Experiment: CRISPR Last edited: May 9, 2023 6:48 PM Owner: Bercin Cenik Type: csv, python, shell Works?: No
add notes about debugging here.
- Make an excel sheet with the following columns:
#1: geneID
#2: 20bp seed sequence
#3: ”CACCG” for seeds not beginning with a “G”, “CACC” for seeds beginning with a “G”
#4: merge of columns 3&2
#5: “AAAC”
#6: reverse complement of seed sequence
#7: ”C” for seeds not beginning with a “G”, blank for seeds beginning with a “G”
#8: merge of columns 5&6&7
Next, populate these columns.
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Column#2: Get your sgRNAs from the Brunello library. Use my “grep sgRNA from list” protocol for this. For example:
cat broadgpp-brunello-library-corrected.txt | grep -iw 'ZC3H13\|KIAA1429\|METTL3\|METTL14\|ELP1\|FOS\|USP49\|SNRNP25\|OGT\|SRF\|AKIRIN2\|MAPK1\|DDX49\|RIOK2' > methylation.sgRNAs.txt
This is assuming you are in a directory that contains the library (”broadgpp-brunello-library-corrected.txt”). The names of genes need to be separated by “|”, this will pipe the output to the subsequent gene, first escaping the pipe character.
The text file will look like this:
AKIRIN2_41635_CTGGTGAAGCTGGTCCACTG CTGGTGAAGCTGGTCCACTG AKIRIN2 AKIRIN2_41636_GCCGCAGAAGTATCTCCGAA GCCGCAGAAGTATCTCCGAA AKIRIN2 AKIRIN2_41637_GGGGACGCCGGGCTCAACAG GGGGACGCCGGGCTCAACAG AKIRIN2 AKIRIN2_41638_TGTGCATCAGAAGTACAACA TGTGCATCAGAAGTACAACA AKIRIN2 #The third column of these include the seed sequence, #which will be pasted into the new excel sheet’s second column (#2: 20bp seed sequence)
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Column#3: This is the 5’ flank of the duplex oligo. If the sgRNA seed sequence begins with a G, type “CACC” here. If it doesn’t, you will need to add a G manually to create a GC-clamp to increase the efficiency of the oligo. For seeds not beginning with a G, type “CACCG” here.
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Column#4: the 5’ oligo, merge the 3th and 2nd columns by using & on excel, in that order.
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Column#5: 3’ flank. This is always a “CAAA”, but since we are writing 5’ to 3’, type “AAAC” in this column for every entry.
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Column #6: ”revcomp of sgRNA”. These are the reverse complements of the seed sequence. Briefly:
original. ACGTACGT reverse. TGCATGCA complement. TGCATGCA reverse complement. ACGTACGT
Run the “## for getting reverse complements” script shown below. This assumes you have a list of sgRNA seed sequences ("sgRNA_list.csv”) in the directory you are working in, and outputs a reverse complement of those (”rc_sgRNA_list.csv”). Paste the contents of rc_sgRNA_list.csv into column#6 (”revcomp of sgRNA”).
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Column #7: clamp. This is a “C” if the seed does not begin with a G, blank if it does (since we are manually adding the GC if it’s absent, etc).
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Column #8: the 3’ oligo, merge the 5th, 6th and 7th columns with the & operator on excel.
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The final sheet will look like this:
"gene","sgRNA sequence","5'flank","5'oligo for ordering","3'flank","revcomp of sgRNA","clamp","3'oligo for ordering" AKIRIN2_sg1,CTGGTGAAGCTGGTCCACTG,CACCG,CACCGCTGGTGAAGCTGGTCCACTG,AAAC,CAGTGGACCAGCTTCACCAG,C,AAACCAGTGGACCAGCTTCACCAGC AKIRIN2_sg2,GCCGCAGAAGTATCTCCGAA,CACC,CACCGCCGCAGAAGTATCTCCGAA,AAAC,TTCGGAGATACTTCTGCGGC,,AAACTTCGGAGATACTTCTGCGGC AKIRIN2_sg3,GGGGACGCCGGGCTCAACAG,CACC,CACCGGGGACGCCGGGCTCAACAG,AAAC,CTGTTGAGCCCGGCGTCCCC,,AAACCTGTTGAGCCCGGCGTCCCC AKIRIN2_sg4,TGTGCATCAGAAGTACAACA,CACCG,CACCGTGTGCATCAGAAGTACAACA,AAAC,TGTTGTACTTCTGATGCACA,C,AAACTGTTGTACTTCTGATGCACAC
The 4th and 8th columns are the sequences of the oligos which will be ordered.
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Run the script “##for reformatting the sgRNA oligos to an order-ready layout” below. This assumes there is an input.csv in your working directory that has the above 8-column format for all guides, and outputs the sequences in tandem (Forward followed by Reverse for each oligo). The list should look like this:
AKIRIN2_sg1_F CACCGCTGGTGAAGCTGGTCCACTG AKIRIN2_sg1_R AAACCAGTGGACCAGCTTCACCAGC AKIRIN2_sg2_F CACCGCCGCAGAAGTATCTCCGAA AKIRIN2_sg2_R AAACTTCGGAGATACTTCTGCGGC AKIRIN2_sg3_F CACCGGGGACGCCGGGCTCAACAG AKIRIN2_sg3_R AAACCTGTTGAGCCCGGCGTCCCC AKIRIN2_sg4_F CACCGTGTGCATCAGAAGTACAACA AKIRIN2_sg4_R AAACTGTTGTACTTCTGATGCACAC
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Order oligos from IDT (25nm, STD).
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Important note: make sure non-target controls are also in your list of oligos to order.
## for getting reverse complements
import csv
def reverse_complement(seq):
complement = {'A': 'T', 'C': 'G', 'G': 'C', 'T': 'A'}
seq = seq.lstrip('\ufeff') # remove UTF-8 BOM character
rev_seq = seq[::-1]
rev_comp_seq = ''.join([complement[base] for base in rev_seq])
return rev_comp_seq
with open('sgRNA_list.csv', 'r') as csvfile:
reader = csv.reader(csvfile)
sgRNA_list = [row[0] for row in reader]
rc_sgRNA_list = [reverse_complement(sgRNA) for sgRNA in sgRNA_list]
with open('rc_sgRNA_list.csv', 'w', newline='') as csvfile:
writer = csv.writer(csvfile)
for sgRNA in rc_sgRNA_list:
writer.writerow([sgRNA])## for only getting reverse sequences:
import csv
def reverse_sequence(seq):
return seq[::-1]
# Read sgRNA sequences from input CSV file
with open('three_five.csv', 'r') as f:
reader = csv.reader(f)
sgRNA_list = [row[0] for row in reader]
# Compute reverse of each sequence
reverse_sgRNA_list = [reverse_sequence(sgRNA) for sgRNA in sgRNA_list]
# Write reversed sequences to output CSV file
with open('reverse_sgRNAs.csv', 'w', newline='') as f:
writer = csv.writer(f)
for sgRNA in reverse_sgRNA_list:
writer.writerow([sgRNA])## for getting reverse complement of the nontarget controls:
def reverse_complement(seq):
complement = {'A': 'T', 'C': 'G', 'G': 'C', 'T': 'A'}
rev_seq = seq[::-1]
rev_comp_seq = ''.join([complement[base] for base in rev_seq])
return rev_comp_seq
sgRNA_list = ['AAAAAGCTTCCGCCTGATGG',
'AAAACAGGACGATGTGCGGC',
'AAAACATCGACCGAAAGCGT',
'AAAATAGCAGTAAACTCAAC']
rc_sgRNA_list = [reverse_complement(sgRNA) for sgRNA in sgRNA_list]
print(rc_sgRNA_list)##for reformatting the sgRNA oligos to an order-ready layout
import csv
with open('input.csv') as infile, open('output2.csv', 'w', newline='') as outfile:
reader = csv.reader(infile)
writer = csv.writer(outfile, delimiter='\t')
for row in reader:
for i in range(0, len(row), 2):
writer.writerow([row[i], row[i+1]])