Skip to main content
bioRxiv
  • Home
  • About
  • Submit
  • ALERTS / RSS
Advanced Search
New Results

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

Ignacio Garcia-Tunon, Veronica Alonso-Perez, Elena Vuelta, Sandra Perez- Ramos, Maria Herrero, Lucia Mendez, Jesus Maria Hernandez-Sanchez, Marta Martin-Izquierdo, Raquel Saldana, Julian Sevilla, Fermin Sanchez-Guijo, Jesus Maria Hernandez-Rivas, View ORCID ProfileManuel Adolfo Sanchez-Martin
doi: https://doi.org/10.1101/532820
Ignacio Garcia-Tunon
IBSAL, Instituto de Investigacion Biomedica de Salamanca, Salamanca;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Veronica Alonso-Perez
IBSAL, Instituto de Investigacion Biomedica de Salamanca, Salamanca;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Elena Vuelta
University of Salamanca;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Sandra Perez- Ramos
University of Salamanca;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Maria Herrero
University of Salamanca;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Lucia Mendez
University of Salamanca;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jesus Maria Hernandez-Sanchez
IBSAL, Instituto de Investigacion Biomedica de Salamanca, Salamanca;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Marta Martin-Izquierdo
IBSAL, Instituto de Investigacion Biomedica de Salamanca, Salamanca;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Raquel Saldana
Servicio de Hematologia, Hospital de Especialidades de Jerez, Zacatecas, Spain;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Julian Sevilla
Hospital Infantil Universitario Nino Jesus, Madrid, Spain;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Fermin Sanchez-Guijo
Servicio de Hematologia, Hospital Universitario de Salamanca, Salamanca, Spain
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Jesus Maria Hernandez-Rivas
Servicio de Hematologia, Hospital Universitario de Salamanca, Salamanca, Spain
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
Manuel Adolfo Sanchez-Martin
University of Salamanca;
  • Find this author on Google Scholar
  • Find this author on PubMed
  • Search for this author on this site
  • ORCID record for Manuel Adolfo Sanchez-Martin
  • For correspondence: adolsan@usal.es
  • Abstract
  • Info/History
  • Metrics
  • Preview PDF
Loading

Abstract

CRISPR/Cas9 enables 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.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. All rights reserved. No reuse allowed without permission.
Back to top
PreviousNext
Posted January 29, 2019.
Download PDF
Email

Thank you for your interest in spreading the word about bioRxiv.

NOTE: Your email address is requested solely to identify you as the sender of this article.

Enter multiple addresses on separate lines or separate them with commas.
Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency
(Your Name) has forwarded a page to you from bioRxiv
(Your Name) thought you would like to see this page from the bioRxiv website.
Share
Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency
Ignacio Garcia-Tunon, Veronica Alonso-Perez, Elena Vuelta, Sandra Perez- Ramos, Maria Herrero, Lucia Mendez, Jesus Maria Hernandez-Sanchez, Marta Martin-Izquierdo, Raquel Saldana, Julian Sevilla, Fermin Sanchez-Guijo, Jesus Maria Hernandez-Rivas, Manuel Adolfo Sanchez-Martin
bioRxiv 532820; doi: https://doi.org/10.1101/532820
Digg logo Reddit logo Twitter logo CiteULike logo Facebook logo Google logo Mendeley logo
Citation Tools
Splice donor site sgRNAs enhance CRISPR/Cas9-mediated knockout efficiency
Ignacio Garcia-Tunon, Veronica Alonso-Perez, Elena Vuelta, Sandra Perez- Ramos, Maria Herrero, Lucia Mendez, Jesus Maria Hernandez-Sanchez, Marta Martin-Izquierdo, Raquel Saldana, Julian Sevilla, Fermin Sanchez-Guijo, Jesus Maria Hernandez-Rivas, Manuel Adolfo Sanchez-Martin
bioRxiv 532820; doi: https://doi.org/10.1101/532820

Citation Manager Formats

  • BibTeX
  • Bookends
  • EasyBib
  • EndNote (tagged)
  • EndNote 8 (xml)
  • Medlars
  • Mendeley
  • Papers
  • RefWorks Tagged
  • Ref Manager
  • RIS
  • Zotero
  • Tweet Widget
  • Facebook Like
  • Google Plus One

Subject Area

  • Molecular Biology
Subject Areas
All Articles
  • Animal Behavior and Cognition (998)
  • Biochemistry (1489)
  • Bioengineering (943)
  • Bioinformatics (6821)
  • Biophysics (2418)
  • Cancer Biology (1784)
  • Cell Biology (2528)
  • Clinical Trials (106)
  • Developmental Biology (1692)
  • Ecology (2564)
  • Epidemiology (1493)
  • Evolutionary Biology (5018)
  • Genetics (3613)
  • Genomics (4625)
  • Immunology (1165)
  • Microbiology (4244)
  • Molecular Biology (1622)
  • Neuroscience (10780)
  • Paleontology (82)
  • Pathology (236)
  • Pharmacology and Toxicology (409)
  • Physiology (555)
  • Plant Biology (1455)
  • Scientific Communication and Education (411)
  • Synthetic Biology (542)
  • Systems Biology (1871)
  • Zoology (259)