Mutant/mutation
The mutant contains a C-terminal quadruple Myc (4Myc) tagged Spcas9 gene introduced into the silent sera1 gene locus.  The Spcas9 gene is under control of the 5'- and 3'-UTR regions of sera1. The mutants does not contain a drug-selectable marker.

Protein (function)
The RNA-guided endonuclease Cas9 has applied as an efficient gene-editing method in malaria parasite Plasmodium. However, the size (4.2 kb) of the commonly used Cas9 from Streptococcus pyogenes (SpCas9) limits its utility for genome editing in the parasites only introduced with cas9 plasmid. To establish the endogenous and constitutive expression of Cas9 protein in the rodent malaria parasite P. yoelii, we replaced the coding region of an endogenous gene sera1 with the intact SpCas9 coding sequence using the CRISPR/Cas9-mediated genome editing method, generating the cas9-knockin parasite (PyCas9ki) of the rodent malaria parasite P. yoelii. The resulted PyCas9ki parasite displays normal progression during the whole life cycle and possesses the Cas9 protein expression in asexual blood stage

Phenotype
The resulted PyCas9ki parasite displays normal progression during the whole life cycle and possesses the Cas9 protein expression in asexual blood stage.

To evaluate the effect of Cas9 expression on the parasite development and differentiation, we performed detailed analyses comparing the life cycle between wildtype and PyCas9ki parasites. PyCas9ki parasites displayed similar asexual proliferation and gametocytes formation in mouse blood stage, ookinete differentiation in vitro, day 7 oocysts per mosquito, day 14 salivary gland sporozoites, and sporozoite infectivity of mice to those of the WT parasite, suggesting that replacement of sera1 with Spcas9 does not affect parasite development.

The expression of 4Myc-tagged Cas9 protein was detected within the nucleus of different asexual blood stages of PyCas9ki parasites, including rings, trophozoites, and schizonts using anti-Myc antibody by immunofluorescence assay (IFA). In addition, no expression of Cas9 protein was detected in gametocytes, ookinetes, midgut oocysts, and salivary gland sporozoites. These results indicated that the promoter of endogenous sera1 gene possesses the transcription activity in the asexual blood stages of P. yoelii parasite.

Additional information
Because the Spcas9 gene is a 4200 bp coding sequence in size, it is challenging to incorporate the Cas9 coding sequence, its promoter and polyadenlyation sequence into the parasite genome together. We decided to replace the coding region of a non-essential gene, sera1, with Spcas9 gene. To confirm that sera1 is not essential for the parasite, we first disrupted the sera1 gene in the 17XNL parasite using CRISPR/Cas9 methods (see RMgm-1095) and obtained two parasite knockout clones (Δsera1c1 and Δsera1c2) with deletion of the whole coding region. Both Δsera1c1 and Δsera1c2 parasite clones displayed normal progression comparable with wildtype parasite in the life cycle, including asexual and sexual gametocytes stages in mice, mosquito stages, and mouse infectivity, suggesting functional redundancy of the sera1 gene in the life cycle of P. yoelii.

Next, we constructed a plasmid pYCm-Cas9 containing an intact Cas9 encoding sequence tagged with quadruple Myc epitope (4Myc) C-terminally and flanked by two homologous regions of sera1 (0.5 kb of the 5′-flanking region and 0.5 kb of the 3′-flanking region). After transfection, drug selection, and parasite cloning, we obtained two parasite clones (PyCas9kic1 and PyCas9kic2) with targeted integration of the Spcas9 gene into the sera1 locus. Correct replacement of sera1 with Spcas9 was further confirmed by RT-PCR analyses showing cas9 transcript, but not that of sera1 in the red blood stages of PyCas9ki parasites. Furthermore, expressions of both Cas9::4Myc protein from integrated gene and 3Flag::Cas9 protein from the episomal pYCm plasmid were detected on western blotting. To remove the episome plasmid in the parasites for next-round genetic modification, we applied the negative selection to the PyCas9ki parasite by using 5-FC and confirmed the successful removal of episomal plasmid as no expression of 3Flag::Cas9 protein was detected in the parasites after 5-FC treatment.

Other mutants