Abstract
CRISPR-Cas9 is an RNA guided endonuclease derived from the bacterium Streptococcus pyogenes. Due to its simplicity, versatility, and high efficiency, it has been widely used for genome editing in a variety of organisms including the protozoan parasite Leishmania, the causative agent of human leishmaniasis. Compared to the traditional homologous recombination gene targeting method, CRISPR-Cas9 has been shown to be a more efficient method to delete or disrupt Leishmania genes, generate point mutations, and add tags to endogenous genes. Notably, the stable CRISPR expression systems were shown to delete multicopy family Leishmania genes and genes present in multiploid chromosomes, identify essential Leishmania genes, and create specific chromosome translocations. In this chapter, we describe detailed procedures on using the stable CRISPR expression system for genome editing in Leishmania. These procedures include CRISPR targeting site selection, gRNA design, cloning single and double gRNA coding sequences into the Leishmania CRISPR vector pLdCN, oligonucleotide donor and drug resistance selection donor design, Leishmania cell transfection, screening, and isolation of CRISPR-edited mutants. As the principles of gene editing are generally similar, many of these procedures could also apply to the transient Leishmania CRISPR systems described by other labs.
Highlights
The commonly used Streptococcus pyogenes CRISPR-Cas9 is an RNA-guided endonuclease that has been shown to facilitate sitespecific DNA cleavage in various organisms [1–6]
Leishmania protozoans are the causative agents of human leishmaniasis, the second most deadly parasitic disease after malaria
Due to the lack of an RNA interference pathway in most of the Leishmania species [17], gene targeting in Leishmania traditionally relied on gene replacement by homologous recombination which requires a minimum of 180 bp flanking sequences and two antibiotic selection markers as Leishmania is diploid [18–21]
Summary
The commonly used Streptococcus pyogenes CRISPR (clustered regularly interspaced short palindromic repeats)-Cas is an RNA-guided endonuclease that has been shown to facilitate sitespecific DNA cleavage in various organisms [1–6]. Gene editing is achieved by transiently cotransfecting the Cas and T7 RNA polymerase expressing promastigotes with gRNA templates and donor DNA containing antibiotic selection markers or fluorescent protein tags [28, 34]. In the case of SaCas, the longer PAM (NNGRRT) sequence requirement could limit the sites available for SaCas targeting [12, 29, 35] How these transient systems will perform on multicopy and essential Leishmania genes remains to be determined. We describe the detailed procedure for carrying out gene editing using the stable plasmid based CRISPR Cas expression system [10–12] This is a versatile system and can be used to delete or disrupt single and multicopy genes, carry out selection with or without antibiotic resistance genes, edit gene codons and establish gene essentiality [10–12]. As the principles of gene editing are similar, many of the procedures described within this chapter apply to the transient systems
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