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Investigation of CRISPR/Cas mediated sequence-specific induction of mutations in Vicia faba

Untersuchung zur CRISPR/Cas vermittelten, sequenzspezifischen Induktion von Mutationen in Vicia faba

  • Vicia faba leaves and calli were transformed using CRISPR Cas RNP. Two kinds of CPP fused SpyCas9 were used with sgRNA7, sgRNA5 or sgRNA13 targeting PDS exon 1, PDS exon 2 or MgCh exon 3 respectively. RNP were applied using high pressure spraying, biolistic delivery, incubation in RNP solution and infiltration of leaf tissue. A PCR and restriction enzyme based approach was used for detection of mutation. Screening of 679 E. coli colonies containing the cloned fragments resulted in detection of 14 mutations. Most of the 14 mutations were deletions of sizes 150, 500 or 730 bp. 5 out of the 14 mutations were point mutations located two to three bp upstream of PAM.

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Author:Jens Gruhne
Advisor:Röbbe Wünschiers, Michael Wallbraun
Document Type:Master's Thesis
Year of Completion:2020
Granting Institution:Hochschule Mittweida
Release Date:2022/06/27
GND Keyword:Ackerbohne; CRISPR/Cas-Methode
Institutes:Angewandte Computer‐ und Bio­wissen­schaften
DDC classes:572.8028 CRISPR/Cas-Methode
Open Access:Frei zugänglich
Licence (German):License LogoUrheberrechtlich geschützt