Abstract:Until 2015, loss-of-function studies to elucidate protein function in Leishmania relied on gene disruption through homologous recombination. Then, the CRISPR/Cas9 revolution reached these protozoan parasites allowing efficient genome editing with one round of transfection. In addition, the development of LeishGEdit, a PCR-based toolkit for generating knockouts and tagged lines using CRISPR/Cas9, allowed a more straightforward and effective genome editing. In this system, the plasmid pTB007 is delivered to Leis… Show more
“…braziliensisthat express the exogenous SpCas9 protein from the episomal pTB007-Viannia plasmid. 17 Therefore, we consider that this immunological tool will facilitate studies based on CRISPR/Cas in this research parasitic model and various other biological samples.…”
The CRISPR/Cas adaptative immune system has been harnessed as an RNA-guided, programmable genome editing tool, allowing for diverse biotechnological applications. The implementation of the system relies on the ability to detect the Cas9 protein in biological samples. This task is facilitated by employing antibodies, which exhibit several advantageous features and applications in the context of tropical neglected diseases. This study reports a one-month immunization scheme with the Cas9 protein fromStreptococcus pyogenes to produce IgY polyclonal antibodies (anti-SpCas9), which can be rapidly isolated by combining yolk de-lipidation with protein salting out using pectin and ammonium sulfate, respectively. Immunodetection assays indicate that the antibodies are highly sensitive, specific, and useful for detecting the SpCas9 protein in promastigotes ofLeishmania braziliensisexpressing exogenous SpCas9. Thus, the simple method for producing anti-SpCas9 IgY antibodies will accelerate CRISPR/Cas-based studies in Leishmania spp. This approach serves as a valuable research tool in this parasite model and holds the potential for wide application in various other biological samples, promoting the implementation of the system. In fact, a bioinformatics approach based on the identification of antigenic determinants in the SpCas9 protein suggests the possibility of using the anti-SpCas9 IgY antibodies in applications such as Prime and Base editing.
“…braziliensisthat express the exogenous SpCas9 protein from the episomal pTB007-Viannia plasmid. 17 Therefore, we consider that this immunological tool will facilitate studies based on CRISPR/Cas in this research parasitic model and various other biological samples.…”
The CRISPR/Cas adaptative immune system has been harnessed as an RNA-guided, programmable genome editing tool, allowing for diverse biotechnological applications. The implementation of the system relies on the ability to detect the Cas9 protein in biological samples. This task is facilitated by employing antibodies, which exhibit several advantageous features and applications in the context of tropical neglected diseases. This study reports a one-month immunization scheme with the Cas9 protein fromStreptococcus pyogenes to produce IgY polyclonal antibodies (anti-SpCas9), which can be rapidly isolated by combining yolk de-lipidation with protein salting out using pectin and ammonium sulfate, respectively. Immunodetection assays indicate that the antibodies are highly sensitive, specific, and useful for detecting the SpCas9 protein in promastigotes ofLeishmania braziliensisexpressing exogenous SpCas9. Thus, the simple method for producing anti-SpCas9 IgY antibodies will accelerate CRISPR/Cas-based studies in Leishmania spp. This approach serves as a valuable research tool in this parasite model and holds the potential for wide application in various other biological samples, promoting the implementation of the system. In fact, a bioinformatics approach based on the identification of antigenic determinants in the SpCas9 protein suggests the possibility of using the anti-SpCas9 IgY antibodies in applications such as Prime and Base editing.
“…Until 2015, double homologous recombination of laboriously generated vectors was the only way to screen for function in Leishmania parasites. However, supplementation of the genome with endogenous ( L. major, L. mexicana ( Beneke et al., 2017 ) and L. braziliensis ( Espada et al., 2021 )) or episomal ( L. mexicana , L. major ( Beneke et al., 2017 ), L. tarentolae ( Turra et al., 2021 ), L. donovani ( Martel et al., 2017 ) and L. braziliensis ( Adaui et al., 2020 ) polymerase expression enabled the use of Cas9 mediated genome editing. Targeting a library of kinases in L. mexicana promastigotes, and using BarSeq to track mutants, showed that 79% of the kinome is dispensable for promastigote growth in culture, while 21% were refractory to gene knockout ( Baker et al., 2021 ).…”
In the age of big data an important question is how to ensure we make the most out of the resources we generate. In this review, we discuss the major methods used in Apicomplexan and Kinetoplastid research to produce big datasets and advance our understanding of Plasmodium, Toxoplasma, Cryptosporidium, Trypanosoma and Leishmania biology. We debate the benefits and limitations of the current technologies, and propose future advancements that may be key to improving our use of these techniques. Finally, we consider the difficulties the field faces when trying to make the most of the abundance of data that has already been, and will continue to be, generated.
“…On the other hand, several limitations include the potential loss of fluorescence during the fixation process and interference with tagged-protein expression, folding, functionality, and/or subcellular targeting ( Chalfie et al., 1994 ; Palmer & Freeman, 2004 ; Swenson et al., 2007 ) (Table 1). In regards to the functional interference, in locus reinsertion of a fluorescent protein-tagged PF16 in the L. braziliensis PF16 knockout line showed only partial recovery of wild-type flagellar motility phenotype ( Espada et al., 2021 ). Likewise, regarding the subcellular targeting interference, the amino acid permease 3 (aap3) was located on the surface membrane of L. donovani promastigotes by tagging aap3 with fluorescent proteins at first ( Shaked-Mishan et al., 2006 ).…”
Section: Enzymatic Systemsmentioning
confidence: 99%
“…Such advancements enabled the investigation of targeted gene expression and function, as well as the subcellular localization of the encoded protein of interest using various tags. However, the results obtained from manipulating heterologous reporter gene must be carefully analyzed since the introduction of exogenous sequences may interfere with the endogenous sequences, which then could result in misleading observations and conclusions ( Zhang et al., 2003 ; Folgueira & Requena, 2007 ; Kovtun et al., 2010 ; Goldman-Pinkovich et al., 2016 ; Espada et al., 2021 ). Fig.…”
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