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ParameterFile_Cas9.txt
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##########################################################
### ###
### GENETARGETER PARAMETER FILE ###
### Contact pablocarderam@gmail.com with any questions ###
### ###
##########################################################
# ALL BOOLEAN PARAMETERS SHOULD BE ENCODED AS True OR False
# ALL PARAMETERS THAT ARE NON-NUMERIC AND NON BOOLEAN SHOULD BE IN QUOTES
### Plasmid vector content ###
plasmidType : 'pSN054_V5' # Vector backbone to be used (either 'custom', or 'pSN054_V5', 'pSN054', 'pSN150', 'pSN150-KO' for 3', 5', and for gene knock-outs, respectively; also 'pSN150-Ter', 'pSN150-KO-Ter' with modified T7 terminator with no Nhe1 sequence) QUOTES ("" or '') NEEDED
basePlasmid : None # File path to custom base plasmid (inside quotes '') if using 'custom' in plasmidType, otherwise None
locationType : '3prime' # Either '5prime', '3prime', or 'center', if using 'custom' in plasmidType
haTag : 'Auto' # Adds HA protein tag to 5' end if using pSN150; if 'Auto', adds only if gene has no reported signal peptide ('Auto'/True/False, QUOTES ("" or '') NEEDED FOR AUTO)
### Plasmid assembly ###
filterCutSites : 'Auto' # Checks designs for restriction enzyme cut sites specified as [ "ggccggcc", "gcgatcgc", "gctagc" ]
# If 'Auto' (QUOTES ("" or '') NEEDED FOR AUTO), checks for predetermined lists according to plasmid type:
# 'pSN054' : FseI, AsiSI, IPpoI, ISceI, AflII, AhdI
# 'pSN150' : FseI,AsiSI,IPpoI,ISceI,AflII,AhdI,BsiWI, NheI
# 'pSN150-KO' : FseI, AsiSI, IPpoI, ISceI, AflII, AhdI, BsiWI, NheI
gibsonHomRange : [20,30,60] # Length in bp of homology on each side used for Gibson assembly primer design [minimum, preferred, maximum]
gibTemp : 50 # Minimum Gibson primer melting temperature in ºC
gibTDif : 5 # Maximum melting temperature difference between Gibson primers in ºC
gBlockDefault : True # Defaults to always using gene fragment synthesis for recoded regions, if any (instead of Klenow oligos for smaller regions) (True, False)
minGBlockSize : 250 # Minimum gene fragment size in bp (extends fragment if under minimum size if gBlockDefault is True, otherwise uses Klenow oligos)
maxGBlockSize : 1000 # Maximum gene fragment size in bp
codonOptimize : 'T. gondii' # Codon usage table used for recodonization when recoding gene fragment region, if any ('P. falciparum 3D7', 'P. vivax', 'T. gondii', 'E. coli K12', 'S. cerevisiae', 'H. sapiens', 'R. norvegicus', 'scramble') QUOTES ("" or '') NEEDED
codonSampling : False # Codon optimization algorithm used to select codons when recodonizing recoded region, if any (True, False for Codon Sampling and CAI maximization, respectively) (http://omicsomics.blogspot.com/2009/04/is-codon-optimization-bunk.html)
### Homologous region design ###
HRannotated : False # Uses gRNA, LHR, and/or RHR already annotated on plasmid rather than software selected (True, False)
lengthLHR : [400,500,750] # Length in bp of Left Homologous Region (LHR) [minimum, preferred, maximum]
lengthRHR : [400,500,750] # Length in bp of Right Homologous Region (LHR) [minimum, preferred, maximum]
optimRangeLHR : [-20,20] # Range in bp over which a higher melting temperature will be searched for selected LHR starts and ends [upstream, downstream]
optimRangeRHR : [-20,20] # Range in bp over which a higher melting temperature will be searched for selected RHR starts and ends [upstream, downstream]
endSizeLHR : 40 # Length in bp of region at start and end of LHR to be taken into account when evaluating optimal starts and ends
endSizeRHR : 40 # Length in bp of region at start and end of RHR to be taken into account when evaluating optimal starts and ends
endTempLHR : 55 # Minimum temperature in ºC that LHR start and end regions must have
endTempRHR : 55 # Minimum temperature in ºC that RHR start and end regions must have
maxDistLHR : 700 # Maximum distance in bp between LHR end and gRNA (if targeting 5') or gene start (if targeting 3' end)
maxDistRHR : 125 # Maximum distance in bp between gene end (if targeting 5') or gRNA (if targeting 3' end) and RHR start
### gRNA Selection ###
enzyme : 'Cas9' # Enzyme type used in plasmid ('Cas9', 'Cas12') QUOTES ("" or '') NEEDED
PAM : 'NGG' # Protospacer-Adjacent Motif (PAM) to be searched for when defining candidate gRNA sequences (most commonly 'NGG' for Cas9 and 'TTTV' for Cas12) QUOTES ("" or '') NEEDED
minGRNAGCContent : 25 # Minimum GC content of gRNA (%)
onTargetMethod : 'azimuth' # Algorithm used to score on-target gRNA activity ('azimuth','cindel'); Doench et al. (2016) scores obtained from gradient-boosted regression trees of SpCas9 data. Kim et al. (2017) scores determined through logistic regression of AsCas12 data.
minOnTargetScore : 35 # Minimum on-target score for gRNAs (numeric, 0 to 100; 35 and 20 are useful cutoffs for azimuth and cindel, respectively)
offTargetMethod : 'cfd' # Algorithm used to score off-target gRNA activity ('cfd','hsu'); CFD scores are more accurate in Cas9-edited mammalian cells (Doench et al., 2016). Hsu et al. (2013) scores are used by crispr.mit.edu and Benchling, and are used as proxies for Cas12. Individual scores are aggregated as per crispr.mit.edu.
minOffTargetScore : 20 # Minimum off-target score for gRNAs (numeric, 0 to 100; 20 and 75 are useful cutoffs for cfd and hsu, respectively)
maxOffTargetHitScore : 50 # Maximum off-target score for gRNAs (numeric, 0 to 100; 50 and 5 are useful cutoffs for cfd and hsu, respectively)
### Misc ###
setCoding : 'Auto' # String defining whether a gene should be treated as 'Coding', 'Noncoding', or 'Auto' (called based on the presence of a stop codon or being notated as an RNA)
### Bulk file handling ###
bulkFile : True # Aggregates all gene outputs into a single file for each output type, containing all gene outputs of that given file type; oligos are sorted by purpose and orientation before gene (True, False)
prefix : '*None*' # If not *None*, adds this prefix to all file, oligo, DNA fragment, and GenBank names (String)
prefixNum : -1 # If prefix is not *None* and a number > -1 is given here, numbers each gene processed consecutively starting at the given value; adds number to prefix on all file, oligo, DNA fragment, and GenBank names (int)