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Details of the target gene |
Gene Model of Rodent Parasite |
PY17X_0210200
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Gene Model P. falciparum ortholog |
PF3D7_0104300
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Gene product | ubiquitin carboxyl-terminal hydrolase 1, putative |
Gene product: Alternative name | UBP1 |
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Details of the genetic modification |
Inducable system used | No |
Additional remarks inducable system |
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Type of plasmid/construct used | CRISPR/Cas9 construct: integration through double strand break repair |
PlasmoGEM (Sanger) construct/vector used | No |
Modified PlasmoGEM construct/vector used | No
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Plasmid/construct map |
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Plasmid/construct sequence |
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Restriction sites to linearize plasmid |
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Partial or complete disruption of the gene | Complete |
Additional remarks partial/complete disruption |
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Selectable marker used to select the mutant parasite | hdhfr/yfcu |
Promoter of the selectable marker | eef1a |
Selection (positive) procedure | pyrimethamine |
Selection (negative) procedure | No |
Additional remarks genetic modification | The CRISPR/Cas9 plasmid pYCm was utilized for gene editing. To construct plasmids for ubp1 allelic replacement at amino acid codon 1560 and 2874 between P. yoelii lines NSS and NSR, four DNA fragments from two regions (R1, from 4393 to 5036; and R2, from 8368 to 9034) were amplified and inserted into the NcoI/XhoI sites of the pYCm vector individually. Three silent nucleotide substitutions were introduced into the single guide RNA (sgRNA) binding sites in the donor templates using synthetic oligonucleotides and PCR amplification to yield the editing plasmids.
To construct plasmids for gene tagging or tag insertion, a fragment (450–800 bp) from the C- or N-terminal of coding regions as left or right homologous arm and a fragment (450–800 bp) from 3’UTR or 5’UTR regions as right or left homologous arm were amplified, respectively. DNA sequence encoding 6HA, 4Myc, mCherry, GFP, APEX2, or 3HA was inserted between the left and right arms in the frame and cloned into the pYCm vector.
To construct the plasmid to delete the coding region of the P. yoelii ubp1 gene, 5’- and 3’- genomic fragments (400–600 bp) were amplified as left and right arms and cloned them into restriction sites of HindIII/NcoI and XhoI/EcoRI in the pYCm vector, respectively. Synthesized sgRNAs were annealed and ligated into the restriction site of BsmBI in the vector. |
Additional remarks selection procedure | |
Primer information: Primers used for amplification of the target sequences 
Primer information: Primers used for amplification of the target sequences
Sequence Primer 1 | |
Additional information primer 1 | |
Sequence Primer 2 | |
Additional information primer 2 | |
Sequence Primer 3 | |
Additional information primer 3 | |
Sequence Primer 4 | |
Additional information primer 4 | |
Sequence Primer 5 | |
Additional information primer 5 | |
Sequence Primer 6 | |
Additional information primer 6 | |
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