Researchers Develop Better CRISPR-Cas9 Using GFP Tagged Protoplasts
June 20, 2019| |
CRISPR-Cas9 is a popular method used to edit genes in various organisms. However, the system may not work properly in the presence of complex high ploidy genomes and inefficient or poorly controlled CRISPR-Cas9 delivery to cells with regenerative potential. Thus, experts from the University of Copenhagen aimed to improve the system's efficiency using Fluorescence Activated Cell Sorting (FACS) of protoplasts. The results are published in BMC Biotechnology.
The researchers used Agrobacterium tumefaciens infiltration in leaves of Nicotiana benthamiana, a tobacco relative, to deliver the improved CRISPR-Cas9 system targeting two locations in the genome. Then they isolated the protoplasts from the infiltrated leaves and subjected them to FACS to select green fluorescent protein (GFP)-enriched protoplast populations. The results showed a 3 to 5-fold increase in mutation frequencies as manifested by various analyses conducted.
Based on the findings, FACS of protoplasts expressing green fluorescent protein-tagged CRISPR-Cas9 delivered through A. tumefaciens infiltration facilitated clear CRISPR-Cas9 mediated mutation enrichment in selected protoplast populations.
Read more about the study in BMC Biotechnology.
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