| First Author | García-Tuñón I | Year | 2019 |
| Journal | PLoS One | Volume | 14 |
| Issue | 5 | Pages | e0216674 |
| PubMed ID | 31071190 | Mgi Jnum | J:275498 |
| Mgi Id | MGI:6305770 | Doi | 10.1371/journal.pone.0216674 |
| Citation | Garcia-Tunon I, et al. (2019) Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency. PLoS One 14(5):e0216674 |
| abstractText | 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. |
