This repository accompanies the work "Discovery, characterization, and application of chromosomal integration sites for stable heterologous gene expression in Rhodotorula toruloides".
Add scripts and ifo0880 folders to code and data folder of "CRISPR-COPIES" repository.
First, run the script to incorporate putative essential genes of Rhodotorula toruloides:
python code/ifo0880/deg_update.py
Add the genome, feature table, and protein sequences of Rhodotorula toruloides strain NBRC 0880 from NCBI Assembly database to data/ifo0880 folder. Utilize the updated database of essential genes to run the CRISPR-COPIES command line tool and obtain genome-wide integration sites-
python code/main_rt.py -g ../data/ifo0880/GCA_000988875.2_ASM98887v2_genomic.fna -t ../data/ifo0880/GCA_000988875.2_ASM98887v2_feature_table.txt -p NGG -o 3prime -l 20 --edit_dist 5 --intspace 400 --distal_end_len 20000 -hr_l 1000 --protein_file ../data/ifo0880/GCA_000988875.2_ASM98887v2_protein.faa --blast_org 'Rhodosporidium toruloides IFO0880' --GC_grna 20,80 --polyG_grna 6 --polyT_grna 6 --RE_grna CCTGCAGG,GGCGCGCC,GTTTAAAC --RE_hr CCTGCAGG,GGCGCGCC,GTTTAAAC -sl 10 -out ../data/ifo0880/output_10_5.csv
Next, run the scripts on the generated output file to integrate transcriptomics information:
python code/ifo0880/transcript.py
To reproduce the results for zero-shot predictions of on-target efficiency for gRNAs characterized in this study (Supplementary Figure S1), run:
python code/ifo0880/on-target.py
python code/Uni-deepSG/PredictionTools/predict.py (Note: Requires CUDA)
If you using these scripts, please cite us:
Xu, H., Shi, L., Boob, A. G., Park, W., Tan, S. I., Tran, V. G., ... & Zhao, H. (2025). Discovery, characterization, and application of chromosomal integration sites for stable heterologous gene expression in Rhodotorula toruloides. Metabolic Engineering.