def delGene(geneName, cutsite): # This function asks user for a chromosomal locus, a region to be deleted, a suitable CRIPSR cutsite # and outputs oligos for cloning of a pL308 Cas9-gRNA vector, and ones for generating a donor DNA # to delete the unwanted chromosomal region. Primers Lup+Rdown produce a 1kb band if deletion was # successful. # part of yCRISPRv3 by [email protected] #GeneName=input("Name, using quotes: ") #cutsite=input("20-mer cut sequence, using quotes: ").upper() locus = genomicData[geneName][0] deletion = genomicData[geneName][1] deletion = Seq(deletion) if deletion.find(cutsite)==-1: if deletion.reverse_complement().find(cutsite)==-1: print ("WARNING: Guide 20-mer sequence not found in deletion region.") locus=Seq(locus) index=locus.find(deletion) # index gives the start position within locus of the string deletion. # now we delete the deletion region to redefine a newlocus: newlocus=locus[0:index]+locus[index+len(deletion):] # note that since index starts at 0, a value of n points to, in the newlocus, # the first nt after the deletion. So we define the newlocus as above. Note too # that a string of len=40 ends at an index of 39--so we pick up at index+len-1. Lup=newlocus[index-500:index-470] Rdown=newlocus[index+469:index+499].reverse_complement() Rtemp1 = newlocus[:index].reverse_complement() Rtemp2 = newlocus[index:].reverse_complement() rPrimer, rLength = getPrimer(Rtemp1) lPrimer, lLength = getPrimer(newlocus[index:]) Rup = getOverhang(Rtemp2, rLength) + rPrimer Ldown = getOverhang(newlocus[:index], lLength) + lPrimer cutSequence=Seq("cgggtggcgaatgggacttt")+cutsite+Seq("gttttagagctagaaatagc") seqprimer=Seq("gacttt")+cutsite print("cut" + GeneName + " " + cutSequence) print("seq" + GeneName + " " + seqprimer) print("Lup" + GeneName + "del" + " " + Lup) print("Rup" + GeneName + "del" + " " + Rup) print("Ldown" + GeneName + "del" + " " + Ldown) print("Rdown" + GeneName + "del" + " " + Rdown) return Ldown, Rup
def cutOligos(GeneName, cutsite, DNA):
# This function asks user for a target cut site, and generates oligos to clone that cut site into
# a CRISPR plasmid, and to screen for positive clones containing that cut site. It may also generate
# universal homology regions to integrate or otherwise alter that target region.
#
# The current cut site architecture is: >>>>> YtRNAp-HDV ribozyme- >20nt< -gRNA <<<<<
GeneName=input("Name, using quotes: ")
cutsite=input("20-mer cut sequence, using quotes: ")
DNA=input("Locus sequence +/- a few kb, using quotes: ")
if DNA.find(cutsite)==-1: # If cutiste sequence found in ANTISENSE
DNA=Seq(DNA).reverse_complement() # then reverse DNA, and turn it into a string
index=DNA.find(cutsite)+16 # index gives the start position of the string, e.g., 0.
# we add 16 since index+0=start of 20-mer, so index+16=cut site,
# 3 nt before last of 20mer
Lup=DNA[index-520:index-490] # This primer binds 500bp upstream of cut site
cutSequence=Seq("cgggtggcgaatgggacttt")+cutsite+Seq("gttttagagctagaaatagc")
seqprimer=Seq("gacttt")+cutsite
print("cut" + GeneName + " " + cutSequence)
print("Lcolony" + GeneName + " " + seqprimer)
print("Lup" + GeneName + " " + Lup)
def getSequences(geneName):
from intermine.webservice import Service
template = service.get_template('Gene_GenomicDNA')
rows = template.rows(
E = {"op": "LOOKUP", "value": geneName, "extra_value": "S. cerevisiae"}
)
count = 0
for row in rows:
geneSeq = Seq(row["sequence.residues"])
locusSeq = Seq(row["chromosome.residues.locus"])
index = locusSeq.find(geneSeq)
locusSeq = locusSeq[index-1000:locusSeq]
# Reduce locusSize so it is only +/- 1 kbp of geneSeq
break
return geneSeq, locusSeq
def insertGene(locusName, cutsite, insertName):
# This function asks user for a target cut site, a target locus, and a DNA sequence to be inserted.
# The function outputs primers to allow one to PCR the insert with homology to the cut site. The final
# integration happens 17nt from the cut site in both direction, such that a 34nt region is deleted.
# The current cut site architecture is: >>>>> YtRNAp-HDV ribozyme- >20nt< -gRNA <<<<<
"""
locusName=input("Locus name, using quotes: ")
cutsite=input("20-mer cut sequence, using quotes: ").upper()
insertName=input("Name of gene to be inserted, using quotes: ")
"""
locus = genomicData[locusName][0]
insert = genomicData[locusName][1]
if locus.find(cutsite)==-1: # If cutsite sequence found in ANTISENSE
locus=Seq(locus).reverse_complement() # then reverse DNA, and turn it into a string
index=locus.find(cutsite) # index gives the start position of cutsite within locus
Rtemp1 = locus[:index].reverse_complement()
Rtemp2 = insert.reverse_complement()
rPrim, rLength = getPrimer(Rtemp1)
lPrim, lLength = getPrimer(locus[index+34:])
Rup = getOverhang(Rtemp2, rLength) + rPrim # This is the sequence to the left of the insertion
Ldown = getOverhang(insert, lLength) + lPrim # This is the sequence to the right of the insertion
Lhom=locus[index-60:index]
Rhom=Seq(locus[index+34:index+94]).reverse_complement()
print("L_" + insertName + "_:" + locusName + " " + Lprimer)
print("R_" + insertName + "_:" + locusName + " " + Rprimer)
print("Lext:" + locusName + " " + Lhom)
print("Rext:" + locusName + " " + Rhom)
return Rup, Ldown
