# PrimedSherlock A python-based command-line script to augment CRISPR-Cas12 and CRISPR-Cas13 Assay generation using RPA Primer Sets. **Contact Author for example datasets used in publication or visit github for Zip files that can be dropped into the program folder.** ### Installation Prior to cloning the repo, ensure your python environment is setup with the following packages ``` Bio Pandas configparser ``` Create a new folder and utilize git to download this into the folder. This is now your PrimedSherlock directory. ``` gh repo clone JamesGerardMann/PrimedSherlock ``` Copy over your background db_fasta (off-target) from PrimedRPA as well as your input.fna (on-target) file. Lastly, copy over your Output_Sets.csv file. ``` background_db.fasta input.fna Output_Sets.csv ``` Lastly, we need to prep the tool to use either the CPU or a specific GPU for Cas-OFFinder. Open a cmd prompt, and navigate to our folder using cd. Then type Cas-offinder.exe An example below ``` cd C:\Users\14man\OneDrive\Desktop\PrimedSherlock\PrimedSherlock Cas-Offinder.exe ``` Under Available Device List find your desired CPU / GPU. Note down the ID #. Open the .py file included with our tool and search for "G0". Change this value wherever you find it to your desired GPU or CPU. For CPU change it to "C1" if its a cpu and # 1. Save the tool, close it and run the script.bat file. ### Parameters ``` MismatchThreshold ``` This sets the tolerance for generated crRNA target to mismatches present in the strains utilized by the tool. It should be somewhere between 0-3. Most of the examples utilized 3, where as the ZIKA example utilized 5. If no crRNA sets are generated you may want to increase this value. ``` Use_PrimedRPA ``` This sets the tool into a mode where it seeks a generated PrimedRPA Output_Sets.csv file. PrimedRPA is the best tool to generate primer sets for our tool. If you set this to No, you will need to provide hand generated primer pairs and ensure they are accurate. ``` Threadcount ``` This sets the amount of CPU threads to use for Cas-Offinder instances. More equals faster data processing. ``` Amplicon_Max_Size ``` Sets limits on the max size of amplicon that the tool will use. This is important as larger amplicons equals slower amplification by RPA polymerase. ``` Amplicon_Min_Size ``` Sets lower limits on the size of amplicon that the tool will use. This is important as smaller amplicons equals faster amplification by RPA polymerase. ``` Max_Background_Identity ``` This is a background identity limit introduced by PrimedRPA. Lower is better, sets a off-target amplification threshold. Hypothetically shouldn't matter with how our tool generates crRNA instead of probes. ``` Off_Target_Mismatch_Score ``` This sets lower limits on how many mismatches are required for the crRNA to be deemed safe from recognition of off-target sequences. ``` Contingency_crRNA_Utilization ``` This is very experimental and should not be used. It recognizes which strains have low affinity to be detected by crRNA targets and generates potential separate pairs for highly conserved, highly mismatched strains. ``` Working_Directory, str(master_dir)) Primed_RPA_Output, str(master_dir)) Input.fna_Location, str(master_dir)) Background_Blast_File, str(master_dir)) ``` This is used for developer troubleshooting. This is automatically set when the code first run. ``` Run_Cas-off ``` This is used for developer troubleshooting. Turning this off will stop the code prior to the Cas-Offinder.exe step. Keep this on. ### Key Output Files PrimedSherlock runs generate the following output file: ``` [RunID]_FinalOutput.csv ``` ### 3rd-Party Software PrimerSherlock incorporates segments of ... PrimedRPA - https://github.com/MatthewHiggins2017/bioconda-PrimedRPA PrimedSherlock includes an executable from... Cas-Offinder http://www.rgenome.net/cas-offinder/ ### Contact If you encounter any bugs please contact me directly at **James_Mann1[at]alumni.baylor.edu**