Mutant/mutation
The mutant contains a gene encoding Cas9 from Streptococcus pyogenes, introduced into the c/d-ssu-rrna gene. This Cas9 nuclease does not cleave double-stranded DNA in the absence of sgRNA and is thus suitable for generating the parasite which expresses it constitutively. It does not contain a drug-selectable marker that has been removed by negative selection

Protein (function)
This Cas9 nuclease does not cleave double stranded DNA in the absence of sgRNA and is thus suitable for generating the parasite which express it constitutively.

Phenotype
No obvious effect on development of all life cycle stages due to constitutive expression of Cas9 and integration of its expression cassette at the c-ssu-rrna locus.

Additional information
The mutant does not contain a drug-selectable marker which has been removed by negative selection.

Whole genome sequencing revealed that the constitutive expression of Cas9 did not cause unexpected mutations in the parasite genome.

Unexpected recombination events were observed when this parasite was used to modify genes with circular donor DNA plasmids:
From the paper: 
'When the target genomic locus is cleaved by the Cas9–sgRNA complex, the molecules responsible for the repair of the doublestrand break are recruited, elevating the recombination activity specifically at the locus. Thus, the observed additional recombination was probably caused by the increase in recombination activity at the target genomic locus. Similar additional recombination was found in P. falciparum when  nonessential genes for erythrocytic development were engineered by the CRISPR/Cas9 system using a plasmid that contained the donor template. Conversely, when an essential gene for erythrocytic development was mutated in our previous study, such recombination was never found despite using a circular plasmid carrying the donor template. However, the additional recombination might be overlooked in this case, because the transgenic parasites in which this recombination occurred died due to the  functional disruption of the essential gene. Thus, whenever a circular plasmid is used for delivering the donor template, an additional recombination will occur between the integrated donor template and another copy of the plasmid as the donor template, resulting in failure or reduced efficiency of the genetic modification. Therefore, this is a serious technical problem of the CRISPR/Cas9 system when using circular plasmid DNA  as the donor template.'

Based on these observations the authors developed linear donor templates ('To solve the technical problem of integrated circular donor DNA, we used a linear donor template DNA for subsequent genetic modifications.')
 

From the Abstract:

'The use of a linear donor template prevented unexpected recombination; in addition, constitutive expression of Cas9 enabled immediate cleavage of the target locus after transfection, allowing efficient integration of the donor template. Furthermore, due to the absence of the cNHEJ pathway, there were no off-target mutations in the resultant parasites. In addition, this developed method could be applied for multiple genetic modifications on different chromosomes and for large scale chromosomal deletion in the subtelomeric region.'

Other mutants