Rapid, Selection-Free, High-Efficiency Genome Editing in Protozoan Parasites Using CRISPR-Cas9 Ribonucleoproteins

Abstract

Trypanosomatids (order Kinetoplastida), including the human pathogens Trypanosoma cruzi (agent of Chagas disease), Trypanosoma brucei, (African sleeping sickness), and Leishmania (leishmaniasis), affect millions of people and animals globally. T. cruzi is considered one of the least studied and most poorly understood tropical disease-causing parasites, in part because of the relative lack of facile genetic engineering tools. This situation has improved recently through the application of clustered regularly interspaced short palindromic repeats–CRISPR-associated protein 9 (CRISPR-Cas9) technology, but a number of limitations remain, including the toxicity of continuous Cas9 expression and the long drug marker selection times. In this study, we show that the delivery of ribonucleoprotein (RNP) complexes composed of recombinant Cas9 from Staphylococcus aureus (SaCas9), but not from the more routinely used Streptococcus pyogenes Cas9 (SpCas9), and in vitro-transcribed single guide RNAs (sgRNAs) results in rapid gene edits in T. cruzi and other kinetoplastids at frequencies approaching 100%. The highly efficient genome editing via SaCas9/sgRNA RNPs was obtained for both reporter and endogenous genes and observed in multiple parasite life cycle stages in various strains of T. cruzi, as well as in T. brucei and Leishmania major. RNP complex delivery was also used to successfully tag proteins at endogenous loci and to assess the biological functions of essential genes. Thus, the use of SaCas9 RNP complexes for gene editing in kinetoplastids provides a simple, rapid, and cloning- and selection-free method to assess gene function in these important human pathogens.

State‐of‐the‐art CRISPR/Cas9 Technology for Genome Editing in Trypanosomatids

“…Using SaCas9 RPNs these authors achieved 37% gene knockdown in the presence of SSOs (HDR mechanism) as previously reported (Soares Medeiros et al. ). However, in the absence of SSOs they did not observe parasites with the expected phenotype (detached flagellum), confirming the low efficiency of MMEJ for DSB repair in T. cruzi .…”

“…; Soares Medeiros et al. ). One of the protocols involves the constitutive expression of SpCas9‐GFP with sgRNAs that were either constitutively expressed or in vitro‐transcribed.…”

“…A third approach for genome editing in T. cruzi involves the delivery of a ribonucleoprotein (RPN) complex composed by the Staphylococcus aureus Cas9 (SaCas9), and in vitro‐transcribed sgRNAs by nucleofection into different stages of the parasite's life cycle (epimastigotes and trypomastigotes) and different T. cruzi strains (Soares Medeiros et al. ). The system was tested with or without providing single stranded repair oligonucleotides to induce HDR, and targeting either exogenous reporter genes (tdTomato, mCherry, and eGFP) or endogenous genes, in order to achieve gene knockout and tagging.…”

“…Interestingly, this was useful for generating T. cruzi parasites exhibiting flagellar and cytokinesis defects in a substantial population of epimastigotes where RPN complexes were delivered to target proven essential genes for the flagellar structure, as observed by bright‐field microscopy (Soares Medeiros et al. ). This group also studied the effect of RPN‐mediated knockdown of genes encoding nitroreductase (NTR), an enzyme involved in the activation of anti‐ T. cruzi compounds benznidazole and nifurtimox, and the target of posaconazole, CYP51.…”

“…After evaluating several clones none of them were null mutants, although a few clones were single knockouts, a result that has been previously observed in attempts to generate knockouts in T. cruzi by conventional methods (Jimenez and Docampo 2015;Li et al 2011). Interestingly, this was useful for generating T. cruzi parasites exhibiting flagellar and cytokinesis defects in a substantial population of epimastigotes where RPN complexes were delivered to target proven essential genes for the flagellar structure, as observed by brightfield microscopy (Soares Medeiros et al 2017). This group also studied the effect of RPN-mediated knockdown of genes encoding nitroreductase (NTR), an enzyme involved in the activation of anti-T. cruzi compounds benznidazole and nifurtimox, and the target of posaconazole, CYP51.…”