Editing GRNA design

for E coli

[https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3748948/ https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3748948/]



[[File:GRNA design.png|600px|GRNA design.png]]

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&nbsp;2637&nbsp; Odin pgRNA ==

use genome browser to find&nbsp;rpsL gene:

>gi|556503834|ref|NC_000913.3|:3474171-3474553 Escherichia coli str. K-12 substr. MG1655, complete genome<br/>AGAACCATTAAGCCTTAGGACGCTTCACGCCATACTTGGAACGAGCCTGCTTACGGTCTTTAACGCCGGA<br/>GCAGTCAAGCGCACCACGTACGGTGTGGTAACGAACACCCGGGAGGTCTTTAACACGACCGCCACGGATC<br/>AGGATCACGGAGTGCTCCTGCAGGTTGTGACCTTCACCACCGATGTAGGAAGTCACTTCGAAACCGTTAG<br/>TCAGACGAACACGGCATACTTTACGCAGCGC'''GGAGTTCGGTTTTTTAGGAG''TGG'''''TAGTATATACACGAGT<br/>ACATACGCCACGTTTTTGCGGGCATGCTTCCAGCGCAGGCACGTTGCTTTTCGCAACTTTGCGAGCACGT<br/>GGTTTGCGTACCAGCTGGTTAACTGTTGCCATT



goto&nbsp;[http://chopchop.cbu.uib.no/ http://chopchop.cbu.uib.no/]

select&nbsp;E. coli str. K12/MG1655

Enter&nbsp;rpsL as&nbsp;Target&nbsp;

search

find #21&nbsp;GGAGTTCGGTTTTTTAGGAG <span style="color:#FF0000;">TGG </span>as sequence for spacer&nbsp;



&nbsp;F&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;Spacer&nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp; &nbsp;F<br/>GTCCTAGGTATAATACTAGT GGAGTTCGGTTTTTTAGGAG GTTTTAGAGCTAGAAATAGC&nbsp;



Forward primer<br/>GTCCTAGGTATAATACTAGT + Spacer + GTTTTAGAGCTAGAAATAGC

Reverse Primer<br/>The reverse primer should just be the Reverse complement of the Forward Primer<br/>GCTATTTCTAGCTCTAAAAC + Spacer + ACTAGTATTATACCTAGGAC





== gRNA Design for inserting GFP gene into&nbsp;E coli ybhH-ybhD region using&nbsp;plasmid&nbsp;2637&nbsp; Odin pgRNA ==

[https://www.the-odin.com/advanced-crispr-gene-editing-in-bacteria/ https://www.the-odin.com/advanced-crispr-gene-editing-in-bacteria/]

Find safe harbor genome&nbsp; location

1, go to NCBI,&nbsp;[https://www.ncbi.nlm.nih.gov/genome/?term=e+coli https://www.ncbi.nlm.nih.gov/genome/?term=e+coli]<br/>2. find&nbsp;[https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3 https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3]<br/>3. click Graphics,&nbsp;[https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3?report=graph https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3?report=graph]<br/>4. Search ybhH, click on the search result, close the pop up window, then scroll left&nbsp;<br/>5. Will see ybhH gene, click on the gene you will see browser reload the&nbsp;ybhH gene<br/>6. Safe harbor is between ybhD & ybhH<br/>7. click zoom in (+) icon, will see sequence<br/>8. select range<br/>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

&nbsp;>gi|556503834|ref|NC_000913.3|:800575-800759 Escherichia coli str. K-12 substr. MG1655 TTATTACTCCGGAAAATGGAAGCGACGA'''TTTTGGGTGGCTGGCCGTTAAAAATTTTAACTGCATTTAG'''CCA'''''ACTTAAATTAATGAAAAAAT''GTTATTAATCGTTGAGCTAA'''AGTCATTAGAGATGCTTTGCCCTTAATGTAACCATATCGCAATAAGTTATGTTTTTAAATTGAGGGCATTATTA

find spacer from&nbsp;[https://www.atum.bio/eCommerce/cas9/input [1]]

Go to&nbsp;[https://www.atum.bio/eCommerce/cas9/input https://www.atum.bio/eCommerce/cas9/input]<br/>select E coli K12 MG1655,&nbsp;<br/>PAM: NGG<br/>Wild-type Cas9<br/>gRNA offset: 4-10<br/>past the above sequence into&nbsp; textbox<br/>click search

<br/><strike>select #1</strike>

<strike>AAA ATGGAAGCGACGATTTTGGG (TGG) CTG<br/>TTT TACCTTCGCTGCTAAAACCC&nbsp; ACC GAC</strike>

<strike>So our primers for rolling cycle PCR amplification gRNA&nbsp;would be&nbsp;</strike>

<strike>Forward Primer ATGGAAGCGACGATTTTGGG<br/>GTCCTAGGTATAATACTAGT + ATGGAAGCGACGATTTTGGG + GTTTTAGAGCTAGAAATAGC</strike>

<strike>Reverse Primer (<br/>GCTATTTCTAGCTCTAAAAC + CCCAAAATCGTCGCTTCCAT + ACTAGTATTATACCTAGGAC</strike>

also need to order a gRNA sequencing primer&nbsp;cacatgcagctcatatgctg

'''Make template DNA for homologous recombination'''

To make inserting sequence, the upstream homology arm can be 40bp and have this sequence

F:&nbsp;TTTTGGGTGGCTGGCCGTTAAAAATTTTAACTGCATTTAG(CACAGCTAACACCACGTCGT<--GFP)

R: TTAGCTCAACGATTAATAACATTTTTTCATTAATTTAAGT ( CAAAAAACCCCTCAAGACCC <--GFP)<br/>&nbsp;(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&nbsp;

Humans Rosa26 or H11 ortholog locus or see [http://www.bushmanlab.org/assets/pdf/publications/22129804.pd http://www.bushmanlab.org/assets/pdf/publications/22129804.pd]



== Youtube Example for knock-in into safe harbor ==

[https://www.youtube.com/watch?v=5Rv6aMdKY40 https://www.youtube.com/watch?v=5Rv6aMdKY40]

Find safe harbor genome&nbsp; location

1, go to NCBI, [https://www.ncbi.nlm.nih.gov/genome/?term=e+coli https://www.ncbi.nlm.nih.gov/genome/?term=e+coli]<br/>2. find [https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3 https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3]<br/>3. click Graphics, [https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3?report=graph https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3?report=graph]<br/>4. Search ybhH, click on the search result, close the pop up window, then scroll left&nbsp;<br/>5. will see ybhD gene<br/>6. Safe harbor is between ybhD & ybhH<br/>7. click zoom in (+) icon, will see sequence<br/>8. select range<br/>9. then download-->Download sequence-->FASTA (all selection)

downloaded file:<br/>>gi|556503834|ref|NC_000913.3|:800575-800763 Escherichia coli str. K-12 substr. MG1655, complete genome<br/>TTATTACTCCGGAAAATGGAAGCGACGA'''TTTTGGGTGGCTGGCCGTTAAAAATTTTAACTGCATTTAG'''CC'''A''ACTTAAATTAATGAAAAAAT''GTTATTAATCGTTGAGCTAA'''GAGGGCATTATTATGAA<br/><br/>11. Go to [https://www.atum.bio/eCommerce/cas9/input https://www.atum.bio/eCommerce/cas9/input]<br/>select E coli K12 MG1655,&nbsp;<br/>PAM: NGG<br/>Wild-type Cas9<br/>gRNA offset: 4-10<br/>pastthe above seqeunce into&nbsp; textbox<br/>click search<br/>select #3

TAG&nbsp; CCA&nbsp; ACTTAAATTAATGAAAAAAT GTT<br/>ATC (GGT) TGAATTTAATTACTTTTTTA CAA<br/>&nbsp; &nbsp; &nbsp;Pam

the rev comp sequence of (TGG) TGAATTTAATTACTTTTTTA&nbsp;<br/>top strand: ACTTAAATTAATGAAAAAAT, search this on Snapgene, no match<br/>make rev of ACTTAAATTAATGAAAAAAT --> TAAAAAAGTAATTAAATTCA, search on Snapgene,&nbsp; it matches<br/>copy bottom stran 5'-->3': ATTTTTTCATTAATTTAAGT<br/>past ATTTTTTCATTAATTTAAGT into the following vector sequence

gcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcatatgctggatccttgacagctagctcagtcctaggtataatactagt

<br/>gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttgaattc

--->yield--->

gcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgacgtctaagaaaccattattatcatgacattaacctataaaaataggcgtatcacgaggccctttcgtctcgcgcgtttcggtgatgacggtgaaaacctctgacacatgcagctcatatgctggatccttgacagctagctcagtcctaggtataatactagt<br/>ATTTTTTCATTAATTTAAGT&nbsp; &nbsp;<br/>gttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgctttttttgaattc

gRNA Primers:<br/>Top: gtcctaggtataatactagtATTTTTTCATTAATTTAAGTgttttagagctagaaatagc<br/>Bottom: gctatttctagctctaaaacACTTAAATTAATGAAAAAATactagtattatacctaggac

<br/>to design knock-in frag with homology arms

F:<br/>TTTTGGGTGGCTGGCCGTTAAAAATTTTAACTGCATTTAG + 21 nt target sequence<br/>R: (3' 21 nt + AACTTAAATTAATGAAAAAATGTTATTAATCGTTGAGCTAA 5')<br/>TTAGCTCAACGATTAATAACATTTTTTCATTAATTTAAGTT + 21 nt target sequence

== Trying to&nbsp;Knock out&nbsp; ackA gene ==

using genome browser to download ackA gene

ATGTCGAGTAAGTTAGTACTGGTTCTGAACTGCGGTAGTTCTTCACTGAAATTTGCCATCATCGATGCAGTAAATGGTGAAGAGTACCTTTCTGGTTTAGCCGAATGTTTCCACCTGC<span style="color:#FF0000;">''CCG''</span>'''AAGCACGTATCAAATGGAAA'''ATGGACGGC'''AATAAACAGGAAGCGGCTTT'''<span style="color:#FF0000;">''AGG''</span>TGCAGGCGCCGCTCACAGCGAAGCGCTCAACTTTATCGTTAATACTATTCTGGCACAAAAACCAGAACTGTCTGCGCAGCTGACTGCTATCGGTCACCGTATCGTACACGGCGGCGAAAAGTATACCAGCTCCGTAGTGATCGATGAGTCTGTTATTCAGGGTATCAAAGATGCAGCTTCTTTTGCACCGCTGCACAACCCGGCTCACCTGATCGGTATCGAAGAAGCTCTGAAATCTTTCCCACAGCTGAAAGACAAAAACGTTGCTGTATTTGACACCGCGTTCCACCAGACTATGCCGGAAGAGTCTTACCTCTACGCCCTGCCTTACAACCTGTACAAAGAGCACGGCATCCGTCGTTACGGCGCGCACGGCACCAGCCACTTCTATGTAACCCAGGAAGCGGCAAAAATGCTGAACAAACCGGTAGAAGAACTGAACATCATCACCTGCCACCTGGGCAACGGTGGTTCCGTTTCTGCTATCCGCAACGGTAAATGCGTTGACACCTCTATGGGCCTGACCCCGCTGGAAGGTCTGGTCATGGGTACCCGTTCTGGTGATATCGATCCGGCGATCATCTTCCACCTGCACGACACCCTGGGCATGAGCGTTGACGCAATCAACAAACTGCTGACCAAAGAGTCTGGCCTGCTGGGTCTGACCGAAGTGACCAGCGACTGCCGCTATGTTGAAGACAACTACGCGACGAAAGAAGACGCGAAGCGCGCAATGGACGTTTACTGCCACCGCCTGGCGAAATACATCGGTGCCTACACTGCGCTGATGGATGGTCGTCTGGACGCTGTTGTATTCACTGGTGGTATCGGTGAAAATGCCGCAATGGTTCGTGAACTGTCTCTGGGCAAACTGGGCGTGCTGGGCTTTGAAGTTGATCATGAACGCAACCTGGCTGCACGTTTCGGCAAATCTGGTTTCATCAACAAAGAAGGTACCCGTCCTGCGGTGGTTATCCCAACCAACGAAGAACTGGTTATCGCGCAAGACGCGAGCCGCCTGACTGCCTGA

find spacers

TGCCCGAAGCACGTATCAAATGGAAAAT...GC'''AATAAACAGGAAGCGGCTTT'''<span style="color:#FF0000;">AGG</span>TGC<br/>ACG<span style="color:#FF0000;">GGC</span>'''TTCGTGCATAGTTTACCTTT'''TA...CGTTATTTGTCCTTCGCCGAAATCCACG



use this one&nbsp;'''AATAAACAGGAAGCGGCTTT&nbsp;'''spacer

>gRNA_for_124_-_167_A<br/>TTTCCATTTGATACGTGCTT<br/>>gRNA_for_167_B<br/>AATAAACAGGAAGCGGCTTT<br/>>gRNA_for_778_-_818_A<br/>TGCGTCAACGCTCATGCCCA<br/>>gRNA_for_818_B<br/>AAACTGCTGACCAAAGAGTC<br/>>gRNA_for_779_-_818_A<br/>TTGCGTCAACGCTCATGCCC<br/>>gRNA_for_818_B<br/>AAACTGCTGACCAAAGAGTC

'''gRNA Primers'''

ackA-gRNA-F

gtcctaggtataatactagtAATAAACAGGAAGCGGCTTTgttttagagctagaaatagc

ackA-gRNA-R

gctatttctagctctaaaaAAAGCCGCTTCCTGTTTATTcactagtattatacctaggac

'''PCR primer[[Category:Protocols]]s:'''

ackA-F

AACTGCGGTAGTTCTTCACTG

ackA-R

GTACGATACGGTGACCGATAG