Efficient In Vivo Introduction of Point Mutations Using ssODN and a Co-CRISPR Approach

Biological Procedures Online
Tgst LeviOffer Gerlitz

Abstract

The generation of point mutations is a major tool for evaluating the roles of specific nucleotides or amino acids within the regulatory or functional landscape. However, examination of these mutations in vivo requires the generation of animals carrying only the relevant point mutations at the endogenous genomic loci, which is technically challenging. The CRISPR-Cas9 based genome editing greatly facilitates the generation of such genetically modified animals; however, most of the described methods use double-strand DNA (dsDNA) as the donor template. The dsDNA plasmids frequently undergo undesired integration events into the targeted genomic locus. The use of a single-strand oligodeoxynucleotide (ssODN) as the donor template prevents this complication and is therefore the preferred choice for introducing point mutations, as well as short sequences such as protein tags. We successfully applied the CRISPR-based white co-conversion strategy with a ssODN template, instead of the originally described dsDNA plasmid, to create genetically modified Drosophila melanogaster strains. We used the technique to easily introduce point mutations in two distinct chromosomes. Using the generated flies, we were able to demonstrate the in vivo impor...Continue Reading

References

Apr 14, 2009·Nature Methods·Daniel G GibsonHamilton O Smith
Jun 30, 2012·Science·Martin JinekEmmanuelle Charpentier
Jan 5, 2013·Science·Le CongFeng Zhang
Feb 8, 2013·G3 : Genes - Genomes - Genetics·John Roote, Andreas Prokop
Aug 3, 2013·Nucleic Acids Research·Luhan YangGeorge Church
Jan 8, 2014·Methods in Molecular Biology·Ariel ErijmanYoav Peleg
Mar 19, 2014·The Journal of Biological Chemistry·Yonathan ZehaviTamar Juven-Gershon
Jun 24, 2014·Scientific Reports·Masafumi InuiShuji Takada
Jul 9, 2014·Proceedings of the National Academy of Sciences of the United States of America·Fillip PortSimon L Bullock
May 3, 2015·Biochimica Et Biophysica Acta·Yehuda M DaninoTamar Juven-Gershon
May 20, 2015·Methods in Molecular Biology·Scott J GratzKate M O'Connor-Giles
Oct 21, 2015·The Journal of Clinical Investigation·Ariella Weinberg-ShukronDavid Zangen
Aug 21, 2016·G3 : Genes - Genomes - Genetics·Daniel Tianfang GePhillip D Zamore
Oct 30, 2016·G3 : Genes - Genomes - Genetics·Nanci S KaneRichard W Padgett
Aug 16, 2017·Genes & Development·Long Vo NgocJames T Kadonaga
Jan 6, 2018·Genetics·Ethan BierJill Wildonger
Jun 16, 2018·Nucleic Acids Research·Sergey V PrykhozhijJason N Berman
Jun 24, 2018·G3 : Genes - Genomes - Genetics·Ben Ewen-Campen, Norbert Perrimon
Jun 28, 2018·Nature Reviews. Molecular Cell Biology·Vanja Haberle, Alexander Stark
May 10, 2019·Nature Communications·Hagar MeltzerOren Schuldiner

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Citations

Jan 15, 2021·Proceedings of the National Academy of Sciences of the United States of America·Justin A BoschNorbert Perrimon

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Methods Mentioned

BETA
Transgenic
PCR
genotyping
dissections

Software Mentioned

ssODNs
ssODN

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