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

Enhanced Efficiency of Rna-guided Cas12a Versus Cas9 Transgene Knock-in and Activity at a Schistosoma Mansoni Genome Safe Harbor

“…Optimized genome editing for schistosomes can be expected to hasten research progress in functional genomics of these helminths. Our recent report defining a panel of genome safe harbors in S. mansoni provided a salient innovation for CRISPR-based functional genomics and transgenesis in S. mansoni 21 . Now, we have also overcome other earlier limitations through deployment of Cas12a as the RNA-guided nuclease and the modification of the donor transgene with chemically protected, diminutive length (micro)homology arms.…”

“…Genomic DNAs were recovered from LE as described 21,26 . In brief, the eggs were homogenized in DNAzol (Molecular Research Center, Inc., Cincinnati, OH) using a motorized pestle (Kimble Chase, Thermo Fisher or PowerMasher II Nippi, Funakoshi, Tokyo, Japan), and concentration and purity assessed using a spectrophotometer.…”

“…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.…”

Multiplexed CRISPR-Cas9 protocol for large transgene integration into the Schistosoma mansoni genome

“… Ensure that the eggs are free of contaminating host tissues, cells and other debris, and maintain the eggs in DMEM medium containing 20% heat-inactivated fetal bovine serum at 37°C, 5% CO 2 . 1 , 3 For transfection of schistosome eggs with CRISPR materials delivered using square wave electroporation, suspend the eggs in Opti-MEM as the electroporation medium at a concentration of 50,000 eggs/mL. Note: The mice from SRC are housed at the Animal Research Facility of George Washington University, which is accredited by the American Association for Accreditation of Laboratory Animal Care (AAALAC no.…”

“…Ensure that the eggs are free of contaminating host tissues, cells and other debris, and maintain the eggs in DMEM medium containing 20% heat-inactivated fetal bovine serum at 37°C, 5% CO 2 . 1 , 3 …”