CRISPr with plasmid pRCC K - MetabolicEngineeringGroupCBMA/MetabolicEngineeringGroupCBMA.github.io GitHub Wiki
We originally asked the pCAS (https://www.addgene.org/60847) vector from the designer ([email protected]). Unfortunately it was "pending" and not available at the time.
This link is for the email exchange https://mail.google.com/mail/u/0/#search/crisp/KtbxLthZkGjqGBfdKMpdktVkRtbMDzfMqB
I want:
- 2 micron origin of replication
- kanMX4 selection marker
- cas9 and sgRNA in the same plasmid
I made a search on addgene:
I found these two:
- pRCC-K 10255 https://www.addgene.org/81191
- pML104-KanMx4 10959 https://www.addgene.org/83476
and an alternative system CRISPR/Cpf1.
This system has a nuclease that is different from cas9, so the scaffold sequences are different as well as some of the properties. Among them, the nuclease produces sticky ends.
Of the two alternatives above pRCC-K seem more interesting to me. It is based on in-vivo gap repair between two primer tails of a PCR product made from the plasmid and a donor DNA 80-mer oligonucleotide. The pML104-KanMx4 is made for ligating the sgRNA encoding DNA, which I think is less practical.
The pRCC-K is described in Generoso 2016 (pubmed, pdf) supplementary material
The paper describe four different plasmids. Two plasmids with two different marker genes, natMX4 and kanMX4 for one or two sgRNA sequences. The plasmids for two sgRNA sequences seem to be tricky to use as it needs digestion with NotI or EcoRI prior to PCR.
This leaves the pRCC-K as the best alternative (sequence).
The paper describes the deletion of ILV1, LEU4 and LEU9 genes. The primers used are available in Table S1:
>CC-Ilv1_Fw Incorporation of ILV1 protospacer in pRCC_K
GTTACTTTACCCGACGTCCCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG
>CC-Ilv1_Rv Incorporation of ILV1 protospacer in pRCC_K
GGGACGTCGGGTAAAGTAACGATCATTTATCTTTCACTGCGGAG
>DR-Ilv1 Donor for ILV1 deletion
CAAGCCACATTTAAACTAAGTCAATTACACAAAGTTAGTGAACCGACAATTTACTTTATAAATTTACGCAACAACTTGTT
>A1-Ilv1 Control PCR for ILV1 deletion (promoter)
AATTCACTAGCGGCTCCTTG
>A4-Ilv1 Control PCR for ILV1 deletion (terminator)
ATGGCTATGTGGAAGAAGTC
>CC-Leu4/9_Fw Incorporation of LEU4/9 protospacer in pRCC_K
TGTCACAATGACCGTGGTTGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG
>CC-Leu4/9_Rv Incorporation of LEU4/9 protospacer in pRCC_K
CAACCACGGTCATTGTGACAGATCATTTATCTTTCACTGCGGAG
>DR-Leu4 Donor for LEU4 deletion
TACTGTAGACTTTTTCCTTACAAAAAGACAAGGAACAATCGAACTTTTCTGTATTTCAGGACTTATTCGCTTCTATTTAT
>DR-Leu9 Donor for LEU9 deletion
GGATAATACTATCGGCACATTATCATTTAGCCGCGTAGCCTAGAAAGGAGTAGCTTATGATTACTCATGTTATATATATA
>A1-Leu4 Control PCR for LEU4deletion (promoter)
TTGTACAGTAACGGCCAGTC
>A4-Leu4 Control PCR for LEU4deletion (terminator)
TTCGTCACTAACCGCCAAAC
>A1-Leu9 Control PCR for LEU9deletion (promoter)
GGTAACGGTCGTAGTGAATG
>A4-Leu9 Control PCR for LEU9deletion (terminator)
TGTTCTCCCTTCACAAAGTC
For targeting the ILV1, primers CC-Ilv1_Fw and CC-Ilv1_Rv were used to amplify the pRCC-K:
They bind to the circular template in this way:
5GTTACTTTACCCGACGTCCCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG3
||||||||||||||||||||||||||||||||||| tm 55.8 (dbd) 67.5
5CGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG...CTCCGCAGTGAAAGATAAATGATCG3
3GCAAAATCTCGATCTTTATCGTTCAATTTTATTCC...GAGGCGTCACTTTCTATTTACTAGC5
||||||||||||||||||||||||| tm 54.5 (dbd) 69.5
3GAGGCGTCACTTTCTATTTACTAGCAATGAAATGGGCTGCAGGG5
The primers form a PCR product that recombine with itself in-vivo to form a circular sequence:
GTTACTTTACCCGACGTCCCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG_____
CAATGAAATGGGCTGCAGGGCAAAATCTCGATCTTTATCGTTCAATTTTATTCC____ |
||
_____CTCCGCAGTGAAAGATAAATGATCGTTACTTTACCCGACGTCCC ||
| ____GAGGCGTCACTTTCTATTTACTAGCAATGAAATGGGCTGCAGGG ||
|| ||
|| SNR52 promoter ---> ======== sgRNA scaffold ========== ||
||______________________________________________________________________________________||
|________________________________________________________________________________________|
GTTACTTTACCCGACGT-CCC
CAATGAAATGGGCTGCA-GGG
|
cut
site
The sequence GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG is the sgRNA scaffold
sequence necessary for the recognition by cas9. This part is always the same.
The tails of the primers is the spacer sequence needed for the recognition of
the gene by the sgRNA molecule. We can find this in the ILV1 sequence below (in
CAPS):
>ILV1 YER086W SGDID:S000000888
atgtcagctactctactaaagcaaccattatgtacggttgttcggcaaggtaaacagtccaaagtgtctggattgaacc
ttttgagactaaaggctcatttgcacagacaacacctgtcaccttccttgataaaactacactctgaattgaaattgga
tgagctgcaaactgataacacccctgattacgtccgtttagttttaaggtcctctgtatacgatgttattaatgaatct
ccaatctctcaaggtgtaggtttgtcttcccgtctaaacacgaatgtcatcttgaaaagagaagatctattgcctgttt
tctctttcaagcttcgtggtgcctataacatgattgccaagttggacgattctcaaagaaaccagggtgttattgcctg
ttcagctgggaatcatgcccaaggtgtggcctttgctgctaaacacttgaaaatacctgctactatcgttatgcctgtt
tgtacaccatctattaagtatcaaaatgtctcgagattagggtctcaagtcgtcctatatggtaacgattttgacgagg
ctaaggctgaatgtgccaaattggctgaagagcgtggcttgacgaacattcctcctttcgatcatccttatgtcattgc
cggtcaaggtactgtagctatggaaatcctaagacaagtacgtaccgctaataagatcggtgctgtctttgttcccgtc
ggcggtggtggtttaattgctggtattggtgcttatttgaaaagggttgctcctcatatcaaaatcattggtgttgaaa
cttacgatgcggccactttacataattccttgcaacgcaaccagagaactcctttacctgtggtgggtacttttgccga
tggtacgtctgtgcgtatgattggtgaagaaacatttagagtcgcccaacaagtggttgatgaagttgttcttgttaac
actgacgaaatctgtgctgcagtaaaggatatttttgaagatactagaagtattgtagaaccatctggtgccctttcag
tagccggtatgaagaaatacatctctaccgtacatccagaaattgaccacactaaaaacacctatgttcccatcctttc
tggtgctaacatgaactttgatagattaagatttgtttccgaacgtgctgttcttggtgaaggaaaggaagtcttcatg
ttaGTTACTTTACCCGACGTCCCtggtgcgttcaagaaaatgcaaaagatcatccacccaagatctgtcactgaattct
----------------||--PAM
cttaccgttacaatgaacatcgtcatgagtcctctagtgaagtgcccaaggcttacatttacacttctttcagcgtcgt
tgacagagaaaaggaaatcaagcaagttatgcaacagttgaatgctttaggttttgaagctgtggatatctccgataac
gaattggctaaatctcatggtagatacttggttggtggtgcttctaaggttcctaatgaaagaattatttcatttgaat
tccctgaaagaccaggtgccttgactaggttccttggaggcctaagcgattcttggaatcttactttattccattatag
aaaccatggtgccgatatcggtaaggttttagctggtatttccgttcctccaagggaaaacttaaccttccaaaaattc
ttggaagatttaggctacacttatcatgatgaaactgataacactgtttatcaaaaattcttgaaatattaa
The cas9 needs there to be a PAM sequence (NGG for cas9) immediately after the spacer sequence. in the sequence above, we can spot "tgg" after the spacer sequence.
The donor DNA for the ILV1 gene is the following 80-mer oligonucleotide.
LOCUS New_DNA 80 bp ds-DNA linear 07-OCT-2019
DEFINITION .
ACCESSION
VERSION
SOURCE .
ORGANISM .
COMMENT
COMMENT ApEinfo:methylated:1
FEATURES Location/Qualifiers
misc_feature 42..80
/locus_tag="New Feature"
/label="New Feature"
/ApEinfo_label="New Feature"
/ApEinfo_fwdcolor="cyan"
/ApEinfo_revcolor="green"
/ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
width 5 offset 0"
misc_feature 1..41
/locus_tag="New Feature(1)"
/label="New Feature(1)"
/ApEinfo_label="New Feature"
/ApEinfo_fwdcolor="#00ff46"
/ApEinfo_revcolor="green"
/ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
width 5 offset 0"
ORIGIN
1 CAAGCCACAT TTAAACTAAG TCAATTACAC AAAGTTAGTG AACCGACAAT TTACTTTATA
61 AATTTACGCA ACAACTTGTT
//
The features marked corresponds to 40 nucelotides before and 40 nucleotides after the ILV1 open reading frame, marked by features in the sequence below.
LOCUS ILV1_YER086W_SGD 1815 bp ds-DNA linear 07-OCT-2019
DEFINITION .
ACCESSION
VERSION
SOURCE .
ORGANISM .
COMMENT >ILV1 YER086W SGDID:S000000888, chrV:328477..330207+/- 1kb
COMMENT ApEinfo:methylated:1
FEATURES Location/Qualifiers
misc_feature 1774..1812
/locus_tag="New Feature"
/label="New Feature"
/ApEinfo_label="New Feature"
/ApEinfo_fwdcolor="cyan"
/ApEinfo_revcolor="green"
/ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
width 5 offset 0"
misc_feature 3..43
/locus_tag="New Feature(1)"
/label="New Feature(1)"
/ApEinfo_label="New Feature"
/ApEinfo_fwdcolor="cyan"
/ApEinfo_revcolor="green"
/ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
width 5 offset 0"
misc_feature 43..1772
/locus_tag="ILV1_orf"
/label="ILV1_orf"
/ApEinfo_label="ILV1_orf"
/ApEinfo_fwdcolor="#c9ff4b"
/ApEinfo_revcolor="green"
/ApEinfo_graphicformat="arrow_data {{0 1 2 0 0 -1} {} 0}
width 5 offset 0"
ORIGIN
1 TACAAGCCAC ATTTAAACTA AGTCAATTAC ACAAAGTTAG TGATGTCAGC TACTCTACTA
61 AAGCAACCAT TATGTACGGT TGTTCGGCAA GGTAAACAGT CCAAAGTGTC TGGATTGAAC
121 CTTTTGAGAC TAAAGGCTCA TTTGCACAGA CAACACCTGT CACCTTCCTT GATAAAACTA
181 CACTCTGAAT TGAAATTGGA TGAGCTGCAA ACTGATAACA CCCCTGATTA CGTCCGTTTA
241 GTTTTAAGGT CCTCTGTATA CGATGTTATT AATGAATCTC CAATCTCTCA AGGTGTAGGT
301 TTGTCTTCCC GTCTAAACAC GAATGTCATC TTGAAAAGAG AAGATCTATT GCCTGTTTTC
361 TCTTTCAAGC TTCGTGGTGC CTATAACATG ATTGCCAAGT TGGACGATTC TCAAAGAAAC
421 CAGGGTGTTA TTGCCTGTTC AGCTGGGAAT CATGCCCAAG GTGTGGCCTT TGCTGCTAAA
481 CACTTGAAAA TACCTGCTAC TATCGTTATG CCTGTTTGTA CACCATCTAT TAAGTATCAA
541 AATGTCTCGA GATTAGGGTC TCAAGTCGTC CTATATGGTA ACGATTTTGA CGAGGCTAAG
601 GCTGAATGTG CCAAATTGGC TGAAGAGCGT GGCTTGACGA ACATTCCTCC TTTCGATCAT
661 CCTTATGTCA TTGCCGGTCA AGGTACTGTA GCTATGGAAA TCCTAAGACA AGTACGTACC
721 GCTAATAAGA TCGGTGCTGT CTTTGTTCCC GTCGGCGGTG GTGGTTTAAT TGCTGGTATT
781 GGTGCTTATT TGAAAAGGGT TGCTCCTCAT ATCAAAATCA TTGGTGTTGA AACTTACGAT
841 GCGGCCACTT TACATAATTC CTTGCAACGC AACCAGAGAA CTCCTTTACC TGTGGTGGGT
901 ACTTTTGCCG ATGGTACGTC TGTGCGTATG ATTGGTGAAG AAACATTTAG AGTCGCCCAA
961 CAAGTGGTTG ATGAAGTTGT TCTTGTTAAC ACTGACGAAA TCTGTGCTGC AGTAAAGGAT
1021 ATTTTTGAAG ATACTAGAAG TATTGTAGAA CCATCTGGTG CCCTTTCAGT AGCCGGTATG
1081 AAGAAATACA TCTCTACCGT ACATCCAGAA ATTGACCACA CTAAAAACAC CTATGTTCCC
1141 ATCCTTTCTG GTGCTAACAT GAACTTTGAT AGATTAAGAT TTGTTTCCGA ACGTGCTGTT
1201 CTTGGTGAAG GAAAGGAAGT CTTCATGTTA GTTACTTTAC CCGACGTCCC TGGTGCGTTC
1261 AAGAAAATGC AAAAGATCAT CCACCCAAGA TCTGTCACTG AATTCTCTTA CCGTTACAAT
1321 GAACATCGTC ATGAGTCCTC TAGTGAAGTG CCCAAGGCTT ACATTTACAC TTCTTTCAGC
1381 GTCGTTGACA GAGAAAAGGA AATCAAGCAA GTTATGCAAC AGTTGAATGC TTTAGGTTTT
1441 GAAGCTGTGG ATATCTCCGA TAACGAATTG GCTAAATCTC ATGGTAGATA CTTGGTTGGT
1501 GGTGCTTCTA AGGTTCCTAA TGAAAGAATT ATTTCATTTG AATTCCCTGA AAGACCAGGT
1561 GCCTTGACTA GGTTCCTTGG AGGCCTAAGC GATTCTTGGA ATCTTACTTT ATTCCATTAT
1621 AGAAACCATG GTGCCGATAT CGGTAAGGTT TTAGCTGGTA TTTCCGTTCC TCCAAGGGAA
1681 AACTTAACCT TCCAAAAATT CTTGGAAGAT TTAGGCTACA CTTATCATGA TGAAACTGAT
1741 AACACTGTTT ATCAAAAATT CTTGAAATAT TAAACCGACA ATTTACTTTA TAAATTTACG
1801 CAACAACTTG TTAGG
//
The constant parts of the primers were extracted below, the tail1 and tail2 needs to be replaced by a spacer sequence specific for ScFAA1.
>CC-FAA1_Fw
tail1-gttttagagctagaaatagcaagttaaaataagg
>CC-FAA1_Rv
tail2-gatcatttatctttcactgcggag
The paper suggests a web based tool for selecting spacer sequences (https://www.dna20.com/eCommerce/cas9/input). There is now a redirection to this url (https://www.atum.bio/eCommerce/cas9/input) which i used. The best result was ATCCCTGGTGGCGGCGCCTT , according to the scoring (100).
The spacer was incorporated with the rest of the primer like this:
>CC-FAA1_Fw 54-mer
ATCCCTGGTGGCGGCGCCTTgttttagagctagaaatagcaagttaaaataagg
>CC-FAA1_Rv 44-mer
AAGGCGCCGCCACCAGGGATgatcatttatctttcactgcggag