*4.3. Analytical Platforms for Gene-Editing Detection*

Different analytical platforms for the detection and identification of CRISPR outcomes are reflected in frequency results as they show different analytical sensitivity. Woo et al. [12] showed a 40% transformation efficiency in tobacco when samples were analyzed by the Illumina sequencing platform. In contrast, the same samples showed a much lower efficiency rate (17%) when analyzed by the T7 cleavage assay. While T7 cleavage assays are nowadays being limited to a qualitative rather than quantitative detection methods, high-throughput sequencing platforms are time-consuming and costly options for screening protocols. Usually, such platforms are available in other labs or through service providers, which require long processing time and high costs for samples and assays that are still at the screening stage. In order to overcome such problems, we have proposed a model that analyzes gene-edited cell pools using data from common Sanger sequencing analysis. More specifically, we use the ICE: *Inference of CRISPR Edits* software, which enable the analysis of mixed populations and strongly correlates with next-generation sequencing of amplicons using Sanger sequencing data [23].

However, the proposed model is focused on providing a simple, cost-efficient analysis of gene-editing outcomes at the screening stage. The model is currently limited to detect mutations with more than 30 bp deletions or more than 14 bp additions. The analysis also does not account for very small mutant populations (<0.1%) neither presents mutations with a substitution of bp [23]. Although our platform does not apply exogenous DNA generating potential integration, it cannot be ruled out that microhomologies with gRNA sequences produce spurious cleavage or larges genomic rearrangements [50]. Therefore, long-run high throughput sequencing analysis is recommended for follow up breeding programs and safety tests [51].
