CRISPR/Cas9-based functional genomics in Trypanosoma cruzi: a systematic review of tools, applications, and biological findings

Abstract

Trypanosoma cruzi, the causative agent of Chagas disease, was for decades one of the most genetically intractable organisms among trypanosomatids, owing to a highly repetitive, multigene-family-rich genome, frequent aneuploidy, absence of a functional non-homologous end-joining repair pathway, and the low efficiency of classical genetic manipulation tools. The introduction of CRISPR/Cas9 in 2014-2015 marked a turning point for its functional genomics, yet no systematic review had specifically synthesized this literature. We aimed to systematically identify and synthesize the published evidence on the use of CRISPR/Cas technologies for functional genomics in T. cruzi from the first report (2014) through August 2026. We followed PRISMA 2020 guidance, searching PubMed/MEDLINE, Scopus, Web of Science, and bioRxiv, supplemented by backward and forward citation searching (snowballing) from six contextual reviews and two foundational studies. Study selection and data extraction were performed by two reviewers; eligibility was restricted to English-language articles. Reporting quality of primary studies was assessed with an ad hoc seven-domain checklist informed by ARRIVE and MIQE principles. Of 116 records identified, 72 primary studies and 6 contextual reviews were included (one further record, initially screened in, was reclassified as excluded after full-text review showed it reported no T. cruzi experiments). Primary studies predominantly used standard plasmid-based CRISPR/Cas9 (81%), followed by ribonucleoprotein delivery (8%). A full-text sub-analysis of delivery strategies, possible for 67 of the 72 primary studies with an applicable, accessible Methods section, identified four principal approaches and a mechanistic shift toward cloning-free, in vivo T7RNAP-transcribed sgRNA delivery dominant since 2023. The dominant thematic focus was parasite differentiation (metacyclogenesis) and host-cell invasion/infectivity. Only 8 of 72 studies (11%) reported a quantified editing efficiency in the abstract, and 38% reported none of the seven quality-reporting elements assessed. Synthesis of the six contextual reviews revealed a consistent narrative arc, from an urgent call for efficient tools (2015) to a mature taxonomy of editing strategies (2023). CRISPR/Cas9 transformed functional genomics in T. cruzi, enabling for the first time efficient disruption of multigene families and conditional strategies for essential genes. Important gaps persist: heterogeneous and often incomplete reporting of editing efficiency, absence of confirmed precision-editing applications (base or prime editing), and a marked thematic concentration on differentiation and invasion relative to other biological processes.