Gene editing by CRISPR/Cas9 in the obligatory outcrossing Medicago sativa

Planta
Ruimin GaoAbdelali Hannoufa

Abstract

The CRISPR/Cas9 technique was successfully used to edit the genome of the obligatory outcrossing plant species Medicago sativa L. (alfalfa). RNA-guided genome engineering using Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)/Cas9 technology enables a variety of applications in plants. Successful application and validation of the CRISPR technique in a multiplex genome, such as that of M. sativa (alfalfa) will ultimately lead to major advances in the improvement of this crop. We used CRISPR/Cas9 technique to mutate squamosa promoter binding protein like 9 (SPL9) gene in alfalfa. Because of the complex features of the alfalfa genome, we first used droplet digital PCR (ddPCR) for high-throughput screening of large populations of CRISPR-modified plants. Based on the results of genome editing rates obtained from the ddPCR screening, plants with relatively high rates were subjected to further analysis by restriction enzyme digestion/PCR amplification analyses. PCR products encompassing the respective small guided RNA target locus were then sub-cloned and sequenced to verify genome editing. In summary, we successfully applied the CRISPR/Cas9 technique to edit the SPL9 gene in a multiplex genome, providing some insig...Continue Reading

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Citations

Sep 28, 2018·Frontiers in Plant Science·Steffi FritscheGlenn Thorlby
Jun 9, 2020·Frontiers in Plant Science·Miroslava HrbáčkováJozef Šamaj
Aug 2, 2018·Frontiers in Plant Science·Deepa JaganathanGayatri Venkataraman
Aug 2, 2019·International Journal of Molecular Sciences·Zahra HajiahmadiQiang Zhuge
May 10, 2018·Frontiers in Plant Science·Nicola M Capstaff, Anthony J Miller
Jan 22, 2021·Plant Cell Reports·Dragana MiladinovicZoe Hilioti
Dec 4, 2020·Biology·Caterina MorciaValeria Terzi

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Methods Mentioned

BETA
genotyping
PCR
amplicon sequencing
gene knock-down
transgenic
gene modification

Software Mentioned

Quantasoft

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