Lipides, Nutrition, Cancer
Intervenant : Dr Boris Skryabin
Mouse genome editing via direct microinjection of the CRISPR/cas9 system in zygotes
Skryabin BV, Rozhdestvensky TS. Core Facility TRAnsgenic Animal and Genetic Engineering Models (TRAM), University of Münster, Münster, Germany. email@example.com
Background: CRISPR (Clustered regularly interspaced short palindromic repeat) system for editing genomes of various organisms was introduced in 2012 and revolutionized research in modern biology and medicine. The CRISPR/cas9 technology has borrowed the defense system of the Streptococcus pyogenes and deliver the codon optimized endonuclease Cas9 to the specific target site of most genomes with the help of short programmable RNA molecule. The creation of specific nuclease for almost any region of DNA using CRISPR, unlike ZFN and TALEN systems, relatively easy and demonstrates high efficiency. TRAM (TRAnsgenic animal and genetic engineering Models) group of the Medical faculty of the University of Münster (Germany) uses the CRISPR/cas9 system in our daily work to create new lines of different kind genome-engineered mice. Among them, the classical and conditional gene knock-out, knock-in at specific mouse loci, and introduction of point mutation or epitope in desired gene regions. The efficiency of gene modification at specific locus using the non-homologous end-joining (NHEJ) mechanism reaches almost 90%, and the efficiency of targeted edition using the homology-directed repair (HDR) mechanism is up to 50%. We will present data on the selection of the target, gRNA (crRNA) design, selection of effective nucleases, characteristics of modified loci and methods of microinjection of CRISPR/cas9 components in murine oocytes. Despite the advantages of CRISPR/Cas9 based genome editing, several potential problems such as genome rearrangements and off-target effects still impede the CRISPR/Cas9 technology for use in biomedical research and further efforts are necessary to overcome these hurdles. Our study examines problems that affect direct knock-in genome targeting. During the generation of six different conditional knock-out mouse models, we discovered that frequently (sometimes solely) HDR and/or NHEJ mechanisms caused multiple unwanted head-to-tail insertions of donor DNA templates. Disturbingly, conventionally applied PCR analysis - in most cases - failed to identify such multiple integration events, which led to a high rate of falsely claimed precisely edited alleles. We caution that comprehensive analyses of modified alleles are essential, and offer practical solutions to correctly identify precisely edited genomic loci. Our findings are important to unlock the full potential of the CRISPR/Cas9-mediated genome editing protocols for the generation of custom-designed gene variants for biomedical research and gene therapy. At the end, we will discuss the advantages and pitfalls of the CRISPR/cas9 system in direct mouse genome engineering.
1. Pristyazhnyuk IE, Minina J, Korablev A, Serova I, Fishman V, Gridina M, Rozhdestvensky TS, Gubar L, Skryabin BV, Serov OL Time origin and structural analysis of the induced CRISPR/cas9 megabase-sized deletions and duplications involving the Cntn6 gene in mice. Sci Rep. 2019 Oct 2;9(1):14161Sci Rep.
2. Mo D, Li X, Raabe CA, Cui D, Vollmar JF, Rozhdestvensky TS, Skryabin BV, Brosius J. A universal approach to investigate circRNA protein coding function. Sci Rep. 2019 Aug 12;9(1):11684. Sci Rep.
3. Klaiber M, Dankworth B, Kruse M, Hartmann M, Nikolaev VO, Yang RB, Völker K, Gassner B, Oberwinkler H, Feil R, Freichel M, Groschner K, Skryabin BV, Frantz S, Birnbaumer L, Pongs O, Kuhn MA Cardiac pathway of cyclic GMP-independent signaling of guanylyl cyclase A, the receptor for atrial natriuretic peptide. Proc Natl Acad Sci U S A. 108:18500-5, 2011 PNAS
Invitation: Oleg Demidov - UMR1231
Le séminaire aura lieu à la faculté de médecine -amphi Martin à 11h00
Renseignements - firstname.lastname@example.org Tel: 03 80 39 34 68