Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency

[EN]CRISPR/Cas9 allows the generation of knockout cell lines and null zygotes by inducing site-specific double-stranded breaks. In most cases the DSB is repaired by non-homologous end joining, resulting in small nucleotide insertions or deletions that can be used to construct knockout alleles. Howev...

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Detalles Bibliográficos
Autores: García-Tuñón, Ignacio, Alonso-Pérez, Verónica, Vuelta Ramos, Elena, Pérez- Ramos, Sandra, Herrero García, María, Méndez, Lucía, Hernández Sánchez, Jesús María, Martín Izquierdo, Marta, Saldaña, Raquel, Sevilla, Julián, Sánchez-Guijo Martín, Fermín, Hernández Rivas, Jesús María, Sánchez Martín, Manuel Adolfo
Tipo de recurso: artículo
Estado:Versión publicada
Fecha de publicación:2019
País:España
Institución:Universidad de Salamanca (USAL)
Repositorio:GREDOS. Repositorio Institucional de la Universidad de Salamanca
OAI Identifier:oai:gredos.usal.es:10366/146291
Acceso en línea:http://hdl.handle.net/10366/146291
Access Level:acceso abierto
Palabra clave:CRISPR/Cas9
Donor
sgRNAs
Cell lines
Genome
Nucleotide
Gene therapy
CRISPR-Cas Systems
Cell Line
2409 Genética
genoma
línea celular
sistemas CRISPR-Cas
Descripción
Sumario:[EN]CRISPR/Cas9 allows the generation of knockout cell lines and null zygotes by inducing site-specific double-stranded breaks. In most cases the DSB is repaired by non-homologous end joining, resulting in small nucleotide insertions or deletions that can be used to construct knockout alleles. However, these mutations do not produce the desired null result in all cases, but instead generate a similar, functionally active protein. This effect could limit the therapeutic efficiency of gene therapy strategies based on abrogating oncogene expression, and therefore needs to be considered carefully. If there is an acceptable degree of efficiency of CRISPR/Cas9 delivery to cells, the key step for success lies in the effectiveness of a specific sgRNA at knocking out the oncogene, when only one sgRNA can be used. This study shows that the null effect could be increased with an sgRNA targeting the splice donor site (SDS) of the chosen exon. Following this strategy, the generation of null alleles would be facilitated in two independent ways: the probability of producing a frameshift mutation and the probability of interrupting the canonical mechanism of pre-mRNA splicing. In these contexts, we propose to improve the loss-of-function yield driving the CRISPR system at the SDS of critical exons.

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