Editing GRNA design (section)

== 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