How to select expression system for CRISPR genome editing?

Thus far, at least five CRISPR systems have been reported for use in Leishmania which include two stable expression systems and three transient systems . In the two stable CRISPR expression systems, the Cas9 nuclease and gRNA are constitutively expressed in the parasites using expression vectors and gRNA transcribed from either the U6 promoter or the ribosomal RNA promoter . Because of the continuous expression of Cas9 and gRNA in the cell, this stable expression system using the ribosomal RNA promoter has been used to delete multicopy family Leishmania genes, determine gene essentiality and perform basic gene editing [10, 11]. In the transient expression system relying on the T7 RNA polymerase, the Cas9 and T7 RNA polymerase genes are integrated into the parasite genome and constitutively expressed . Gene editing is then achieved by transiently cotransfecting the Cas9 and T7 RNA polymerase expressing promastigotes with gRNA templates and donor DNA containing antibiotic selection markers or fluorescent protein tags.

Referencias:

• Zhang WW, Matlashewski G (2015) CRISPR-Cas9-mediated genome editing in Leishmania donovani. MBio 6(4):e00861
  
  
• Zhang WW, Lypaczewski P, Matlashewski G (2017) Optimized CRISPR-Cas9 genome editing for Leishmania and its use to target a multigene family, induce chromosomal translocation and study DNA break repair mechanisms. mSphere 2(1):e00340–e00316.  https://doi.org/10.1128/mSphere.00340-16
  
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  Zhang WW, Matlashewski G (2019) Single-Strand Annealing Plays a Major Role in Double-Strand DNA Break Repair following CRISPR-Cas9 Cleavage in Leishmania. mSphere 4:e00408–19.  https://doi.org/10.1128/mSphere.00408-19