A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing.
Int J Mol Sci
; 24(9)2023 May 05.
Article
en En
| MEDLINE
| ID: mdl-37175977
ABSTRACT
CRISPR/Cas systems are some of the most promising tools for therapeutic genome editing. The use of these systems is contingent on the optimal designs of guides and homology-directed repair (HDR) templates. While this design can be achieved in silico, validation and further optimization are usually performed with the help of reporter systems. Here, we describe a novel reporter system, termed BETLE, that allows for the fast, sensitive, and cell-specific detection of genome editing and template-specific HDR by encoding multiple reporter proteins in different open-reading frames. Out-of-frame non-homologous end joining (NHEJ) leads to the expression of either secretable NanoLuc luciferase, enabling a highly sensitive and low-cost analysis of editing, or fluorescent mTagBFP2, allowing for the enumeration and tissue-specific localization of genome-edited cells. BETLE includes a site to validate CRISPR/Cas systems for a sequence-of-interest, making it broadly adaptable. We evaluated BETLE using a defective moxGFP with a 39-base-pair deletion and showed spCas9, saCas9, and asCas12a editing as well as sequence-specific HDR and the repair of moxGFP in cell lines with single and multiple reporter integrants. Taken together, these data show that BETLE allows for the rapid detection and optimization of CRISPR/Cas genome editing and HDR in vitro and represents a state-of the art tool for future applications in vivo.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Roturas del ADN de Doble Cadena
/
Sistemas CRISPR-Cas
Idioma:
En
Revista:
Int J Mol Sci
Año:
2023
Tipo del documento:
Article
País de afiliación:
Estados Unidos