Assessment of two CRISPR-Cas9 genome editing protocols for rapid generation of Trypanosoma cruzi gene knockout mutants

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

“…; Zhang and Matlashewski ) or flagellar detachment (Burle‐Caldas et al. ). However, for genes encoding proteins of unknown function, the inclusion of a resistance marker or reporter gene in the DNA donor template is the best option to efficiently achieve genome editing in trypanosomatids (Beneke et al.…”

“…(i) The method involves the use of a parental cell line expressing Cas9 and T7 RNA polymerase (T7 RNA Pol) for nucleofection of three PCR products: two donor DNAs with different resistance markers (RM 1 and RM 2) and a DNA fragment holding the specific sgRNA sequence downstream the T7 promoter for in vivo transcription; (ii) In the nucleus of the cell transiently expressed sgRNA assembles with Cas9; (iii) Cas9 targets both alleles of the gene, which are simultaneously replaced by two different resistance markers. more efficient than MMEJ in trypanosomatids Burle-Caldas et al 2018;Chiurillo et al 2016;Lander et al 2015;Sollelis et al 2015;Vasquez et al 2018;Zhang and Matlashewski 2015). Other factors could affect the efficiency of the system, such as the DNA delivery system (nucleofection is a more efficient electroporation method than transfection); the possibility of performing cell sorting during the selection period, to enrich the population of genetically modified parasites or even get mutant clones; and the presence of a resistance marker in the DNA donor template to ensure the selection of parasites with properly edited genomes.…”

“…Other factors could affect the efficiency of the system, such as the DNA delivery system (nucleofection is a more efficient electroporation method than transfection); the possibility of performing cell sorting during the selection period, to enrich the population of genetically modified parasites or even get mutant clones; and the presence of a resistance marker in the DNA donor template to ensure the selection of parasites with properly edited genomes. Selection-free methods are useful when there is an expected phenotype associated to the deletion of a gene, as for example drug resistance (Rico et al 2018;Soares Medeiros et al 2017;Zhang and Matlashewski 2015) or flagellar detachment (Burle-Caldas et al 2018). However, for genes encoding proteins of unknown function, the inclusion of a resistance marker or reporter gene in the DNA donor template is the best option to efficiently achieve genome editing in trypanosomatids Bertolini et al 2019;Chiurillo et al 2017Chiurillo et al , 2019Costa et al 2018;Cruz-Bustos et al 2018a,b;Lander et al 2015Lander et al , 2016bLander et al , 2018Liu et al 2019;Martel et al 2017).…”

“…In a comparative approach, Burle‐Caldas et al. () assessed two genome editing protocols modified from previous studies (Lander et al. ; Soares Medeiros et al.…”

“…The main advantage of this method is the possibility of transfecting different sgRNAs simultaneously, which could be useful for targeting several genes. However, in the absence of a DNA donor template to induce HDR, the double-strand break produced by Cas9 would be repair by MMEJ, which is a less efficient DNA repair mechanism in trypanosomatids Burle-Caldas et al 2018;Chiurillo et al 2016;Lander et al 2015;Sollelis et al 2015;Vasquez et al 2018;Zhang and Matlashewski 2015). In addition, the transient expression of in vitro-transcribed sgRNAs could reduce the efficiency of the method.…”