GRNA design
for E coli
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3748948/
Digest pCas9: Buffer: 1.5 ul pCRISPR (clone ID 977) 200~300 ng BsaI 0.5 ul ddH2O up to 15 ul
Digest for 2 hours at 37°C.
Gel purification of digested pCRISPR
Purify using Qiaquick Gel Extraction Kit.
Cleaning eluted DNA with isopropanol precipitation to remove salts is recommended.
Resuspend DNA in H2O Store in freezer at -20°C.
Oligos:
Oligos synthesised:
5’- AAACNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNG -3’ ||||||||||||||||||||||||||||||| 3’- NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNCAAAA -5’
Resuspend in water, as TE buffer can interfere with ligase.
5’ Phosphorylation of Oligos: Same reaction for both oligos:
Oligo stock 100uM 1 ul of each
10x T4 DNA ligase buffer 5 ul
Sterile water 42 ul
T4 Polynucleotide Kinase 1ul
Incubate at 37°C for 1 hour.
Heat inactivate PNK at 65°C for 20 minutes.
Store phosphorylated oligos at -20°C.
Use fresh ligase buffer if possible.
Annealing Oligos: Add 2.5ul of 1M NaCl to phosphorylation reaction (acts as annealing buffer). Heat at 95°C for 4-5 minutes in heat block. Turn off heat block and allow to cool to room temperature. Dilute annealed oligos 10x (check concentration in nanodrop). Store products at -20°C.
Ligation: Annealed oligos 1ul (water in control)
BsaI digested pCRISP ~20ng
10x T4 DNA ligase buffer 2ul
T4 ligase 1ul
ddH2O up to 20ul
Incubate at RT for 2 Hours or 16°C overnight.
Transform into DH5alpha cells.
gRNA Design for E coli for knockout rpsL gene using plasmid 2637 Odin pgRNA
[edit]use genome browser to find rpsL gene:
>gi|556503834|ref|NC_000913.3|:3474171-3474553 Escherichia coli str. K-12 substr. MG1655, complete genome
AGAACCATTAAGCCTTAGGACGCTTCACGCCATACTTGGAACGAGCCTGCTTACGGTCTTTAACGCCGGA
GCAGTCAAGCGCACCACGTACGGTGTGGTAACGAACACCCGGGAGGTCTTTAACACGACCGCCACGGATC
AGGATCACGGAGTGCTCCTGCAGGTTGTGACCTTCACCACCGATGTAGGAAGTCACTTCGAAACCGTTAG
TCAGACGAACACGGCATACTTTACGCAGCGCGGAGTTCGGTTTTTTAGGAGTGGTAGTATATACACGAGT
ACATACGCCACGTTTTTGCGGGCATGCTTCCAGCGCAGGCACGTTGCTTTTCGCAACTTTGCGAGCACGT
GGTTTGCGTACCAGCTGGTTAACTGTTGCCATT
goto http://chopchop.cbu.uib.no/
select E. coli str. K12/MG1655
Enter rpsL as Target
search
find #21 GGAGTTCGGTTTTTTAGGAG TGG as sequence for spacer
F Spacer F
GTCCTAGGTATAATACTAGT GGAGTTCGGTTTTTTAGGAG GTTTTAGAGCTAGAAATAGC
Forward primer
GTCCTAGGTATAATACTAGT + Spacer + GTTTTAGAGCTAGAAATAGC
Reverse Primer
The reverse primer should just be the Reverse complement of the Forward Primer
GCTATTTCTAGCTCTAAAAC + Spacer + ACTAGTATTATACCTAGGAC
gRNA Design for inserting GFP gene into E coli ybhH-ybhD region using plasmid 2637 Odin pgRNA
[edit]https://www.the-odin.com/advanced-crispr-gene-editing-in-bacteria/
Find safe harbor genome location
1, go to NCBI, https://www.ncbi.nlm.nih.gov/genome/?term=e+coli
2. find https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3
3. click Graphics, https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3?report=graph
4. Search ybhH, click on the search result, close the pop up window, then scroll left
5. Will see ybhH gene, click on the gene you will see browser reload the ybhH gene
6. Safe harbor is between ybhD & ybhH
7. click zoom in (+) icon, will see sequence
8. select range
9. then download-->Download sequence-->FASTA (all selection)
downloaded file:
ybhH-ybhD region we can identify a number of targets let’s pick something in the middle
>gi|556503834|ref|NC_000913.3|:800575-800759 Escherichia coli str. K-12 substr. MG1655 TTATTACTCCGGAAAATGGAAGCGACGATTTTGGGTGGCTGGCCGTTAAAAATTTTAACTGCATTTAGCCAACTTAAATTAATGAAAAAATGTTATTAATCGTTGAGCTAAAGTCATTAGAGATGCTTTGCCCTTAATGTAACCATATCGCAATAAGTTATGTTTTTAAATTGAGGGCATTATTA
find spacer from [1]
Go to https://www.atum.bio/eCommerce/cas9/input
select E coli K12 MG1655,
PAM: NGG
Wild-type Cas9
gRNA offset: 4-10
past the above sequence into textbox
click search
select #1
AAA ATGGAAGCGACGATTTTGGG (TGG) CTG
TTT TACCTTCGCTGCTAAAACCC ACC GAC
So our primers for rolling cycle PCR amplification gRNA would be
Forward Primer ATGGAAGCGACGATTTTGGG
GTCCTAGGTATAATACTAGT + ATGGAAGCGACGATTTTGGG + GTTTTAGAGCTAGAAATAGC
Reverse Primer (
GCTATTTCTAGCTCTAAAAC + CCCAAAATCGTCGCTTCCAT + ACTAGTATTATACCTAGGAC
also need to order a gRNA sequencing primer cacatgcagctcatatgctg
Make template DNA for homologous recombination
To make inserting sequence, the upstream homology arm can be 40bp and have this sequence
F: TTTTGGGTGGCTGGCCGTTAAAAATTTTAACTGCATTTAG(CACAGCTAACACCACGTCGT<--GFP)
R: TTAGCTCAACGATTAATAACATTTTTTCATTAATTTAAGT ( CAAAAAACCCCTCAAGACCC <--GFP)
(ACTTAAATTAATGAAAAAATGTTATTAATCGTTGAGCTAA)
For bacteria and yeast homology arms on each side of 40bp-100bp work pretty well if you are inserting a few kb of DNA. If you are only making single point mutations or changing one or two bases you can go as low as 20bp-30bp. The homology arms work best when there are within 100bp of the cut site but even better if they are within 10bp. If you include the spacer and NGG(CCN) PAM beware because the CRISPR system will also cut your template.
Mouse Rosa26 or H11 locus
Humans Rosa26 or H11 ortholog locus or see http://www.bushmanlab.org/assets/pdf/publications/22129804.pd
Youtube Example for knock-in into safe harbor
[edit]https://www.youtube.com/watch?v=5Rv6aMdKY40
Find safe harbor genome location
1, go to NCBI, https://www.ncbi.nlm.nih.gov/genome/?term=e+coli
2. find https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3
3. click Graphics, https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3?report=graph
4. Search ybhH, click on the search result, close the pop up window, then scroll left
5. will see ybhD gene
6. Safe harbor is between ybhD & ybhH
7. click zoom in (+) icon, will see sequence
8. select range
9. then download-->Download sequence-->FASTA (all selection)
downloaded file:
>gi|556503834|ref|NC_000913.3|:800575-800763 Escherichia coli str. K-12 substr. MG1655, complete genome
TTATTACTCCGGAAAATGGAAGCGACGATTTTGGGTGGCTGGCCGTTAAAAATTTTAACTGCATTTAGCCAACTTAAATTAATGAAAAAATGTTATTAATCGTTGAGCTAAGAGGGCATTATTATGAA
11. Go to https://www.atum.bio/eCommerce/cas9/input
select E coli K12 MG1655,
PAM: NGG
Wild-type Cas9
gRNA offset: 4-10
pastthe above seqeunce into textbox
click search
select #3
TAG CCA ACTTAAATTAATGAAAAAAT GTT
ATC (GGT) TGAATTTAATTACTTTTTTA CAA
Pam
the rev comp sequence of (TGG) TGAATTTAATTACTTTTTTA
top strand: ACTTAAATTAATGAAAAAAT, search this on Snapgene, no match
make rev of ACTTAAATTAATGAAAAAAT --> TAAAAAAGTAATTAAATTCA, search on Snapgene, it matches
copy bottom stran 5'-->3': ATTTTTTCATTAATTTAAGT
past ATTTTTTCATTAATTTAAGT into the following vector sequence
gcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcatatgctggatccttgacagctagctcagtcctaggtataatactagt
gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttgaattc
--->yield--->
gcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcatatgctggatccttgacagctagctcagtcctaggtataatactagt
ATTTTTTCATTAATTTAAGT
gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttgaattc
gRNA Primers:
Top: gtcctaggtataatactagtATTTTTTCATTAATTTAAGTgttttagagctagaaatagc
Bottom: gctatttctagctctaaaacACTTAAATTAATGAAAAAATactagtattatacctaggac
to design knock-in frag with homology arms
F:
TTTTGGGTGGCTGGCCGTTAAAAATTTTAACTGCATTTAG + 21 nt target sequence
R: (3' 21 nt + AACTTAAATTAATGAAAAAATGTTATTAATCGTTGAGCTAA 5')
TTAGCTCAACGATTAATAACATTTTTTCATTAATTTAAGTT + 21 nt target sequence
Trying to Knock out ackA gene
[edit]using genome browser to download ackA gene
ATGTCGAGTAAGTTAGTACTGGTTCTGAACTGCGGTAGTTCTTCACTGAAATTTGCCATCATCGATGCAGTAAATGGTGAAGAGTACCTTTCTGGTTTAGCCGAATGTTTCCACCTGCCCGAAGCACGTATCAAATGGAAAATGGACGGCAATAAACAGGAAGCGGCTTTAGGTGCAGGCGCCGCTCACAGCGAAGCGCTCAACTTTATCGTTAATACTATTCTGGCACAAAAACCAGAACTGTCTGCGCAGCTGACTGCTATCGGTCACCGTATCGTACACGGCGGCGAAAAGTATACCAGCTCCGTAGTGATCGATGAGTCTGTTATTCAGGGTATCAAAGATGCAGCTTCTTTTGCACCGCTGCACAACCCGGCTCACCTGATCGGTATCGAAGAAGCTCTGAAATCTTTCCCACAGCTGAAAGACAAAAACGTTGCTGTATTTGACACCGCGTTCCACCAGACTATGCCGGAAGAGTCTTACCTCTACGCCCTGCCTTACAACCTGTACAAAGAGCACGGCATCCGTCGTTACGGCGCGCACGGCACCAGCCACTTCTATGTAACCCAGGAAGCGGCAAAAATGCTGAACAAACCGGTAGAAGAACTGAACATCATCACCTGCCACCTGGGCAACGGTGGTTCCGTTTCTGCTATCCGCAACGGTAAATGCGTTGACACCTCTATGGGCCTGACCCCGCTGGAAGGTCTGGTCATGGGTACCCGTTCTGGTGATATCGATCCGGCGATCATCTTCCACCTGCACGACACCCTGGGCATGAGCGTTGACGCAATCAACAAACTGCTGACCAAAGAGTCTGGCCTGCTGGGTCTGACCGAAGTGACCAGCGACTGCCGCTATGTTGAAGACAACTACGCGACGAAAGAAGACGCGAAGCGCGCAATGGACGTTTACTGCCACCGCCTGGCGAAATACATCGGTGCCTACACTGCGCTGATGGATGGTCGTCTGGACGCTGTTGTATTCACTGGTGGTATCGGTGAAAATGCCGCAATGGTTCGTGAACTGTCTCTGGGCAAACTGGGCGTGCTGGGCTTTGAAGTTGATCATGAACGCAACCTGGCTGCACGTTTCGGCAAATCTGGTTTCATCAACAAAGAAGGTACCCGTCCTGCGGTGGTTATCCCAACCAACGAAGAACTGGTTATCGCGCAAGACGCGAGCCGCCTGACTGCCTGA
find spacers
TGCCCGAAGCACGTATCAAATGGAAAAT...GCAATAAACAGGAAGCGGCTTTAGGTGC
ACGGGCTTCGTGCATAGTTTACCTTTTA...CGTTATTTGTCCTTCGCCGAAATCCACG
use this one AATAAACAGGAAGCGGCTTT spacer
>gRNA_for_124_-_167_A
TTTCCATTTGATACGTGCTT
>gRNA_for_167_B
AATAAACAGGAAGCGGCTTT
>gRNA_for_778_-_818_A
TGCGTCAACGCTCATGCCCA
>gRNA_for_818_B
AAACTGCTGACCAAAGAGTC
>gRNA_for_779_-_818_A
TTGCGTCAACGCTCATGCCC
>gRNA_for_818_B
AAACTGCTGACCAAAGAGTC
gRNA Primers
ackA-gRNA-F
gtcctaggtataatactagtAATAAACAGGAAGCGGCTTTgttttagagctagaaatagc
ackA-gRNA-R
gctatttctagctctaaaaAAAGCCGCTTCCTGTTTATTcactagtattatacctaggac
PCR primers:
ackA-F
AACTGCGGTAGTTCTTCACTG
ackA-R
GTACGATACGGTGACCGATAG