Editing GRNA design (section)

== 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]