• Deletion of PUF4 RNA-binding protein in Leishmania major through CRISPR/Cas9 technique
  • Elaheh Davarpanah,1 Tahereh Taheri,2,*
    1. Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran
    2. Department of Immunotherapy and Leishmania Vaccine Research, Pasteur Institute of Iran


  • Introduction: Leishmaniasis is a neglected tropical disease that is caused by Leishmania (L.) parasites that transmits by the bite of infected sandflies. As a next generation of live vaccines, genetically attenuated parasites are more attention. PUFs are conserved RNA-binding proteins in eukaryotes that are responsible for regulation of RNA processing. Leishmania has 11 members of PUFs that lead to regulate gene expression post-transcriptionally. The main goal of this study is disruption of puf4 gene in L. major and generation an attenuated vaccine through CRISPR/Cas9 system.
  • Methods: In the first step, sgRNA and donor DNA primers have designed using different software (LeishGEdit, EuPaGDT, CCTop, CRISPOR and CHOPCHOP) to increase the accuracy of designed primers.
  • Results: Between 100 bp region from the beginning of the puf4 gene, four suitable sgRNA sequences (including: 5'- AAGACACAGGTGCAGATGCA-3', 5'-CAGCTCTTCGCTCTCGGATT-3', 5'-GCTCTCGGATTGGGCAAACA-3' and 5’-AGGTGCAGATGCACGGATGA-3’) can be considered to have the highest efficiency in cutting the 5' of the puf4 gene. In addition, four appropriate sgRNAs for 3'-end cutting of gene have recognized (including: 5’-GCACGCATGCAAAATGCTAA-3’, 5’-AGACGGAGCTCGCCTTGCTG-3’, 5’-TAGTTGCGCGAGCTCAGCAG-3’ and 5’-GACGGAGCTCGCCTTGCTGA-3’). GC content of primers is ranging from 33 to 64.8. In addition, to increase MMEJ (Microhomology-Mediated End Joining) rate and substitution of puf4 gene with antibiotic markers, two specific short nucleotide sequences (~30 nt) in upstream and downstream of the gene (5’-ACGTTGTGCAGCTCCCTTCACCGTCATCCG-3’ and 5’-GAGTGCAGCGGCCGGGAACGGTGGCCACAC-3’, with GC content 60% and 73%, respectively) have selected.
  • Conclusion: In the next step, sgRNA and donor DNA sequences will be used to create DSB within flanking puf4 target region and replace of two alleles with two drug resistance genes.
  • Keywords: Live vaccine, Leishmania major, PUF4, CRISPR/Cas9.