Transformation of Arabidopsis Thaliana with CRISPR/Cas9 -modified AtHMA4 (Heavy-metal ATPase-4) Using Floral Dip to Increase Zn2+ Tolerance
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This study utilizes recent plasmid-designing technology to customize guide-RNA fragments and full plasmids encoded with the CRISPR/Cas9 system in order to artificially increase zinc tolerance or ‘levels’ in plants to decrease malnutrition via cereal grains. The CRISPR/Cas9 technology is used to alter the C-terminus of the heavy-metal ATPase 4 enzyme (AtHMA4) in Arabidopsis thaliana plants. Truncation of the C-terminus of the AtHMA4 enzyme has been shown increase zinc tolerance when transformed into yeast cells. Thus, to see its effects on the plants themselves, A. thaliana plants were grown over the course of two months while preparing two separate plasmids (pFULL_6805/ pFULL_6757) containing the CRISPR/Cas9 system. Using the floral dip method, A. thaliana plants were transformed with CRISPR/Cas9-equipped plasmids via a PCR golden gate assembly. The CRISPR/Cas9-containing seeds were successfully harvested in order to grow a second generation of plants to observe the zinc tolerance of these A. thaliana plants. This experiment is conducted with hopes to determine a method of enriching cereal grains around the world to better global nutrition.