Difference between revisions of "Part:BBa K2060000:Design"
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===Design Notes=== | ===Design Notes=== | ||
− | One of the Cardiff_Wales iGEM instructors Daniel Pass, designed a [https://github.com/passdan/scriptdrop/blob/master/cas9_targeter.py bioinformatic tool]for the selection of appropriate guide RNAs that adhered to the following rules: | + | One of the Cardiff_Wales iGEM instructors Daniel Pass, designed a [https://github.com/passdan/scriptdrop/blob/master/cas9_targeter.py bioinformatic tool]for the selection of appropriate guide RNAs from <i>E.coli</i> ribosomal 16S RNA that adhered to the following rules: |
<html> | <html> | ||
<p> - The 3' end of the DNA target sequence must have a proto-spacer adjacent motif (PAM) sequence (5'-NGG-3'). | <p> - The 3' end of the DNA target sequence must have a proto-spacer adjacent motif (PAM) sequence (5'-NGG-3'). |
Revision as of 16:55, 17 October 2016
CRISPR-Cas9 guide RNA targeting to 16S RNA
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal AgeI site found at 48
- 1000COMPATIBLE WITH RFC[1000]
Design Notes
One of the Cardiff_Wales iGEM instructors Daniel Pass, designed a bioinformatic toolfor the selection of appropriate guide RNAs from E.coli ribosomal 16S RNA that adhered to the following rules:
- The 3' end of the DNA target sequence must have a proto-spacer adjacent motif (PAM) sequence (5'-NGG-3').
- The 20 nucleotides upstream of the PAM sequence will be your targeting sequence (crRNA) and Cas9 nuclease will cleave approximately 3 bases upstream of the PAM.
- The PAM sequence itself is absolutely required for cleavage, but it is NOT part of the sgRNA sequence and therefore should not be included in the sgRNA.
- The target sequence can be on either DNA strand.
From this program we identified the R1 guide sequence which is: GGTGGGGTAACGGCTCACCA
This was added to a conserved DNA scaffold sequence: GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTT
This was synthesised as a single fragment and directly cloned into pSB1C3 using EcoRI and PstI.
Source
E.coli 16S ribosomal RNA