Desenho in silico de sgRNAs e donor DNAs para deleção de enzimas relacionadas ao fenótipo de resistência do Trypanosoma cruzi ao benzonidazol
Resumo
A doença de Chagas, causada pelo protozoário Trypanosoma cruzi, afeta milhões de pessoas, principalmente na América Latina. Seu tratamento é baseado no uso clínico do benzonidazol (BZ) ou nifurtimox e possui limitações, como baixa eficácia de cura principalmente na fase crônica da doença e ocorrência de parasitos resistentes a ambos compostos. Várias enzimas têm sido relacionadas com o fenótipo de resistência dos parasitos a esses fármacos, tais como a nitroredutase 1, a aldo‐keto redutase e a álcool desidrogenase. A fim de melhor compreender o papel dessas enzimas no fenótipo de resistência do T. cruzi ao BZ, propomos utilizar o CRISPR/Cas9 para realizar a deleção dessas enzimas. Foram realizadas análises in silico para o desenho dos sgRNAs e donor DNAs por meio da avaliação do número de cópias de cada gene na cepa CL Brener usando o tBLASTn, a construção dos sgRNAs e dos donor DNAs pela ferramenta EuPaGDT e o alinhamento global das cópias pelo Needle (EMBOSS). Foram encontradas duas sequências do gene NTR-1, duas para a ADH e pelomenos nove para a AKR, sendo escolhidos três sgRNAs para reconhecimento específico de cada uma das cópias. Devido às características dos sgRNAs selecionados e eficiência do protocolo de CRISPR/Cas9, acreditamos que será possível realizar a deleção dos genes NTR-1 e ADH na cepa CL Brener de T. cruzi utilizando essa metodologia caso não sejam genes essenciais para o parasito. Já no caso da AKR mais análises serão necessárias para conhecer o número exato de cópias do gene.
Palavras-chave: Trypanosoma cruzi. Resistência ao benzonidazol. CRISPR/Cas9. Deleção gênica.
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