Paul.xu at 20:32, 1 November 2019

2019-11-01T20:32:46Z

New page

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 2637  Odin pgRNA ==

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 TCAGACGAACACGGCATACTTTACGCAGCGC'''GGAGTTCGGTTTTTTAGGAG''TGG'''''TAGTATATACACGAGT ACATACGCCACGTTTTTGCGGGCATGCTTCCAGCGCAGGCACGTTGCTTTTCGCAACTTTGCGAGCACGT GGTTTGCGTACCAGCTGGTTAACTGTTGCCATT

goto [http://chopchop.cbu.uib.no/ 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 ==

[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  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] 2. find [https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3 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 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 TTATTACTCCGGAAAATGGAAGCGACGA'''TTTTGGGTGGCTGGCCGTTAAAAATTTTAACTGCATTTAG'''CCA'''''ACTTAAATTAATGAAAAAAT''GTTATTAATCGTTGAGCTAA'''AGTCATTAGAGATGCTTTGCCCTTAATGTAACCATATCGCAATAAGTTATGTTTTTAAATTGAGGGCATTATTA

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

Go to [https://www.atum.bio/eCommerce/cas9/input 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

 <strike>select #1</strike>

<strike>AAA ATGGAAGCGACGATTTTGGG (TGG) CTG TTT TACCTTCGCTGCTAAAACCC  ACC GAC</strike>

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

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

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

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 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  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] 2. find [https://www.ncbi.nlm.nih.gov/nuccore/NC_000913.3 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 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 TTATTACTCCGGAAAATGGAAGCGACGA'''TTTTGGGTGGCTGGCCGTTAAAAATTTTAACTGCATTTAG'''CC'''A''ACTTAAATTAATGAAAAAAT''GTTATTAATCGTTGAGCTAA'''GAGGGCATTATTATGAA 11. Go to [https://www.atum.bio/eCommerce/cas9/input 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 ==

using genome browser to download ackA gene

ATGTCGAGTAAGTTAGTACTGGTTCTGAACTGCGGTAGTTCTTCACTGAAATTTGCCATCATCGATGCAGTAAATGGTGAAGAGTACCTTTCTGGTTTAGCCGAATGTTTCCACCTGC''CCG'''''AAGCACGTATCAAATGGAAA'''ATGGACGGC'''AATAAACAGGAAGCGGCTTT'''''AGG''TGCAGGCGCCGCTCACAGCGAAGCGCTCAACTTTATCGTTAATACTATTCTGGCACAAAAACCAGAACTGTCTGCGCAGCTGACTGCTATCGGTCACCGTATCGTACACGGCGGCGAAAAGTATACCAGCTCCGTAGTGATCGATGAGTCTGTTATTCAGGGTATCAAAGATGCAGCTTCTTTTGCACCGCTGCACAACCCGGCTCACCTGATCGGTATCGAAGAAGCTCTGAAATCTTTCCCACAGCTGAAAGACAAAAACGTTGCTGTATTTGACACCGCGTTCCACCAGACTATGCCGGAAGAGTCTTACCTCTACGCCCTGCCTTACAACCTGTACAAAGAGCACGGCATCCGTCGTTACGGCGCGCACGGCACCAGCCACTTCTATGTAACCCAGGAAGCGGCAAAAATGCTGAACAAACCGGTAGAAGAACTGAACATCATCACCTGCCACCTGGGCAACGGTGGTTCCGTTTCTGCTATCCGCAACGGTAAATGCGTTGACACCTCTATGGGCCTGACCCCGCTGGAAGGTCTGGTCATGGGTACCCGTTCTGGTGATATCGATCCGGCGATCATCTTCCACCTGCACGACACCCTGGGCATGAGCGTTGACGCAATCAACAAACTGCTGACCAAAGAGTCTGGCCTGCTGGGTCTGACCGAAGTGACCAGCGACTGCCGCTATGTTGAAGACAACTACGCGACGAAAGAAGACGCGAAGCGCGCAATGGACGTTTACTGCCACCGCCTGGCGAAATACATCGGTGCCTACACTGCGCTGATGGATGGTCGTCTGGACGCTGTTGTATTCACTGGTGGTATCGGTGAAAATGCCGCAATGGTTCGTGAACTGTCTCTGGGCAAACTGGGCGTGCTGGGCTTTGAAGTTGATCATGAACGCAACCTGGCTGCACGTTTCGGCAAATCTGGTTTCATCAACAAAGAAGGTACCCGTCCTGCGGTGGTTATCCCAACCAACGAAGAACTGGTTATCGCGCAAGACGCGAGCCGCCTGACTGCCTGA

find spacers

TGCCCGAAGCACGTATCAAATGGAAAAT...GC'''AATAAACAGGAAGCGGCTTT'''AGGTGC ACGGGC'''TTCGTGCATAGTTTACCTTT'''TA...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 primer[[Category:Protocols]]s:'''

ackA-F

AACTGCGGTAGTTCTTCACTG

ackA-R

GTACGATACGGTGACCGATAG

GRNA design - Revision history

Paul.xu at 20:32, 1 November 2019