Mutant/mutation
A microhomology-mediated end joining (MMEJ)-based CRISPR/Cas9 (mCRISPR) strategy was used to generate multiple mutant parasites simultaneously in the repetitive region of CSP. Several mutants have been generated that express mutated forms of CSP that contain shorter versions of the central repeat region (CRR) of CSP.

Protein (function)
The CS protein is the major protein on the surface of sporozoites and is critical for development of sporozoites within the oocysts and is involved in motility and invasion of both the salivary gland of the mosquito and the liver cells. The protein is also found on the oocyst plasma membrane and on the inner surface of the oocyst capsule. Specific motifs in CS are involved in sporozoite binding to mosquito salivary glands and in sporozoite attachment to heparan sulfate proteoglycans in the liver of the mammalian host. During substrate-dependent locomotion of sporozoites, CS is secreted at the sporozoite anterior pole, translocated along the sporozoite axis and released on the substrate at the sporozoite posterior pole. Following sporozoite invasion of hepatocytes, the CS is released in the host cell cytoplasm.

Phenotype
In this study several mutants have been generated that express mutated forms of CSP that contain shorter versions of the central repeat region (CRR) of CSP.
Monitoring mixed parasite populations and individual parasites with different sizes of CSP-CRR showed that the CSP-CRR plays a role in the development of mosquito stages, with severe developmental defects in parasites with large deletions in the repeat region. However, the majority of the csp mutant parasite clones grew similarly to the wild type Plasmodium yoelii 17XL parasite in mice.

Additional information
Three major DSB repair mechanisms have been described in eukaryotic organisms, including non-homologous end joining (NHEJ), homologous recombination (HR), and microhomology-mediated end joining (MMEJ). Because no genes encoding enzymes for the NHEJ pathway can be found in the genomes of malaria parasites, the major DSB repair pathway in Plasmodium parasites is HR, which has been experimentally verified by numerous HR-based genetic modifications. An alternative end joining pathway was also described in the human parasite Plasmodium falciparum, which is likely to be mediated through MMEJ. The MMEJ pathway may be responsible for the high rates of small indel mutations in AT-rich malaria genome sequences and elevated recombination events associated with G-quadruplex motifs.
The CRISPR/Cas9 system generally employs a prokaryotic RNA programmable nuclease that can introduce DSBs at a target site identified by sequence-specific guide RNA (sgRNA). This technique has been successfully adapted to edit malaria parasite genomes. Unfortunately, because malaria parasites lack the critical components of the NHEJ DNA repair pathway such as Ku70/80 and DNA Ligase IV9, HR is considered to be the primary DSB repair pathway in this parasite. Therefore, CRISPR/Cas9 methods for malaria parasites also require homologous DNA templates flanking the targeted DSB site that are typically provided in a plasmid. The requirement of template DNAs can greatly increase the workload in plasmid construction, particularly when editing a large number of genes or members of a gene family.
One aim of this study was to test the principle of the Plasmodium MMEJ pathway in repairing CRISPR/Cas9-mediated DSBs and to develop a simple MMEJ-mediated CRISPR/Cas9 method (mCRISPR) for parasite genome editing.

The CSP of 17XL parasite contains two types of tandem repeats [(QGPGAP)₂₅ and (PPQQ)₇] in the central repeat region (CRR). We designed four guide RNAs (sgRNA) from the CRR region and constructed four plasmids that transcribes each of the sgRNAs and the Cas9 enzymes without homologous DNA templates to generate targeted DSBs.
sgRNA-2 is expected to cleave three sites at the CRR because it has three corresponding target sequences, whereas the other three sgRNAs have one cleavage site. Without the presence of homologous DNA templates,overlapping repetitive sequences may anneal randomly, leading to cleavages of overhanging sequences and sealing of the gaps. Target DNA samples were extracted from the 17XL parasite in mouse blood when the parasitemia reached 1-5% after transfection and pyrimethamine selection, and were amplified using primer pairs F1/R1 (or R2) flanking the PyCSP-CRR. Multiple bands were detected in parasites transfected with sgRNA-2 and sgRNA-3, whereas a major band similar to that of 17XL WT was observed in the parasites transfected with sgRNA-1 and sgRNA-4.

Four sgRNAs were designed to target multiple sites in the CRR of the Pycsp gene (PlasmoDB accession no. PY17X_0405400, 1284 bp). Cas9-sgRNA plasmids without homologous donor templates were generated based on the pYC vector that contains the gene encoding Cas9 enzyme from Streptococcus pyogenes (Zhang et al., 2017; RMgm-4069). Plasmid without sgRNA which served as a control was generated as well. Transfection and selection of transformed parasites with pyrimethamine were performed as described previously (Nair et al., 2017; Zhang et al., 2017)

Other mutants