Supplementary Data
REPLACR-mutagenesis, a one-step method for site-directed mutagenesis by recombineering
Ashutosh Trehan 1, Michał Kiełbus 4, Jakub Czapinski 4, Andrzej Stepulak 4, Ilpo Huhtaniemi 1,2 and Adolfo Rivero-Müller 1,3,4*
1
Department of Physiology, Institute of Biomedicine, University of Turku, Turku,
Finland 2
Department of Surgery and Cancer, Institute of Reproductive and Developmental
Biology, Hammersmith Campus, Imperial College London, London, United Kingdom 3
Faculty of Natural Sciences and Technology, Åbo Akademi University, Turku,
Finland 4
Department of Biochemistry and Molecular Biology, Medical University of Lublin,
Lublin, Poland.
*Corresponding author Email:
[email protected] (ARM)
1
Homology 2 bp
ScaI_digested PCR product
_
_
_
_
+
_
_
_
_
_ 366 bp 190 bp
_
_
+
+
_
_
_ 366 bp 190 bp
366 bp 190 bp _
_
+
+
_
+
+ 366 bp 190 bp
366 bp 190 bp _
17 bp
_
366 bp 190 bp
_ 14 bp
_
366 bp 190 bp
_ 11 bp
_
366 bp 190 bp _
8 bp
Molecular Weight Marker 366 bp 190 bp
366 bp 190 bp _
5 bp
Undigested PCR product
+
+
_
+
_
+
_
366 bp 190 bp
366 bp 190 bp +
+
_
+
+
+
+
+
Supplementary Figure S1. PCR products with homology at their termini ranging from 2 bp to 17 bp were transformed in Red/ET bacteria and the resulting bacterial colonies were picked for colony PCR. The mutated plasmids should yield 366 bp long PCR products (not digested by ScaI), whereas the wild-type (WT) background should yield 364 bp long PCR products which upon ScaI digestion gives 190 bp and 174 bp products (indistiguishable as a single band). The mutated plasmids are marked as “+” while the LHCGR_WT background is marked as “-”. The data is presented for one representative experiment with three independent repeats.
2
LHCGR_Asn291Ser LHCGR_Val454Ile FSHR_Ala444Thr Flexible domain (60 nt addition)
REPLACR-mutagenesis Efficiency (%)
100
β2AR_Asp130Asn β2AR_Tyr350Ala
84 %
75
LHCGR_1850delG LHCGR_Lys12-Leu15del CRY2_NLS (45 nt addition)
50
25
0
2
5
8
11
14
17
20
Base pair homology
23
27
30
Supplementary Figure S2. Mutants made using homology longer than 17 bp (shown in red) by REPLACR-mutagenesis show very similar efficiencies to those made using a 17 bp homology (mean ± SEM), thereby demostrating that a 17 bp homology at the ends of PCR termini is sufficient for mutagenesis. The mutants with 20, 23 and 30 bp homology are marked with an arrow and their efficiencies are mentioned in Table 1. The data for 2 bp to 17 bp homology is the same as mentioned in Figure 2a.
3
14 bp
ScaI_digested PCR product
+
+
+
_
+
_
+ 366 bp 190 bp
366 bp 190 bp + 366 bp 190 bp
17 bp
366 bp 190 bp
+
+
+
+
+
+
+ 366 bp 190 bp
+
+
+
+
+
+
+
+ 366 bp 190 bp
+
_
+
+
+
+
_
_
GeneArt Seamless Cloning
14 bp
Molecular Weight Marker 366 bp 190 bp
366 bp 190 bp +
17 bp
Undigested PCR product
Gibson Assembly
Molecular Weight Marker
Homology
Supplementary Figure S3. PCR products with 14 bp and 17 bp homology at their ends were used for recombination using Gibson asembly and GeneArt Seamless cloning commercial kits. The resulting bacterial colonies containing circular plasmids were used for colony PCR. The expected PCR products (366 bp for plasmids with mutated ScaI site and 364 bp for LHCGR_WT background with intact ScaI site) were subjected to ScaI digestion. The PCR products from mutated plasmids (366bp) are not digested (marked as “+”) while LHCGR_WT background results in 190 bp and 174 bp products (seen as a single band and marked as “-”). The presented data is for one representative experiment that has been independently repeated three times.
4
a. LHCGR_Asn291Ser mutation [A −> G]
b. LHCGR_Val454Ile mutation [G −> A].
LHCGR_WT
T T GC C A A C A A A A G A A C A G A A T T T T T C A C A T T C C A T T
LHCGR_WT
C A A G T G A A C T T T C T GT C T A C A C C C T C A C C G T C A T C A 10
LHCGR_Asn291Ser
LHCGR_Val454Ile
T T G C CA A C A A A A G A A C A G A G T T T T T CA C A T T C CA T T
c. FSHR_Ala444Thr mutation [G −> A]
CAA GT GA A CT T T CT AT C T A CA C C C T CAC C G T C AT C A F S
d. FSHR_Gly70Ala mutation [G −> C]
FSHR_WT
FSHR_WT
A A C T G G G GC A G G C T G T G AT G C T G C T G G C T T T T T C A C
T GC A T T T T CA G G A T T T G G G G A C C T G G A G A A A A T A G A
FSHR_Gly70Ala
FSHR_Ala444Thr
T GC A T T T T CA G G A T T T G C G G A C C T G G A G A A A A T A G A
A A C T G G G G C A G G C T G T G A T A C T G C T G G C T T T T T CA C
f. β2AR_Asp130Asn mutation [G −> A]
e. β2AR_Asp79Asn mutation [G −> A]
β2AR_WT
β2AR_WT
C A C T G G C C T GT G C T G A T C T G GT C A T G G G C C T G G C A G
T G T G C GT G A T C GC A G T G G AT C G C T A C T T T G C CA T T A
β2AR_Asp79Asn
β2AR_Asp130Asn
C A C T G G C C T G T G C T A A T C T G G T CA T G G G C C T G G C A G W 10
T G T G C G T G A T C GC A G T G A A T C GC T A C T T T G C CA T T A
g. β2AR_Cys341Gly mutation [T −> G]
h. β2AR_Tyr350Ala mutation [TA −> GC] β2AR_WT
β2AR_WT
C G C A G GT C T T C T T T G A A G G C C T A T G G G A A T G G C T A C
G C C T T C C A G G A G C T T C T GT G C C T G C G C A G GT C T T C T
β2AR_Tyr350Ala
β2AR_Cys341Gly
C G C A G GT C T T C T T T G A A G G C C G C T G G G A A T G GC T A C
G C C T T C C A G G A G C T T C T G G G C C T G C G C A G GT C T T C T
Supplementary Figure S4. Point substitutions made using REPLACR-mutagenesis. The nucleotides changed are marked with an arrow and are highlighted. The original wild type (WT) sequences are on top and the mutated sequences in the bottom row, with gene name followed by the associated mutation. 5
a.
Deleted Nucleotide T A T
C C CA T
C A A T
T
C T
T
G T
LHCGR_WT
G C CA A T
C CA T
T
T
C T
GT
A T
G
LHCGR_1850delG T A T
C C CA
T
C A A T
T
C T
T
T
G C C A A T
C CA T
T
T
C T
G T A T
G
Sequence after deletion
b.
Deleted Region
LHCGR_WT
G C G C T GC A G C T G C T G A A G C T G C T GC T GC T GC T GC A G C C GC C G C T G C C A
LHCGR_Lys12-Leu15del GC GC T GCAGC T GC T GC T GC T GCA GC C GC C GC T G CCA
c.
Site of Deletion 144 kb
A A G C C G C AG A A GC C C A G T T C GC C G G C C AT G G AC G T C G G T AC C G G T G T T T C T C T G AC C C T
Supplementary Figure S5. Deletion of nucleotides by REPLACR-mutagenesis. (a) Deletion of one nucleotide [G] in LHCGR_WT to create LHCGR_1850delG mutant. (b) Deletion of 12 nucleotides in LHCGR_WT for creating LHCGR_Lys12-Leu15del mutant. (c) A 144 kb DNA sequence was deleted from a human LHCGR BAC clone (RPCI-11-186L7) and the resulting circularized vector was verified for the correct recombination site by sequencing. The site of deletion is marked in the chromatogram.
6
a.
LHCGR_WT T GC A G C T G C T G A A G C T G C T GC T GC T GC T GC A G C C GC C G
LHCGR_Leu10-Gln17Dup A G C T G C T G A A GC T GC T G C T G C T G C T G C A G C T G C T G A A G C T G C T G C T G C T GC T GC A G C C
b.
Duplicated Sequence
Duplicated Sequence
CRY2_NLS
T G CA A A C C T A A A A A G A A A A G A A A A GT A G A C C C G A A G A A A A A G A G G A A G GT T G A AT T C
c.
45 nucleotide addition
C C A A T T C T T T A T A C G G A T T A T A T T C A T G G T G G C G G T G G C T C T G G A G G T G G T G G GT C C G G
60 nucleotide addition
A G G A G G C G G C C G CA A G A TG GA CA A A A A G A C TA TA
Supplementary Figure S6. Additions using REPLACR-mutagenesis. (a) Duplication/addition of 27 nucleotides in wild type (WT) LHCGR to create LHCGR_Leu10-Gln17Dup mutant. (b) Addition of 45 nucleotides (CCTAAAAAGAAAAGAAAAGTAGACCCGAAGAAAAAGAGGAAGGTT) containing a nuclear localization signal (NLS) to the CRY2 gene. (c) 60 nucleotide addition of a flexible domain (ACGGATTATATTCATGGTGGCGGTGGCTCTGGAGGTGGTGGGTCCGGAGGAGGCGGCCGC) to CRY2 gene. 7
MfeI restriction site
Wnt1_Original Vector
A A C A T A A A A T G A A T G C A A T T G T T G T T GT T A A C T
Site of addition
Wnt1_Mutated Vector
A A CA T A A A A T G A A T G C A A T G T T G T T G T T G T T A A C T
Added nucleotides Supplementary Figure S7. Addition of two nucleotides (TG) in the middle of MfeI restriction site (CAATTG, marked with triangle; upper row) in the original Wnt1 targeting vector to generate a mutated Wnt1 targeting vector (CAATGTTG).
8
Deleted Sequence: AATTAGTTGTTTTCAAAGCAAATGAACTAGCGATTAGTCGCTATGACTTAACGGAGCATGAAACCAAGCTAATTTTATGCTGTGTGGCACT ACTCAACCCCACGATTGAAAACCCTACAAGGAAAGAACGGACGGTATCGTTCACTTATAACCAATACGCTCAGATGATGAACATCAGTAG GGAAAATGCTTATGGTGTATTAGCTAAAGCAACCAGAGAGCTGATGACGAGAACTGTGGAAATCAGGAATCCTTTGGTTAAAGGCTTTGA GATTTTCCAGTGGACAAACTATGCCAAGTTCTCAAGCGAAAAATTAGAATTAGTTTTTAGTGAAGAGATATTGCCTTATCTTTTCCAGTTAA AAAAATTCATAAAATATAATCTGGAACATGTTAAGTCTTTTGAAAACAAATACTCTATGAGGATTTATGAGTGGTTATTAAAAGAACTAACAC AAAAGAAAACTCACAAGGCAAATATAGAGATTAGCCTTGATGAATTTAAGTTCATGTTAATGCTTGAAAATAACTACCATGAGTTTAAAAGG CTTAACCAATGGGTTTTGAAACCAATAAGTAAAGATTTAAACACTTACAGCAATATGAAATTGGTGGTTGATAAGCGAGGCCGCCCGACT GATACGTTGATTTTCCAAGTTGAACTAGATAGACAAATGGATCTCGTAACCGAACTTGAGAACAACCAGATAAAAATGAATGGTGACAAAA TACCAACAACCATTACATCAGATTCCTACCTACGTAACGGACTAAGAAAAACACTACACGATGCTTTAACTGCAAAAATTCAGCTCACCAG TTTTGAGGCAAAATTTTTGAGTGACATGCAAAGTAAGCATGATCTCAATGGTTCGTTCTCATGGCTCACGCAAAAACAACGAACCACACT AGAGAACATACTGGCTAAATACGGAAGGATCTGA Site of 944 bp deletion A A AT C A C C T A G A C CA A T T G A G AT G T C C A T G G T C T G G G T T C T T AT G G C T C T T G TA T C T AT CA
Supplementary Figure S8. A 944 bp DNA sequence encoding RepA protein (temperature sensitive repressor) was deleted (sequence specified in rectangular box) in Red/ET plasmid using REPLACR-mutagenesis. The site of deletion is marked in the chromatogram and the resulting sequence was correct, as expected.
9
Supplementary Table S1. Primers used to determine the length of optimal base pair (bp) homology in REPLACR-mutagenesis. The homologous base pairs are highlighted in bold. All primers are 23 bp in length. Homology Forward primer (5’-3’) (bp) 2 ATACTATAACCATGCCATA GACT 5 ATACTATAACCATGCCATA GACT 8 ATACTATAACCATGCCATA GACT 11 ATACTATAACCATGCCATA GACT 14 ATACTATAACCATGCCATA GACT 17 ATACTATAACCATGCCAT AGACT 20 ATACTATAACCATGCCAT AGACT 23 ATACTATAACCATGCCAT AGACT
Reverse primer (5’-3’) ATACTGGCCCTTGGTTTGGGA AT AGTATACTGGCCCTTGGTTTG GG TATAGTATACTGGCCCTTGGT TT GGTTATAGTATACTGGCCCTT GG CATGGTTATAGTATACTGGC CCT TGGCATGGTTATAGTATACT GGC CTATGGCATGGTTATAGTAT ACT AGTCTATGGCATGGTTATAG TAT
Supplementary Table S2. PCR products with homology ends ranging from 2 bp to 17 bp after REPLACR-mutagenesis resulted in below-mentioned efficiencies. The data is presented for one experiment, with three independent repeats. Homology (bp) 2 5 8 11 14 17
Colonies Obtained 13 77 >100 >100 >100 >100
Colonies Screened 7 8 8 8 8 8
Correct Colonies 0 1 2 4 4 7
Efficiency (%) 0 13 25 50 50 88
Supplementary Table S3. Colony PCR conditions for bacterial colonies obtained using PCR products with 2-17 bp homology. Temperature Time Initial denaturation 94 °C 5 min Denaturation 98 °C 30 s Annealing 50 °C 30 s Extension 72 °C 1 min (30 cycles) Final extension 72 °C 5 min
10
Supplementary Table S4. Efficiency comparison of REPLACR-mutagenesis with commercial kits (Gibson Assembly and GeneArt Seamless cloning). PCR products with 14 bp homology at their termini results in lowest efficiency for REPLACR mutagenesis among the methods compared whereas PCR products with 17 bp homology yields similar efficiencies for all the methods. The presented table is for one representative experiment only, with three independent repeats. Homology
Mutagenesis method Gibson assembly
14 bp
GeneArt Seamless Cloning REPLACRmutagenesis Gibson assembly
17 bp
GeneArt Seamless Cloning REPLACRmutagenesis
Colonies Colonies obtained screened
Correct colonies
Efficiency (%)
>100
8
6
75
85
8
8
100
>100
8
4
50
>100
8
8
100
21
8
5
63
>100
8
7
88
Supplementary Table S5. Primers used in this study. Mutated nucleotides in the original plasmids are shown in bold and the homology regions are underlined. Gene and Mutation LHCGR_Asn291Ser
LHCGR_Val454Ile
FSHR_Ala444Thr
FSHR_Gly70Ala
β2AR_Asp79Asn
β2AR_Asp130Asn
Forward (5´-3´) CAAAAGAACAGAGT TTTTCACATTCCATT TCTGAA TATTCGCAAGTGAA CTTTCTATCTACACC CTCACCGTCATCAC
Reverse (5´-3´) AGAAATGGAATGTGAAAA AC
CTGGCAAACTGGGG CAGGCTGTGATACT GCTGGCTTTTTCACT GTCTTTG ATTTTCAGGATTTGC GGACCTGGAGAAAA TAGAGATC TGTGCTAATCTGGTC ATGGGCCTGGCAGT GGTGC GCAGTGAATCGCTA CTTTGCCATTACTTC
ACAGTGAAAAAGCCAGCA GT
11
ATGACGGTGAGGGTGTAG AT
TCTCTATTTTCTCCAGGTC CG CACTGCCAGGCCCATGAC CAGATTAGCACAGGCCAG TGAAGTAATGGCAAAGTA GCGATTCACTGCGATCAC
β2AR_Cys341Gly
β2AR_Tyr350Ala
ACCTTTC CTTCTGGGCCTGCGC AGGTCTTCTTTGAAG GCCTAT AAGGCCGCTGGGAA TGGCTACTCCAGCA ACGGCAAC
LHCGR_1850delG
ATCCCATCAATTCTT TGCCAATCCATTTCT GTATGCA LHCGR_Lys12CAGCTGCTGCTGCTG Leu15del CAGCCGCCG LHCGR_deletion_144kb ATGGACGTCGGTAC (RPCI-11-186L7) CGGTGTTTCTCTGAC CCTGTTTC LHCGR_Leu10TGCTGCTGCTGCAG Gln17Dup CCGCCGCTGCCACG AGCGTAC CRY2_NLS (45 nt addition)
Flexible domain (60 nt addition)
Wnt1 targeting vector
ET/Red Deletion_944bp
AAGAAAAGTAGACC CGAAGAAAAAGAGG AAGGTTGAATTCGG CAGTGGAGAGGGCA CGGTGGCTCTGGAG GTGGTGGGTCCGGA GGAGGCGGCCGCAA GATGGACAAAAAGA CTATAGTT TGTTGTTGTTGTTAA CTTGTTTATTGCAGC TTATAATG GATGTCCATGGTCT GGGTTCTTATGGCTC TTGTATCT
CTTCAAAGAAGACCTGCG CAGGCCCAGAAGCTCCTG GTTGCTGGAGTAGCCATT CCCAGCGGCCTTCAAAGA
TGCATACAGAAATGGATT GGCAA GGCGGCTGCAGCAGCAGC AGCTGCAGCGCCGAGAAC CCGGTACCGACGTCCATG GCCGGCGAACTGGGCTTC T GCTGCAGCAGCAGCAGC AGCTTCAGCAGCTGCAG CAGCAGCAGCAGCTTCAG CA TTTTCTTCGGGTCTACTTT TCTTTTCTTTTTAGGTTTG CAACCATTTTTTCCCAAA CACCACCTCCAGAGCCAC CGCCACCATGAATATAAT CCGTATAAAGAATTGGAG CGTAAGTCTG AGTTAACAACAACAACAT TGCATTCATTTTATGTTTC CAGACCATGGACATCTCA ATTGGTCTAGGTGA
Supplementary Table S6. Sequencing primers for checking the final mutant plasmids. Gene and Mutation Forward Sequencing Reverse Sequencing primer (5´-3´) Primer (5´-3´) ATATAAATTCTGGCTGG TGAAAGCTTGAGATG • LHCGR_Asn291Ser CGTGG GGATCAC • LHCGR_Leu10Gln17Dup • LHCGR_Lys12Leu15del
12
• LHCGR_1850delG • LHCGR_Val454Ile • FSHR_Gly70Ala • FSHR_Ala444Thr • • • • •
β2AR_Asp79Asn β2AR_Asp130Asn β2AR_Cys341Gly β2AR_Tyr350Ala LHCGR_deletion_144 kb (RPCI-11-186L7)
• Wnt1 targeting vector • ET/Red Deletion_944bp
AATTGCTATGTTGCCCCT TG GCTAATTGCCACGTCAT CCT TACAAAGATGATGATGA TAAG AGGCTAGGGGTCAGAGA TCC CTCTCATCGTCCTGGCCA TC
GCCTACATACCTCGCT CTGC TGGGGAAGCAAATAC TGACC CTCAGAGATTTGCCGT CTCC ACCTTGAGGGAGGCA GAAAT ATGATCACCCGGGCCT TATT
GCCCAGATTTCAGGATT GCC GCAGATGGTAGGGGACA AGA
GATGGCCCACAAAGT CTTCC GCTATGTGGCTGTTGG GATT
CGGTCGCTACCATTACC AGT AGCGGAATTTACAGAGG GTCT
GTGGACATCTCTTGGG CACT AGGCTGTCTATGTGTG ACTGT
Supplementary Table S7. PCR conditions for deletion of 144 kb from human LHCGR BAC (RPCI-11-186L7).
Polymerase activation Denaturation Annealing Extension
Temperature 94 °C 98 °C 54 °C 68 °C
Time 2 min 30 s 30 s 36 min (20 cycles)
Supplementary Table S8. PCR conditions for Wnt1 targeting plasmid
Polymerase activation Denaturation Annealing Extension
Temperature 94 °C 98 °C 62 °C 68 °C
Time 2 min 10 s 30 s 27 min (20 cycles)
Supplementary Table S9. PCR conditions for deletion of 944 bp from Red/ET plasmid
Polymerase activation Denaturation Annealing Extension
Temperature 94 °C 98 °C 61 °C 68 °C
13
Time 2 min 10 s 30 s 10 min (25 cycles)
Supplementary Table S10. PCR conditions for β2AR_Asp130Asn
Polymerase activation Denaturation Annealing Extension
Temperature 94 °C 98 °C 64 °C 68 °C
Time 2 min 10 s 30 s 8 min (30 cycles)
Supplementary Table S11. PCR conditions for β2AR_Asp79Asn, β2AR_Cys341Gly and β2AR_Tyr350Ala
Polymerase activation Denaturation Annealing/ Extension Denaturation Annealing/ Extension Denaturation Annealing Extension
Temperature 94 °C 98 °C 70 °C 98 °C 68 °C 98 °C 66 °C 68 °C
Time 2 min 10 s 8 min (5 cycles) 10 s 8 min (5 cycles) 10 s 30 s 8 min (20 cycles)
Supplementary Table S12. PCR conditions for all other mutants
Polymerase activation Denaturation Annealing Extension
Temperature 98 °C 98 °C 56 °C 68 °C
14
Time 2 min 10 s 30 s 1 min/Kb (30 cycles)
