Abstract
ABSTRACTTrypanosomatids (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.
Highlights
Trypanosomatids, including the human pathogens Trypanosoma cruzi, Trypanosoma brucei, (African sleeping sickness), and Leishmania, affect millions of people and animals globally
We show for the first time that RNP complexes of Cas9/gRNA can be delivered into epimastigotes and trypomastigotes of T. cruzi, with or without singlestranded repair oligonucleotides, to knock out or tag target genes with very high efficiency
To test the efficiency of gene editing of RNP complexes composed of SaCas9/single guide RNAs (sgRNAs) in T. cruzi, epimastigotes from T. cruzi strain CL expressing either enhanced green fluorescent protein or the tdTomato red fluorescent protein were transfected with RNPs targeting these transgenes, and fluorescent protein expression was assessed 7 days later
Summary
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 Cas expression and the long drug marker selection times. We describe new methods that are available to essentially any laboratory and applicable to any parasite isolate for and rapidly editing the genomes of kinetoplastid parasites We demonstrate that these methods provide the means to quickly assess function, including that of the products of essential genes and potential targets of drugs, and to tag gene products at their endogenous loci. The rapid turnover of the RNP complexes within cells makes this approach transient, both overcoming the Cas toxicity issue [4, 9] and minimizing potential off-target edits in the genome [10]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
