In vitro cleavage of COL1A1 gene by CRISPR/CAS9: Fundaments for the generation of experimental models.
Name: DANIEL MOREIRA MELO
Publication date: 18/12/2025
Examining board:
Name |
Role |
|---|---|
| AGNES LUMI NISHIMURA | Examinador Externo |
| ELDAMARIA DE VARGAS WOLFGRAMM DOS SANTOS | Examinador Interno |
| FLAVIA DE PAULA | Presidente |
Summary: Gene editing mediated by the CRISPR/Cas9 system has become a central element of modern biotechnology, enabling genome modification in a rapid, accessible and highly precise manner. Among its applications, the generation of cellular models of genetic diseases is a key area, in which a critical step is the prior evaluation of the cleavage efficiency of Cas9/gRNA complexes before their use in living cells, functioning as an initial filter to select guide RNAs with higher functional potential. Among clinically relevant genes used as experimental models, COL1A1 stands out due to its essential role in type I collagen formation and because it harbors most of the mutations associated with Osteogenesis Imperfecta, a hereditary disease characterized by bone fragility and a broad clinical spectrum. The in vitro cleavage efficiency of the CRISPR/Cas9 system targeting the COL1A1 gene was investigated using DNA from five human cell lines of different origins. Guide RNAs were designed and selected
based on computational predictions of efficiency and specificity, and the fragment corresponding to exon 2 and its respective guide RNA was used in the cleavage assays. The analyses revealed intermediate and consistent efficiencies among the
tested cell lines, ranging from approximately 31% to 47%. The results demonstrate that the evaluated guide exhibits reproducible activity in different genomic contexts and support the use of this workflow as a preliminary step in the generation of
genetically edited cellular models. Overall, the data reinforce the importance of in vitro validation prior to cellular editing and provide an experimental basis for future studies aimed at modeling COL1A1 mutations and investigating the molecular mechanisms of Osteogenesis Imperfecta.
