I have been trying this for weeks but I do not observe transgenic parasites emerge after drug selection. My protocol in brief is as follows:
(1) Designed gRNAs as 5'-G-N19-NGG-3' to be inserted into a pUF1-Cas9 "suicide vector" which kills the essential gene. (NGG is not included in actual gRNA sequence)
(2) Synthetic Donor plasmid containing left and right Homology arms (with tag included) and PAM sequence shielded by mutation.
(3) Circular plasmids are electroporated into Red Blood Cells (310 V, 1575 Ohm Resistance, and 950 uF capacitance).
(4) DNA-loaded Red Cells are infected with Plasmodium falciparum 3D7 for 2-3 generations.
(5) Drug selection (1.5 uM DSM1) is applied which selects only for Cas9 expression.
Please suggest any flaw in the procedure or suggest any better protocol to do this.
Thanks in Advance.
Arnish
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