Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency
| Autor: | Fermín Sánchez-Guijo, Jesús María Hernández-Rivas, Jesus M Hernández-Sánchez, Ignacio García-Tuñón, Marta Martín-Izquierdo, Sandra Pérez Ramos, Lucía Méndez, Elena Vuelta, Manuel Sánchez-Martín, Verónica Alonso-Pérez, María de las Nieves Sánchez González de Herrero, Raquel Saldaña, Julián Sevilla |
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| Přispěvatelé: | Ministerio de Economía y Competitividad (España), European Commission, Junta de Castilla y León, Fundacion de la Sociedad Española de Hematología y Hemoterapia |
| Rok vydání: | 2019 |
| Předmět: |
0301 basic medicine
Embryology sistemas CRISPR-Cas Molecular biology Genetic enhancement Ataxia Telangiectasia Mutated Proteins sgRNAs Synthetic Genome Editing Genome Engineering Exon Gene Knockout Techniques Mice Database and Informatics Methods 0302 clinical medicine Sequencing techniques CRISPR DNA sequencing Proto-Oncogene Proteins c-abl Frameshift Mutation Gene Editing Multidisciplinary Genome Mammalian Genomics Monophenol Monooxygenase Crispr línea celular Exons Genomics Null allele RNA splicing Medicine Cell lines Engineering and Technology Synthetic Biology Donor Nucleotide Transcriptome Analysis Sequence Analysis RNA Guide Kinetoplastida Research Article Next-Generation Sequencing Bioinformatics Science Bioengineering Computational biology Biology Frameshift mutation Cell Line 03 medical and health sciences Gene therapy Genetics Cancer Genetics Animals Humans CRISPR/Cas9 Alleles genoma 2409 Genética Cas9 Null (mathematics) Embryos Biology and Life Sciences Computational Biology Oncogenes Synthetic Genomics Genome Analysis Research and analysis methods 030104 developmental biology Molecular biology techniques Synthetic Bioengineering Animal Genomics Mutation RNA Splice Sites CRISPR-Cas Systems K562 Cells 030217 neurology & neurosurgery Cloning Developmental Biology |
| Zdroj: | PLoS ONE GREDOS: Repositorio Institucional de la Universidad de Salamanca Universidad de Salamanca (USAL) Digital.CSIC. Repositorio Institucional del CSIC instname PLoS ONE, Vol 14, Iss 5, p e0216674 (2019) GREDOS. Repositorio Institucional de la Universidad de Salamanca |
| ISSN: | 1932-6203 |
| Popis: | 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. This work was mainly supported by a grant from the Fondo de Investigaciones Sanitarias (FIS) of the Spanish Ministry of Economy and Competitiveness and the European Regional Development Fund (ERDF) “Una manera de hacer Europa” [grant PI17/01895 to IGT and MSM.]; Junta de Castilla y León, Fondos FEDER [SA085U16 to JMHR]; Novartis grant; and by the Fundación “Jabones para Daniel”. JM Hernández-Sánchez was supported by a research grant from Fundación Española de Hematología y Hemoterapia (FEHH). |
| Databáze: | OpenAIRE |
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