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RSS FeedsIJMS, Vol. 22, Pages 12611: Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis (International Journal of Molecular Sciences)

 
 

22 november 2021 17:58:20

 
IJMS, Vol. 22, Pages 12611: Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis (International Journal of Molecular Sciences)
 


The use of the novel CRISPR/Cas12a system is advantageous, as it expands the possibilities for genome editing (GE) applications due to its different features compared to the commonly used CRISPR/Cas9 system. In this work, the CRISPR/Cas12a system was applied for the first time to apple to investigate its general usability for GE applications. Efficient guide RNAs targeting different exons of the endogenous reporter gene MdPDS, whose disruption leads to the albino phenotype, were pre-selected by in vitro cleavage assays. A construct was transferred to apple encoding for a CRISPR/Cas12a system that simultaneously targets two loci in MdPDS. Using fluorescent PCR capillary electrophoresis and amplicon deep sequencing, all identified GE events of regenerated albino shoots were characterized as deletions. Large deletions between the two neighboring target sites were not observed. Furthermore, a chimeric composition of regenerates and shoots that exhibited multiple GE events was observed frequently. By comparing both analytical methods, it was shown that fluorescent PCR capillary gel electrophoresis is a sensitive high-throughput genotyping method that allows accurate predictions of the size and proportion of indel mutations for multiple loci simultaneously. Especially for species exhibiting high frequencies of chimerism, it can be recommended as a cost-effective method for efficient selection of homohistont GE lines.


 
160 viewsCategory: Biochemistry, Biophysics, Molecular Biology
 
IJMS, Vol. 22, Pages 12598: RNA-Seq Analysis Reveals an Essential Role of the Tyrosine Metabolic Pathway and Inflammation in Myopia-Induced Retinal Degeneration in Guinea Pigs (International Journal of Molecular Sciences)
IJMS, Vol. 22, Pages 12612: Identification of the Capsicum baccatum NLR Protein CbAR9 Conferring Disease Resistance to Anthracnose (International Journal of Molecular Sciences)
 
 
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