 nuclease. Lane A+C = amplicon incubated with CAS9 nuclease alone. Lane A+R = amplicon incubated with the sgRNA alone.

Fig 3

Gaussia luciferase activity secreted by individual F1 microfilaria: Microfilariae were collected from two gerbils that were independently infected with transfected molting L3 and cultured overnight, and one gerbil infected with untransfected L3. The media for the cultured individual microfilaria were assayed for Gaussia luciferase activity, as described in Materials and Methods. The number of microfilariae producing luciferase activity corresponding to each interval on the X axis are represented by the bars. The Y axis is presented using a logarithmic scale for clarity. #mf = number of mf secreting the indicated range of Gaussia luciferase activity into the culture medium. RLU = Relative light units.

Fig 4

Hemi-nested PCR analysis of insertion sites in individual microfilaria secreting Gaussia luciferase: Results shown are 12 representatives of those obtained from all 46 luciferase positive microfilariae recovered. Lanes labeled mf+ = DNA prepared from individual microfilaria positive for luciferase activity. Lane mf- = DNA prepared from an individual microfilaria recovered from the animals that did not secrete luciferase. Lane DNA = genomic DNA prepared from untransfected adult female B. malayi DNA.

Fig 5

Adaptor mediated hemi-nested PCR assay for off target insertion sites: Results shown are representatives from 14 individual transgenic microfilariae and 13 non-transgenic microfilariae, all of which gave results identical to those shown here. Lanes labeled mf- = non-transgenic microfilaria and those labeled mf+ = transgenic microfilaria.