Mutant/mutation
The mutant lacks expression of CDPK3 ( and expresses a C-terminal mCherry tagged P28 protein.

The cdpk3 gene was disrupted in mutant RMgm-4071 that expresses a C-terminal mCherry tagged P28 protein.

Both for tagging P28 and for disruption of CDPK3 the CRISPR/Cas9 method was used as described for mutant RMgm-1095  with a slight modification of the pYCm vector. In this vector the original hdhfr slectable marker was replaced by the hdhfr-yfcu selectable marker. This allows for killing mutated parasites that still contain remaining episomal plasmids by 5-FC treatment of mice. Removal of the episomal plasmid is essential for subsequent mutations in the same parasite using the CRISPR/Cas9 method.

Protein (function)
CDPK3 belongs to an expanded family of Ca²⁺ dependent protein kinases (CDPKs). CDPKs combine an amino-terminal serine/threonine kinase domain and a carboxy-terminal calmodulin-like domain, composed of four EF hands, in the same molecule. The protein plays a role in ookinetes. Ookinetes lacking expression of CDPK3 fail to bind and traverse the cell of the midgut wall.

Phenotype
Ookinetes show strongly reduced forward gliding. No oocyst are formed.

Additional information
See mutant RMgm-1095 for a detailed description of the CRISPR/Cas9 method.
Both for tagging P28 and for disruption of CDPK3 the CRISPR/Cas9 method was used as described for mutant RMgm-1095  with a slight modification of the pYCm vector. In this vector the original hdhfr slectable marker was replaced by the hdhfr-yfcu selectable marker. This allows for killing mutated parasites that still contain remaining episomal plasmids by 5-FC treatment of mice. Removal of the episomal plasmid is essential for subsequent mutations in the same parasite using the CRISPR/Cas9 method.

Other mutants