Supporting Information
Supplementary Sequences
Sequence S1. Sequence of the SV40NLS-FLAG-bStCas9 construct
ATGGGACCTAAGAAAAAGAGGAAGGTGGCGGCCGCTGACTACAAGGATGACGACGATA
AATCTAGAATGACTAAGCCATACTCAATTGGACTTGATATTGGAACGAATAGTGTTGGAT
GGGCTGTAACAACTGATAATTACAAGGTTCCGTCTAAAAAAATGAAAGTCTTAGGAAATA
CGAGTAAAAAGTATATCAAAAAGAACCTGTTAGGTGTATTACTCTTTGACTCTGGAATCAC
AGCAGAAGGAAGAAGATTGAAGCGTACTGCAAGAAGACGTTATACTAGACGCCGTAATC
GTATCCTTTATTTGCAGGAAATTTTTAGCACAGAGATGGCTACATTAGATGATGCTTTCTT
TCAAAGACTTGACGATTCGTTTTTAGTTCCTGATGATAAACGTGATAGTAAGTATCCGATA
TTTGGAAACTTAGTAGAAGAAAAAGCCTATCATGATGAATTTCCAACTATCTATCATTTAA
GGAAATATTTAGCAGATAGTACTAAAAAAGCAGATTTGCGTCTAGTTTATCTTGCATTGGC
TCATATGATTAAATATAGAGGTCACTTCTTAATTGAAGGAGAGTTTAATTCAAAAAATAAT
GATATTCAGAAGAATTTTCAAGACTTTTTGGACACTTATAATGCTATTTTTGAATCGGATTT
ATCACTTGAGAATAGTAAACAACTTGAGGAAATTGTTAAAGATAAGATTAGTAAATTAGAA
AAGAAAGATCGTATTTTAAAACTCTTCCCTGGGGAGAAGAATTCGGGGATTTTTTCAGAG
TTTCTAAAGTTGATTGTAGGAAATCAAGCTGATTTTAGGAAATGTTTTAATTTAGACGAAA
AAGCCTCCTTACATTTTTCCAAAGAAAGCTATGATGAAGATTTAGAGACTTTGTTAGGTTA
TATTGGAGATGATTACAGTGATGTCTTTCTCAAAGCAAAGAAACTTTATGATGCTATTCTT
TTATCGGGTTTTCTGACTGTAACTGATAATGAGACAGAAGCACCTCTCTCTTCTGCTATG
ATAAAGCGATATAATGAACACAAAGAAGATTTAGCGTTACTAAAGGAATATATAAGAAATA
TTTCACTAAAAACGTATAATGAAGTATTTAAAGATGACACCAAAAATGGTTATGCTGGTTA
TATTGATGGAAAAACAAATCAGGAAGATTTCTACGTATATCTAAAAAAACTATTGGCTAAA
TTTGAAGGTGCGGATTATTTTCTTGAAAAAATTGATCGAGAAGATTTTTTGAGAAAGCAAC
GTACATTTGACAATGGTTCGATACCATATCAGATTCATCTTCAAGAAATGAGAGCAATTCT
TGATAAGCAAGCTAAATTTTATCCTTTCTTGGCTAAAAATAAAGAAAGAATCGAGAAGATT
TTAACCTTCCGAATTCCTTATTATGTAGGTCCACTTGCGAGAGGGAATAGTGATTTTGCC
TGGTCAATAAGAAAACGAAATGAAAAAATTACACCTTGGAATTTTGAGGACGTTATTGACA
AAGAATCTTCGGCAGAGGCCTTCATTAATCGAATGACTAGTTTTGATTTGTATTTGCCAGA
1
AGAGAAGGTACTTCCAAAGCATAGTCTCTTATACGAAACTTTTAATGTATATAATGAATTA
ACAAAAGTTAGATTTATTGCCGAAAGTATGAGAGATTATCAATTTTTAGATAGTAAGCAGA
AGAAAGATATTGTTAGACTTTATTTTAAAGATAAAAGGAAAGTTACTGATAAGGATATTATT
GAATATTTACATGCAATTTATGGGTATGATGGAATTGAATTAAAAGGCATAGAGAAACAGT
TTAATTCTAGTTTATCTACTTATCACGATCTTTTAAATATTATTAATGATAAAGAGTTTTTGG
ATGATAGTTCAAATGAAGCGATTATCGAAGAAATTATCCATACTTTGACAATTTTTGAAGA
TAGAGAGATGATAAAACAACGTCTTTCAAAATTTGAGAATATATTCGATAAATCCGTTTTG
AAAAAGTTATCTCGTAGACATTACACTGGCTGGGGTAAGTTATCTGCTAAGCTTATTAATG
GTATTCGAGATGAAAAATCTGGTAATACTATTCTTGATTACTTAATTGATGATGGTATTTCT
AACCGTAATTTCATGCAACTTATTCACGATGATGCTCTTTCTTTTAAAAAGAAGATACAGA
AAGCACAAATTATTGGTGACGAAGATAAAGGTAATATTAAAGAGGTCGTTAAGTCTTTGC
CAGGTAGTCCTGCGATTAAAAAAGGTATTTTACAAAGCATAAAAATTGTAGATGAATTGGT
CAAAGTAATGGGAGGAAGAAAACCCGAGTCAATTGTTGTTGAGATGGCTCGTGAAAATC
AATATACCAATCAAGGTAAGTCTAATTCCCAACAACGCTTGAAACGTTTAGAAAAATCTCT
CAAAGAGTTAGGTAGTAAGATACTTAAGGAAAATATTCCTGCAAAACTTTCTAAAATAGAC
AATAACGCACTTCAAAATGATCGACTTTACTTATACTATCTTCAAAATGGAAAAGATATGT
ATACCGGAGATGATTTAGATATTGATAGATTAAGTAATTATGATATTGATCATATTATTCCT
CAAGCTTTTTTGAAAGATAATTCTATTGACAATAAAGTACTTGTTTCATCTGCTAGTAACC
GTGGTAAATCAGATGATGTTCCAAGTTTAGAGGTTGTCAAAAAAAGAAAGACATTTTGGT
ATCAATTATTGAAATCAAAATTAATTTCTCAACGAAAATTTGATAATCTGACAAAAGCTGAA
CGGGGAGGATTGTCACCTGAGGACAAAGCTGGTTTTATTCAACGCCAGTTGGTTGAAAC
ACGTCAAATAACAAAACATGTAGCTCGTTTACTTGATGAGAAATTTAATAATAAAAAAGAT
GAAAATAATAGAGCGGTACGAACAGTAAAAATTATTACCTTGAAATCTACCTTAGTTTCTC
AATTTCGTAAGGATTTTGAACTTTATAAAGTTCGTGAAATCAATGATTTTCATCATGCTCAT
GATGCTTACTTGAATGCCGTTGTAGCAAGTGCTTTACTTAAGAAATACCCTAAACTAGAG
CCAGAATTTGTGTACGGTGATTATCCAAAATACAATAGTTTTAGAGAAAGAAAGTCCGCT
ACAGAAAAGGTATATTTCTATTCAAATATCATGAATATCTTTAAAAAATCTATTTCTTTAGC
TGATGGTAGAGTTATTGAAAGACCACTTATTGAGGTAAATGAGGAGACCGGCGAATCCG
TTTGGAATAAAGAATCTGATTTAGCAACTGTAAGGAGAGTACTCTCTTATCCGCAAGTAAA
2
TGTTGTGAAAAAAGTTGAGGAACAGAATCACGGATTGGATAGAGGAAAACCAAAGGGAT
TGTTTAATGCAAATCTTTCCTCAAAGCCAAAACCAAATAGTAATGAAAATTTAGTAGGTGC
TAAAGAGTATCTTGACCCCAAAAAGTATGGGGGGTATGCTGGAATTTCTAATTCTTTTGC
TGTTCTTGTTAAAGGGACAATTGAAAAAGGTGCTAAGAAAAAAATAACAAATGTACTAGAA
TTTCAAGGTATTTCTATTTTAGATAGGATTAATTATAGAAAAGATAAACTTAATTTTTTACTT
GAAAAAGGTTATAAAGATATTGAGTTAATTATTGAACTACCTAAATATAGTTTATTTGAACT
TTCAGATGGTTCACGTCGTATGTTGGCTAGTATTTTGTCAACGAATAATAAGAGGGGAGA
GATTCACAAAGGAAATCAGATTTTTCTTTCACAGAAGTTTGTGAAATTACTTTATCATGCT
AAGAGAATAAGTAACACAATTAATGAGAATCATAGAAAATATGTTGAGAACCATAAAAAAG
AGTTTGAAGAATTATTTTACTACATTCTTGAGTTTAATGAGAATTATGTTGGAGCTAAAAA
GAATGGTAAACTTTTAAACTCTGCCTTTCAATCTTGGCAAAATCATAGTATAGATGAACTC
TGTAGTAGTTTTATAGGACCTACCGGAAGTGAAAGAAAGGGGCTATTTGAATTAACCTCT
CGTGGAAGTGCTGCTGATTTTGAATTTTTAGGTGTTAAAATTCCAAGGTATAGAGACTATA
CCCCATCATCCCTATTAAAAGATGCCACACTTATTCATCAATCTGTTACAGGCCTCTATGA
AACACGAATAGACCTTGCCAAACTAGGAGAGGGGTAA
Compared with the online Streptococcus thermophilus CRSPR3 Cas9 sequence of strain
DGCC 7710 (GenBank: HQ712120.1), point mutations are indicated with red bases and
codons of missense mutations and synonymous mutations are indicated with yellow and green
colors respectively. The sequences for SV40NLS and FLAG are underlined.
Sequence S2. The RPR1 RNA expression construct in pTrp1
GGTACCGATCTGCCAATTGAACATAACATGGTAGTTACATATACTAGTAATATGGTTCGG
CACACATTAAAAGTATAAAAACTATCTGAATTACGAATTACATATATTGGTCATAAAAATCA
ATCAATCATCGTGTGTTTTATATGTCTCTTATCTAAGTATAAGAATATCCATAGTTAATATT
CACTTACGCTACCTTTTAACCTGTAATCATTGTCAACAGGATATGTTAACGACCCACATTG
ATAAACGCTAGTATTTCTTTTTCCTCTTCTTATTGGCCGGCTGTCTCTATACTCCCCTATA
GTCTGTTTCTTTTCGTTTCGATTGTTTTACGTTTGAGGCCTCGTGGCGCACATGGTACGC
TGTGGTGCTCGCGGCTGGGAACGAAACTCTGGGAGCTGCGATTGGCAGCTCGAGGTCG
ACGGTATCGATAAGCTTGATATCGAATTCCCCCATATCCAACTTCCAATTTAATCTTTCTT
TTTTAATTTTCACTTATTTGCGATACAGAAAGAAAAAAGCGATAGTAACTATTGAATTTTGT
3
TTGGATTTGGTTAGATTAGATATGGTTTCTCTTTATATTTACATGCTAAAAATGGGCTACA
CCAGAGATACATAATTAGATATATATACGCCAGTACACCTTATCGGCCCAAGCCTTGTCC
CAAGGCAGCGTTTTGTTCTTGGAAACGCTGCCCTACACGTTCGCTATGCTTCAAGAACTT
TTCTGAGCACTTCATGATGCATGTTTGTTCCTTATTGGTTAGCTTTGATGTTGTGAAGTCA
TTGACACAGTCTGTGAAACATCTTTCTACCAGATTAGAGTACAAACGCATGAAATCCTTCA
TTTGCTTTTGTTCCACTACTTTTTGGAACTCTTGTTGTTCTTTGGAGTTCAATGCGTCCAT
CTTTACAGTCCTGTCTTATTGTTCTTGATTTGTGCCCCGTAAAATACTGTTACTTGGTTCT
GGCGAGGTATTGGATAGTTCCTTTTTATAAAGGCCATGAAGCTTGAGCTC
Restriction enzyme sites used are indicated with yellow background; the +1 position and
the A, B box are indicated with green color. The RPR1 promoter and terminator are underlined
and the leader sequence is red colored.
Sequence S3. The sequence of Gal4BD-AD expression cassette in pRep.30.SP1
ATGAAGCTACTGTCTTCTATCGAACAAGCATGCGATATTTGCCGACTTAAAAAGCTCAAG
TGCTCCAAAGAAAAACCGAAGTGCGCCAAGTGTCTGAAGAACAACTGGGAGTGTCGCTA
CTCTCCCAAAACCAAAAGGTCTCCGCTGACTAGGGCACATCTGACAGAAGTGGAATCAA
GGCTAGAAAGACTGGAACAGCTATTTCTACTGATTTTTCCTCGAGAAGACCTTGACATGA
TTTTGAAAATGGATTCTTTACAGGATATAAAAGCATTGTTAACAGGATTATTTGTACAAGA
TAATGTGAATAAAGATGCCGTCACAGATAGATTGGCTTCAGTGGAGACTGATATGCCTCT
AACATTGAGACAGCATAGAATAAGTGCGACATCATCATCGGAAGAGAGTAGTAACAAAG
GTCAAAGACAGTTGACTGTATCGCCGGAATTTGTAATACGACTCACTATAGGGCGAGCC
GCCATCATGGAGGAGCAGAAGCTGATCTCAGAGGAGGACCTGCATATGGCCATGGAGG
CCGAATTCCCGGGGATCTAGTCGACCTGCGGCCGCAAATTCTAAACGCTAAAGAGGAAG
AGGACAGGGATCCGACCTGCATATGGCCATGGAGGCCGAATTCATGGATAAAGCGGAA
TTAATTCCCGAGCCTCCAAAAAAGAAGAGAAAGGTCGAATTGGGTACCGCCGCCAATTTT
AATCAAAGTGGGAATATTGCTGATAGCTCATTGTCCTTCACTTTCACTAACAGTAGCAAC
GGTCCGAACCTCATAACAACTCAAACAAATTCTCAAGCGCTTTCACAACCAATTGCCTCC
TCTAACGTTCATGATAACTTCATGAATAATGAAATCACGGCTAGTAAAATTGATGATGGTA
ATAATTCAAAACCACTGTCACCTGGTTGGACGGACCAAACTGCGTATAACGCGTTTGGAA
4
TCACTACAGGGATGTTTAATACCACTACAATGGATGATGTATATAACTATCTATTCGATGA
TGAAGATACCCCACCAAACCCAAAAAAAGAGATTTAA
Not I and BamH I restriction enzyme sites are indicated with yellow background; Gal4 BD
and AD sequences are underlined; the SP1 protospacer and the PAM sequences are red and
blue colored respectively, while the 30 bp homologous SSA arms are indicated with green
background.
Sequence S4. The sequence of humanized StCas9 (hStCas9) with codon optimization
ATGGCGGACTATAAGGACCACGACGGAGACTACAAGGATCATGATATTGATTACAAAGA
CGATGACGATAAGCCCGGGCCAAAGAAGAAGCGGAAGGTCCTACCGGTCACTAAACCA
TACAGCATTGGACTGGACATTGGAACAAACAGCGTGGGGTGGGCCGTGACTACTGACAA
CTACAAAGTGCCATCAAAGAAAATGAAGGTCCTGGGGAACACTTCCAAGAAATACATCAA
GAAAAACCTGCTGGGAGTGCTGCTGTTCGACTCTGGGATTACCGCAGAGGGACGGAGA
CTGAAGAGGACAGCCAGGCGCCGATATACTCGGAGAAGGAACCGCATCCTGTACCTCC
AGGAGATCTTCAGCACAGAAATGGCTACTCTGGACGATGCATTCTTTCAGCGGCTGGAC
GATTCTTTCCTGGTCCCCGACGATAAAAGAGACAGTAAGTATCCTATCTTCGGCAATCTG
GTGGAGGAAAAAGCCTACCACGATGAGTTTCCTACAATCTACCATCTGAGGAAGTACCT
GGCTGACTCCACTAAGAAAGCAGATCTGCGCCTGGTGTATCTGGCACTGGCCCACATGA
TCAAGTACCGGGGCCATTTCCTGATTGAGGGGGAGTTCAACTCTAAGAACAATGACATC
CAGAAAAACTTCCAGGACTTTCTGGATACCTACAATGCCATTTTTGAGTCTGATCTGAGT
CTGGAAAACTCAAAACAGCTGGAGGAAATCGTGAAGGACAAAATTAGCAAGCTGGAGAA
GAAAGATCGCATCCTGAAGCTGTTCCCAGGCGAGAAAAATTCTGGGATCTTCAGTGAATT
TCTGAAGCTGATTGTGGGGAACCAGGCCGACTTCCGGAAGTGCTTTAATCTGGATGAGA
AAGCCAGCCTGCACTTCTCAAAGGAAAGCTACGACGAGGATCTGGAAACTCTGCTGGGA
TATATCGGCGACGATTACAGTGACGTGTTCCTGAAGGCCAAGAAACTGTATGATGCTATT
CTGCTGTCAGGCTTTCTGACCGTGACAGACAACGAGACCGAAGCCCCCCTGAGCTCCG
CTATGATCAAGCGGTACAATGAGCATAAAGAAGATCTGGCTCTGCTGAAAGAGTATATCC
GGAATATTTCCCTGAAGACTTACAACGAAGTGTTTAAAGACGATACCAAGAACGGGTACG
CAGGATATATCGACGGCAAAACAAATCAGGAGGATTTCTACGTGTATCTGAAGAAACTGC
TGGCAAAGTTCGAAGGGGCCGACTATTTTCTGGAGAAAATTGACAGAGAAGATTTCCTG
5
CGGAAGCAGAGAACTTTTGACAACGGCAGCATCCCATACCAGATTCACCTCCAGGAGAT
GAGGGCAATCCTGGATAAACAGGCCAAGTTCTATCCCTTTCTGGCCAAGAACAAAGAGC
GCATCGAAAAGATCCTGACCTTCAGAATCCCCTACTATGTGGGACCTCTGGCTAGAGGC
AATTCTGACTTTGCATGGAGTATCCGAAAACGGAATGAGAAGATTACTCCATGGAACTTC
GAAGACGTGATCGATAAGGAGTCTAGTGCTGAAGCATTCATTAACAGGATGACCTCATTT
GATCTGTACCTGCCAGAGGAAAAAGTGCTGCCCAAGCATAGCCTGCTGTACGAGACCTT
CAACGTGTACAACGAACTGACAAAGGTCCGATTCATCGCCGAGAGCATGCGGGACTATC
AGTTTCTGGATTCCAAGCAGAAGAAAGACATTGTCAGACTGTACTTCAAGGATAAAAGGA
AGGTGACAGACAAGGATATCATTGAGTATCTGCACGCTATCTACGGCTATGACGGGATC
GAGCTGAAAGGCATTGAAAAGCAGTTTAACTCAAGCCTGTCCACTTACCATGACCTGCTG
AATATCATTAACGATAAGGAGTTCCTGGACGATTCCTCTAATGAAGCCATCATTGAGGAA
ATCATTCACACTCTGACCATCTTCGAGGACCGGGAAATGATTAAGCAGAGACTGAGCAA
ATTCGAGAACATCTTTGATAAGTCTGTGCTGAAGAAACTGAGTCGCCGACATTACACCGG
CTGGGGGAAACTGTCCGCCAAGCTGATCAACGGCATCCGGGACGAGAAGTCTGGAAAT
ACAATCCTGGATTATCTGATCGACGATGGCATTAGTAACAGGAATTTCATGCAGCTGATC
CACGACGATGCCCTGAGCTTCAAGAAGAAGATCCAGAAGGCTCAGATCATTGGGGACGA
GGATAAAGGAAACATCAAGGAAGTGGTCAAATCACTGCCAGGCAGCCCCGCCATCAAAA
AGGGCATCCTCCAGTCTATCAAGATTGTGGACGAGCTGGTGAAGGTCATGGGCGGCAG
AAAACCTGAAAGCATCGTGGTCGAGATGGCTAGAGAAAATCAGTACACCAACCAGGGGA
AATCCAACTCTCAGCAGAGGCTGAAGCGCCTGGAGAAATCACTGAAGGAACTGGGAAG
CAAAATCCTGAAGGAGAATATTCCAGCTAAACTGTCCAAGATCGACAACAATGCACTCCA
GAACGATCGCCTGTATCTGTACTACCTCCAGAATGGAAAGGACATGTACACAGGCGACG
ATCTGGACATCGATCGGCTGAGTAACTACGACATTGATCACATCATTCCCCAGGCATTTC
TGAAAGACAATTCCATCGATAACAAGGTGCTGGTCAGTTCAGCCTCAAATAGAGGAAAAA
GCGACGATGTGCCTTCCCTGGAGGTGGTCAAAAAGAGGAAGACATTCTGGTATCAGCTG
CTGAAATCCAAGCTGATCTCTCAGCGAAAGTTTGACAACCTGACTAAAGCTGAGAGAGG
AGGCCTGAGCCCCGAAGATAAGGCAGGCTTTATCCAGAGGCAGCTGGTCGAGACCCGC
CAGATTACAAAACACGTGGCTAGACTGCTGGACGAGAAGTTCAACAACAAGAAGGATGA
AAACAATCGAGCAGTGCGGACCGTCAAGATCATTACTCTGAAAAGTACCCTGGTCTCAC
6
AGTTCCGGAAGGACTTTGAGCTGTACAAAGTGAGAGAAATCAACGACTTCCACCATGCA
CATGATGCCTATCTGAATGCCGTGGTCGCCTCTGCTCTGCTGAAAAAGTACCCTAAACTG
GAGCCAGAGTTCGTGTACGGCGACTATCCAAAGTACAACTCCTTTAGAGAGAGGAAGTC
TGCAACAGAAAAGGTGTACTTCTATAGCAACATCATGAACATCTTCAAGAAGAGCATCTC
ACTGGCCGACGGCAGAGTGATCGAGAGGCCCCTGATTGAAGTCAACGAGGAAACCGGG
GAGAGCGTGTGGAATAAGGAATCAGATCTGGCCACAGTGCGGAGAGTCCTGTCCTACC
CTCAGGTGAACGTGGTCAAAAAGGTCGAGGAACAGAATCACGGGCTGGACCGCGGAAA
ACCAAAGGGCCTGTTTAACGCCAATCTGAGCTCCAAACCCAAGCCTAACTCTAATGAGAA
CCTGGTGGGAGCTAAGGAATACCTGGACCCCAAGAAGTATGGGGGATACGCCGGCATC
AGCAACTCCTTCGCTGTGCTGGTCAAGGGCACCATCGAGAAAGGGGCCAAAAAGAAAAT
TACAAATGTGCTGGAATTTCAGGGAATCAGCATTCTGGACCGCATCAATTACCGAAAAGA
TAAGCTGAACTTCCTGCTGGAGAAAGGCTATAAGGACATTGAGCTGATCATTGAACTGCC
TAAGTACTCCCTGTTCGAGCTGTCCGATGGGTCTAGGCGCATGCTGGCCAGTATCCTGT
CAACCAACAATAAGCGGGGAGAGATCCACAAAGGCAACCAGATTTTCCTGAGCCAGAAG
TTTGTGAAGCTGCTGTACCATGCTAAAAGGATCTCCAACACAATTAATGAGAACCACCGC
AAGTATGTGGAGAATCATAAGAAAGAGTTCGAGGAACTGTTTTACTACATCCTGGAGTTT
AATGAAAACTACGTGGGGGCAAAGAAAAATGGAAAGCTGCTGAACAGCGCCTTCCAGTC
CTGGCAGAATCACTCTATCGACGAGCTGTGCAGCAGCTTCATCGGACCTACTGGCAGTG
AGAGGAAGGGACTGTTTGAACTGACCAGCCGCGGCTCCGCCGCTGATTTCGAGTTTCTG
GGCGTGAAAATCCCTCGCTATCGAGACTACACACCATCAAGCCTGCTGAAGGATGCCAC
TCTGATTCATCAGAGCGTGACTGGGCTGTATGAGACTCGGATTGACCTGGCTAAACTGG
GAGAAGGCAAGCGTCCTGCTGCTACTAAGAAAGCTGGTCAAGCTAAGAAAAAGAAATAA
The sequence for the 3xFLAG tag is underlined and sequences for nuclear localization
signals (NLS) are indicated with green background.
Sequence S5. The sequence of the interrupted eGFP expression cassette in pRep.eGFP
and pInt.eGFP
ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTG
GACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCC
ACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCT
7
GGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGA
CCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAG
CGCAGCGGCCGCGAATTCGGGGATCCTAAGTGACTAACAAGTTCAGCGTGTCCGGCGA
GGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGC
AAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCT
TCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAA
GGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCG
CCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGA
CTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCAC
AACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCG
CCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCC
ATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCC
TGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGC
CGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTCCGGACTCAGATCCACCGGA
TCTAGATAA
The homologous SSA arms of 215 bp eGFP gene sequence are underlined; Not I and
BamH I restriction enzyme sites are indicated with yellow background while the stop codons in
the middle with red background. Positions of eGFP.F/R primers are red colored.
8
Supplementary Figures
Figure S1. Secondary structure of the short chimeric RNA (scRNA) design (Jinek et al.,
2012). The structure was drawn by RNAstructure Version 4.6, not considering the influence of
the guide sequence.
Figure S2. Adaption of the StCas9 system for yeast assay. (A) Western blotting analysis
for the StCas9 expression in yeast. Western blotting assay for the StCas9 protein from yeast
cells was conducted with primary antibody against FLAG. (B) Spontaneous repair frequencies
9
of the yeast surrogate reporters with different SSA arms (20 and 30 bp). Transformation assay
was conducted with the single reporter plasmids only. (C) Examining the engineered StCas9
system in yeast with different surrogate reporters. Transformation assay was conducted with
different plasmid groups. pLeu2: StCas9 expression parental vector; pLeu2-bSthCas9: Cas9
expression vector; pTrp1: sgRNA expression parental vector; pTrp1-SP1.sgRNA.WT: SP1
sgRNA expression vector; pRep.20.SP1 and pRep.30.SP1: reporter vector with 20 and 30 bp
of SSA arms respectively.
Figure S3. Confirmation of the expression of the reporter genes and the restoration of
the Gal4 gene sequence. (A) Confirmation of the reporter genes HIS3 and ADE2 expression
with yeast surrogate reporter pRep.20.SP1. (B) Confirmation of the reporter genes HIS3 and
ADE2 expression with yeast surrogate reporter pRep.30.SP1. For both (A) and (B), left plate:
SD without histidine and adenine; right plate: SD with supplement of histidine and adenine. P:
positive control transformed with pGal4 plasmid; N: negative control transformed with parental
vectors. (i), colonies transformed with the plasmids expressing StCas9 (bStCas9), sgRNA
(sgRNA.WT) and reporter vector with either 20 (A) or 30 (B) bp of SSA arms. (ii), colonies
transformed with the parental vector (pLeu2) of bStCas9 gene expression vector. (iii), colonies
transformed with the parental vector (pTrp1) of sgRNA expression vector. (iv), colonies
10
transformed with both parental vectors (pLeu2 and pTrp1) of bStCas9 gene and sgRNA. The
plates were maintained at 30℃ for 3 days and photographed by ChemiDoc™ MP System. (C)
Confirmation of the reporter gene LacZ expression by β-galactosidase assay. (D) PCR
identification of SSA-mediated Gal4 reporter gene repair in yeast. PCR product was subjected
Not I/BamH I digestion. The PCR templates used in the assay include the plasmid of two
reporter vectors (pRep.20.SP1 and pRep.30.SP1) and pGal4 vector, and yeast DNA extracts
from co-transformation of pRep.20.SP1or pRep.30.SP1 with bStCas9, SP1.gRNA.WT
expression vectors. The two reporter vectors contains Not I and BamH I sites in the middle of
Gal4 gene. Therefore, PCR product from these two reporter vectors after digestion will yield
two bands, while the wild type of Gal4 gene on pGal4 vector generates only one band. The
PCR product of digestion with one band derived from yeast DNA extract suggests
SSA-mediated recombination.
Figure S4. The design of different series of sgRNA mutants for the optimization assay in
yeast. (A) The mutant sequences of the tracrRNA. (B) The sequences for different Loop
patterns. (C) The mutants of the Match II motif sequence. (D) The sequences of sgRNA
11
mutants without Bulge structure. (E) The mutants of the guide sequences. (F) The single
mismatch mutants of guide sequences. Transformation assays for these sgRNA mutants were
carried out with the bStCas9 expression vector and the reporter vector pRep.20.SP1.
Figure S5. Investigation of the Loop and Match II motifs in yeast assay. (A) Effect of
different Loop patterns on the StCas9 activity. (B) Effect of Match II mutants on the StCas9
activity.
Figure S6. Variable Bulge motif patterns may be available for guiding the StCas9
activity. (A) (B) Secondary structures of the sgRNA.Mat-18/21 designs not accounting for the
12
influence of the guide sequence. (C) (D) (E) Secondary structures of the
SP1.sgRNA.Mat-8/21/WT designs. All the structures were drawn by RNAstructure Version 4.6.
Figure S7. Target sequences used in yeast assays for the StCas9 system. (A) Target
sequences derived from different species. For all the targets, surrogate reporters were
constructed with protospacers with 30 bp immediately adjacent to the PAM patterns. (B) The
mutant sequences used for target preference experiments.
13
Figure S8. The StCas9 system functions on targets integrated in yeast genome. (A)
Schematic for the integration of the reporter construct at the HO locus. Primers used for
verification of integration are indicated with arrows. (B) Verification for the integration of
SP1.20/150 reporter constructs. (C) Verification of the integration of ERG6/TPD1/YAP1.20
reporter constructs. PCR analyses were conducted using the primers 5’AD/3’HO with
corresponding genome DNA of positive colonies from different AH109 reporter strains. (D)
Examination of the engineered StCas9 system targeting chromosome locus in the integrated
yeast reporter strains. (E) PCR assay for the SSA-mediated DSB repair of the Gal4 sequence
within the genome. The assay was conducted as explained in Figure S3D.
AH109.SP1.20/150* represent the PCR assay was carried out with genome DNA prepared.
14
Figure S9. Schematic of mammalian cell assay for the engineered StCas9 system. (A)
Illustration of mammalian cell reporter assays for the StCas9 system. The eGFP.F/R primers
used for detection of SSA-mediated repair are indicated with arrows. (B) Strategies used to
express sgRNA in mammalian cells.
15
Figure S10. Functional analysis of the StCas9 nucleases designed for mouse ROSA26
locus targeting. (A) Target sequences from mouse ROSA26 locus. (B) Validation of the
StCas9 nuclease activity on mouse ROSA26 targets in yeast assay. (C) Validation of the
StCas9 nuclease activity on mouse ROSA26 targets in 293T cells. The designed sgRNA.Opti
was tested with both bacterial origin and humanized Cas9.
Figure S11. T7EI nuclease assay for endogenous target mutation induced by the StCas9
system in human 293T cells. 293T cells were transfected with the hStCas9 and sgRNA.Opti
expression vectors and corresponding SSA-RPG selecting reporter vectors, treated with
puromycin for selection and enrichment, and subjected to T7EI nuclease assay. Arrow
16
indicates the expected positions of DNA bands cleaved by T7EI nuclease. Mutation
frequencies (indels (%)) were calculated by measuring the band intensities.
Figure S12. Alignment of different Cas9 orthologs. Including the proteins derived from S.
thermophilus DDGC7710 CRSPR3, S. thermophilus LMD-9 CRISPR3 and S. pyogenes
SF370 (Use by Jinek et al.,2013).
Supplementary Tables
Table S1.
Nucleotide frequencies of the 12 spacer sequences (SP1-12) within the S.
thermophilus DGCC7710 CRISPR3/Cas locus
Position in target
30
29
28
27
26
25
24
23
22
21
20
19
18
16
15
14
13
12
11
10
9
8
7
6
5
4
3
2
Base preference
T
A
T
T
T/G
C
A/T
A/T
A
T
T
A
A/T A/G T/G
A/T
A
A
A
A
T/C
A
A
T/G
T
G/A
T
A
G
A
A
0.3
0.8
0.3
0.0
0.2
0.3
0.5
0.3
0.6
0.3
0.3
0.8
0.4
0.3
0.2
0.4
0.4
0.4
0.4
0.4
0.2
0.4
0.5
0.1
0.1
0.4
0.3
0.4
0.3
0.6
Proportion in SPn
sequences
Wildtype SP1 protospacer
1
T
0.5
0.2
0.5
0.4
0.3
0.1
0.4
0.3
0.1
0.4
0.4
0.1
0.4
0.2
0.3
0.4
0.3
0.3
0.2
0.2
0.3
0.3
0.3
0.4
0.5
0.0
0.3
0.3
0.2
0.2
G
0.1
0.1
0.0
0.3
0.3
0.2
0.0
0.3
0.3
0.3
0.3
0.1
0.1
0.3
0.3
0.0
0.2
0.3
0.3
0.3
0.3
0.3
0.1
0.4
0.2
0.5
0.2
0.2
0.5
0.2
C
0.2
0.0
0.2
0.3
0.2
0.5
0.1
0.1
0.1
0.0
0.0
0.1
0.1
0.2
0.2
0.2
0.1
0.0
0.2
0.1
0.3
0.0
0.1
0.1
0.3
0.1
0.3
0.1
0.1
0.1
A
A
A
T
T
C
T
A
A
A
C
G
C
T
A
A
A
G
A
G
G
A
A
G
A
G
G
A
C
A
T
A
A
T
A
T
Up mutations
Down mutations
17
G
C
C
A
A
T
G
C
C
C
G
G
C
C
C
The nucleotide frequencies occurred with or more than 40% are red colored.
17
T
C
G
A
T
T
G
C
C
Table S2.
Investigation of endogenous target sequences in human 293T cells
Surrogate
Endogenous
reporter
Locus
Target sequence
PCR primers (5’-3’) for
target mutation
repair
T7EI nuclease assay
frequency*
frequency
AAVS1
5’-GGGGCCACTAGGGACAGGATTGGTG-3’
A1.F: GTCTGTGCTAGCTCTTCCAGC
28.43%
15.46%
A1.R: TCTCCCTCCCAGGATCCTCTC
CCR5 a
5’-CACACTTGTCACCACCCCAAAGGTG-3’
30.11%
20.84%
CCR5 b
5’-GACAAGTGTGATCACTTGGGTGGTG-3’
35.19%
26.68%
C5.F: CATCTCTGACCTGTTTTTCCTTC
C5.R: TCATTTCGACACCGAAGCAGAG
PAM sequences within the targets are underlined.
* The endogenous target modification frequencies were generated from the T7EI nuclease
assay of the enriched cell samples with the SSA-RPG surrogate reporter (Refer to Figure
S11).
Table S3. Primers used in the study
Usage
Primer
Sequence (5’-3’)
For yeast assay system:
Cloning bStCas9 for
Y.bStCas9.F
TGCTCTAGAATGACTAAGCCATACTCAATTGG
Y.bStCas9.R
CTAGCGcgtctcGAATTCTTAACCCTCTCCTAGTTTGGC
yeast expression vector
construction
P.RPR1.F
TTGGGTACCGATCTGCCAATTGAACATAACATGG
promoter and terminator for
P.RPR1.R
CCCCTCGAGCTGCCAATCGCAGCTCCCAGAG
yeast RNA expression vector
T.RPR1.F
TCGGAATTCCCCCATATCCAACTTCCAATTTAATC
construction
T.RPR1.R
GTGGAGCTCAAGCTTCATGGCCTTTATAAAAAG
Cloning the RPR1
TCGAGCATCCTGATAAACTGCAAAAGTTTTAGAGCTAGAA
Annealing primers for
CCR5.ScR.F
ATAGCAAGTTAAAATAAGGCTAGTCCGG
generating CCR5.ScRNA
expression sequence
AATTCCGGACTAGCCTTATTTTAACTTGCTATTTCTAGCT
CCR5.ScR.R
fragment
CTAAAACTTTTGCAGTTTATCAGGATGC
Cloning SP1.ScRNA
CAGCTCGAGAAATTCTAAACGCTAAAGAGGAAGAGGACAG
expression sequence
SP1.ScR.F
TTTTAGAGCTAGAAATAGCAAG
(Paired with T.RPR1.R)
CAGCTCGAGGCGTCATCCTCATCCTGATAAACTGCAAAAG
CCR5.sgRwt.F
TTTTAGAGCTGTGTTGTTTC
Cloning sgRNA.WT
CAGCTCGAGTTCCTAGCCACGTCGACATGGAACATCTTTG
expression sequences
ERG6.sgRwt.F
TTTTAGAGCTGTGTTGTTTC
bearing different guide
CAGCTCGAGATGGTATGCTGGCAGTGACGAGATCGTTAGG
sequences instead of SP1
TPD1.sgRwt.F
TTTTAGAGCTGTGTTGTTTC
(Paired with T.RPR1.R)
CAGCTCGAGATTTTGGGCAGTCCTCTTCTGCTTAGTTTCG
YAP1.sgRwt.F
TTTTAGAGCTGTGTTGTTTC
18
Cloning sgRNA mutant
traR-10.R
GGGGAATTCCCGAATCGGTGCCACCTTTTC
traR-20.R
GGGGAATTCGCCACCTTTTCAAGTTGAGTAC
traR-30.R
GGGGAATTCCAAGTTGAGTACGGACTAAGCC
traR-40.R
GGGGAATTCACGGACTAAGCCTTATTTTAAC
traR-50.R
GGGGAATTCCCTTATTTTAACTCGCTGTGTTG
expression sequences with
different tracrRNA length
(Paired with P.RPR1.F)
GAACTGCAGTGTTTTGGAACCATTCGAAACAAC
PstIa.R1
(Paired with P.RPR1.F)
GAACTGCAGTAACCATTCGAAACAACACAGCTC
PstIb.R1
(Paired with P.RPR1.F)
Cloning sgRNA mutant
GAACTGCAGAAACCATTCGAAACAACACAGCG
expression sequences with
PstI.F2
(Paired with T.RPR1.R)
different
Loop patterns
CGCGGATCCTAACCATTCGAAACAACACAGCTC
BamHI.R1
(Paired with P.RPR1.F)
CGCGGATCCAAACCATTCGAAACAACACAGCG
BamHI.F2
(Paired with T.RPR1.R)
Cloning sgRNA mutant
expression sequences with
decreased Match II
Match-03.R1
GAACTGCAGTGTTTTGGCATTCGAAACAACACAGCTC
Match-03.F2
GAACTGCAGACATTCGAAACAACACAGCGAG
Match-06.R1
GAACTGCAGTGTTTTGGTCGAAACAACACAGCTCTAAAACTG
Match-06.F2
GAACTGCAGATCGAAACAACACAGCGAGTTAAAAT
Match-09.R1
GAACTGCAGTGTTTTGGAAACAACACAGCTCTAAAACTGTC
Match-09.F2
GAACTGCAGAAAACAACACAGCGAGTTAAAATAAG
Match-12.R1
GAACTGCAGTGTTTTGGCAACACAGCTCTAAAACTGTCC
Match-12.F2
GAACTGCAGACAACACAGCGAGTTAAAATAAGG
Match-15.R1
GAACTGCAGTGTTTTGGCACAGCTCTAAAACTGTCCTC
Match-15.F2
GAACTGCAGACACAGCGAGTTAAAATAAGGC
Match-18.R1
GAACTGCAGTGTTTTGGAGCTCTAAAACTGTCCTCTTCC
Match-18.F2
GAACTGCAGAAGCGAGTTAAAATAAGGCTTAG
Match-21.R1
GAACTGCAGTGTTTTGGTCTAAAACTGTCCTCTTCCTC
Match-21.F2
GAACTGCAGAGAGTTAAAATAAGGCTTAGTCCG
Motifs
(Match-n.R1 paired with
P.RPR1.F;
Match-n.F2 paired with
T.RPR1.R)
Cloning sgRNA mutant
expression sequences
without Bulge structure
NoB-1.R1
CGCGGATCCTCTAAAACTGTCCTCTTCCTCTTTAG
NoB-1.F2
CGCGGATCCATTAAAATAAGGCTTAGTCCGTAC
NoB-2.R1
CGCGGATCCTTAAAACTGTCCTCTTCCTCTTTAG
NoB-2.F2
CGCGGATCCATAAGGCTTAGTCCGTACTCAAC
NoB-3.R1
CGCGGATCCTTAGCGAGCTAAAACTGTCCTCTTCCTCTTTAG
NoB-3.F2
CGCGGATCCATAGCGAGTTAAAACAAGGCTTAGTC
(NoB-n.R1 paired with
P.RPR1.F;
NoB-n.F2 paired with
T.RPR1.R)
F27.F
CAGCTCGAGTTCTAAACGCTAAAGAGGAAG
Cloning sgRNA mutant
F24.F
CAGCTCGAGTAAACGCTAAAGAGGAAGAGGAC
expression sequences with
F21.F
CAGCTCGAGACGCTAAAGAGGAAGAGGACAG
decreased SP1 guide
F19.F1
CAGCTCGAGGCTAAAGAGGAAGAGGACAG
sequences
F19.F2
CAGCTCGAGCTAAAGAGGAAGAGGACAGTTTTAG
F18.F
CAGCTCGAGCCTAAAGAGGAAGAGGACAGTTTTAG
F17.F
CAGCTCGAGTAAAGAGGAAGAGGACAGTTTTAG
(Paired with T.RPR1.R)
19
Cloning sgRNA mutant
F16.F
CAGCTCGAGAAAGAGGAAGAGGACAGTTTTAG
F15.F
CAGCTCGAGAAGAGGAAGAGGACAGTTTTAGAG
Mis-01.F
CAGCTCGAGAAATTCTAAACGCTAAAGAGGAAGAGGACCGTTTTAG
Mis-02.F
CAGCTCGAGAAATTCTAAACGCTAAAGAGGAAGAGGAGAGTTTTAG
Mis-03.F
CAGCTCGAGAAATTCTAAACGCTAAAGAGGAAGAGGCCAGTTTTAG
Mis-04.F
CAGCTCGAGAAATTCTAAACGCTAAAGAGGAAGAGTAC
Mis-05.F
CAGCTCGAGAAATTCTAAACGCTAAAGAGGAAGATGAC
Mis-06.F
CAGCTCGAGAAATTCTAAACGCTAAAGAGGAAGTGG
Mis-07.F
CAGCTCGAGAAATTCTAAACGCTAAAGAGGAAAAGG
Mis-08.F
CAGCTCGAGAAATTCTAAACGCTAAAGAGGAGGAG
Mis-09.F
CAGCTCGAGAAATTCTAAACGCTAAAGAGGCAGAG
Mis-10.F
CAGCTCGAGAAATTCTAAACGCTAAAGAGTAAG
Mis-11.F
CAGCTCGAGAAATTCTAAACGCTAAAGAAGAAG
Mis-12.F
CAGCTCGAGAAATTCTAAACGCTAAAGCGG
Mis-13.F
CAGCTCGAGAAATTCTAAACGCTAAACAGG
Mis-14.F
CAGCTCGAGAAATTCTAAACGCTAACGAG
Mis-15.F
CAGCTCGAGAAATTCTAAACGCTAGAGAG
Mis-16.F
CAGCTCGAGAAATTCTAAACGCTTAAG
Mis-17.F
CAGCTCGAGAAATTCTAAACGCAAAAG
Mis-18.F
CAGCTCGAGAAATTCTAAACGGTAAAG
expression sequences with
single mismatch in the SP1
guide sequence
(Paired with T.RPR1.R)
SP1F19.sgRopti.F
TCGAGCTAAAGAGGAAGAGGACAGTTTTAGAGCTGTAG
SP1F19.sgRopti.R
GATCCTACAGCTCTAAAACTGTCCTCTTCCTCTTTAGC
ERG6.sgRopti.F
TCGAGGTCGACATGGAACATCTTTGTTTTAGAGCTGTAG
ERG6.sgRopti.R
GATCCTACAGCTCTAAAACAAAGATGTTCCATGTCGACC
TPD1.sgRopti.F
TCGAGGCAGTGACGAGATCGTTAGGTTTTAGAGCTGTAG
TPD1.sgRopti.R
GATCCTACAGCTCTAAAACCTAACGATCTCGTCACTGCC
sgRNA.Opti designs
YAP1.sgRopti.F
TCGAGTCCTCTTCTGCTTAGTTTCGTTTTAGAGCTGTAG
bearing different guide
YAP1.sgRopti.R
GATCCTACAGCTCTAAAACGAAACTAAGCAGAAGAGGAC
sequences instead of
R26A.sgRopti.F
TCGAGACATTGCATGGATTATACAGTTTTAGAGCTGTAG
SP1(G04T)
R26A.sgRopti.R
GATCCTACAGCTCTAAAACTGTATAATCCATGCAATGTC
R26B.sgRopti.F
TCGAGTCTTTCTAGAAGATGGGCGTTTTAGAGCTGTAG
R26B.sgRopti.R
GATCCTACAGCTCTAAAACGCCCATCTTCTAGAAAGAC
R26C.sgRopti.F
TCGAGTGGGCCTATTCTCAGTCCAGTTTTAGAGCTGTAG
R26C.sgRopti.R
GATCCTACAGCTCTAAAACTGGACTGAGAATAGGCCCAC
Annealing primers for
generating
P.ADH1.F1
PCR primers for
BD.R1
CCCGAGCTCATCCTTTTGTTGTTTCCG
CCCGTCGACTAGATCCCCGGGAATTCGGC
generating the
BD.150.F2
CCCGTCGACCTGCGGCCGCaaGGATCCTCATCGGAAGAGAGTAGT
PADH1-Gal4BD-AD-TADH1
Over.150.R2
ATTAATTCCGCTTTATCCATGAATTCGGCCTCCATGGCCA
Over.AD.F3
TGGCCATGGAGGCCGAATTCATGGATAAAGCGGAATTAAT
T.ADH1.R3
CCCGGGCCCAAGCTTGCATGCCGGTAG
reporter construct with SSA
arms of 150 bp
CGCGGATCCTGGCCATGGAGGCCGAATTC
PCR primers for
BD.20.F
(Paired with T.ADH1.R3)
generating Gal4AD-TADH1
constructs with SSA arms of
BD.30.F
CGCGGATCCGACCTGCATATGGCCATG
20
20 or 30 bp
Annealing primers for
generating
(Paired with T.ADH1.R3)
SP1.WT.F
GGCCGCAAATTCTAAACGCTAAAGAGGAAGAGGACAGG
SP1.WT.R
GATCCCTGTCCTCTTCCTCTTTAGCGTTTAGAATTTGC
SP1.P5M4.F
GGCCGCAAATTCTAAACGCTAAAGAGGAAGAGTACAGGGTG
SP1.P5M4.R
GATCCACCCTGTACTCTTCCTCTTTAGCGTTTAGAATTTGC
CCR5.TS.F
GGCCGCGTCATCCTCATCCTGATAAACTGCAAAAGG
CCR5.TS.R
GATCCCTTTTGCAGTTTATCAGGATGAGGATGACGC
ERG6.TS.F
GGCCGCTTCCTAGCCACGTCGACATGGAACATCTTTGG
ERG6.TS.R
GATCCCAAAGATGTTCCATGTCGACGTGGCTAGGAAGC
TPD1.TS.F
GGCCGCATGGTATGCTGGCAGTGACGAGATCGTTAGGG
TPD1.TS.R
GATCCCCTAACGATCTCGTCACTGCCAGCATACCATGC
YAP1.TS.F
GGCCGCATTTTGGGCAGTCCTCTTCTGCTTAGTTTCAG
YAP1.TS.R
GATCCTGAAACTAAGCAGAAGAGGACTGCCCAAAATGC
R26A.TS.F
GGCCGCAATTGCATAACACATTGCATGGATTATACAAGGTG
R26A.TS.R
GATCCACCTTGTATAATCCATGCAATGTGTTATGCAATTGC
R26B.TS.F
GGCCGCTGCAACTCCAGTCTTTCTAGAAGATGGGCGGGAG
R26B.TS.R
GATCCTCCCGCCCATCTTCTAGAAAGACTGGAGTTGCAGC
R26C.TS.F
GGCCGCTGTTCCACATTTGGGCCTATTCTCAGTCCAGGGAG
R26C.TS.R
GATCCTCCCTGGACTGAGAATAGGCCCAAATGTGGAACAGC
fragments of different
target sequences with PAMs
for construction of the
corresponding reporter
vectors
Annealing primers for
generating
SP1.Mu-01.F
GGCCGCAAATTCTAAACGCTAAAGAGGAAGAGGACCGG
SP1.Mu-01.R
GATCCCGGTCCTCTTCCTCTTTAGCGTTTAGAATTTGC
SP1.Mu-02.F
GGCCGCAAATTCTAAACGCTAAAGAGGAAGAGGAGAGG
SP1.Mu-02.R
GATCCCTCTCCTCTTCCTCTTTAGCGTTTAGAATTTGC
SP1.Mu-03.F
GGCCGCAAATTCTAAACGCTAAAGAGGAAGAGGCCAGG
SP1.Mu-03.R
GATCCCTGGCCTCTTCCTCTTTAGCGTTTAGAATTTGC
SP1.Mu-04.F
GGCCGCAAATTCTAAACGCTAAAGAGGAAGAGTACAGG
SP1.Mu-04.R
GATCCCTGTACTCTTCCTCTTTAGCGTTTAGAATTTGC
SP1.Mu-05.F
GGCCGCAAATTCTAAACGCTAAAGAGGAAGATGACAGG
SP1.Mu-05.R
GATCCCTGTCATCTTCCTCTTTAGCGTTTAGAATTTGC
SP1.Mu-06.F
GGCCGCAAATTCTAAACGCTAAAGAGGAAGTGGACAGG
SP1.Mu-06.R
GATCCCTGTCCACTTCCTCTTTAGCGTTTAGAATTTGC
SP1.Mu-07.F
GGCCGCAAATTCTAAACGCTAAAGAGGAAAAGGACAGG
SP1.Mu-07.R
GATCCCTGTCCTTTTCCTCTTTAGCGTTTAGAATTTGC
SP1.Mu-08.F
GGCCGCAAATTCTAAACGCTAAAGAGGAGGAGGACAGG
SP1.Mu-08.R
GATCCCTGTCCTCCTCCTCTTTAGCGTTTAGAATTTGC
SP1.Mu-09.F
GGCCGCAAATTCTAAACGCTAAAGAGGCAGAGGACAGG
SP1.Mu-09.R
GATCCCTGTCCTCTGCCTCTTTAGCGTTTAGAATTTGC
SP1.Mu-10.F
GGCCGCAAATTCTAAACGCTAAAGAGTAAGAGGACAGG
SP1.Mu-10.R
GATCCCTGTCCTCTTACTCTTTAGCGTTTAGAATTTGC
SP1.Mu-11.F
GGCCGCAAATTCTAAACGCTAAAGAAGAAGAGGACAGG
SP1.Mu-11.R
GATCCCTGTCCTCTTCTTCTTTAGCGTTTAGAATTTGC
SP1.Mu-12.F
GGCCGCAAATTCTAAACGCTAAAGCGGAAGAGGACAGG
SP1.Mu-12.R
GATCCCTGTCCTCTTCCGCTTTAGCGTTTAGAATTTGC
fragments of different
SP1 mutant sequences for
construction of the
corresponding reporter
vectors
21
SP1.Mu-13.F
GGCCGCAAATTCTAAACGCTAAACAGGAAGAGGACAGG
SP1.Mu-13.R
GATCCCTGTCCTCTTCCTGTTTAGCGTTTAGAATTTGC
SP1.Mu-14.F
GGCCGCAAATTCTAAACGCTAACGAGGAAGAGGACAGG
SP1.Mu-14.R
GATCCCTGTCCTCTTCCTCGTTAGCGTTTAGAATTTGC
SP1.Mu-15.F
GGCCGCAAATTCTAAACGCTAGAGAGGAAGAGGACAGG
SP1.Mu-15.R
GATCCCTGTCCTCTTCCTCTCTAGCGTTTAGAATTTGC
SP1.Mu-16.F
GGCCGCAAATTCTAAACGCTTAAGAGGAAGAGGACAGG
SP1.Mu-16.R
GATCCCTGTCCTCTTCCTCTTAAGCGTTTAGAATTTGC
SP1.Mu-17.F
GGCCGCAAATTCTAAACGCAAAAGAGGAAGAGGACAGG
SP1.Mu-17.R
GATCCCTGTCCTCTTCCTCTTTTGCGTTTAGAATTTGC
SP1.Mu-18.F
GGCCGCAAATTCTAAACGGTAAAGAGGAAGAGGACAGG
SP1.Mu-18.R
GATCCCTGTCCTCTTCCTCTTTACCGTTTAGAATTTGC
SP1.PAM-1.F
GGCCGCAAATTCTAAACGCTAAAGAGGAAGAGGACAAG
SP1.PAM-1.R
GATCCTTGTCCTCTTCCTCTTTAGCGTTTAGAATTTGC
SP1.PAM-2.F
GGCCGCAAATTCTAAACGCTAAAGAGGAAGAGGACAGAG
SP1.PAM-2.R
GATCCTCTGTCCTCTTCCTCTTTAGCGTTTAGAATTTGC
SP1.PAM-3.F
GGCCGCAAATTCTAAACGCTAAAGAGGAAGAGGACAGGAG
protospacer sequence with
SP1.PAM-3.R
GATCCTCCTGTCCTCTTCCTCTTTAGCGTTTAGAATTTGC
different PAM patterns for
SP1.PAM-4.F
GGCCGCAAATTCTAAACGCTAAAGAGGAAGAGGACAGAAG
Annealing primers for
generating
fragments of SP1
construction of the
SP1.PAM-4.R
GATCCTTCTGTCCTCTTCCTCTTTAGCGTTTAGAATTTGC
corresponding reporter
SP1.PAM-5.F
GGCCGCAAATTCTAAACGCTAAAGAGGAAGAGGACAGGGTG
vectors
SP1.PAM-5.R
GATCCACCCTGTCCTCTTCCTCTTTAGCGTTTAGAATTTGC
SP1.PAM-6.F
GGCCGCAAATTCTAAACGCTAAAGAGGAAGAGGACAGGGAG
SP1.PAM-6.R
GATCCTCCCTGTCCTCTTCCTCTTTAGCGTTTAGAATTTGC
PCR primers for
ADH1P.F
ATCCTTTTGTTGTTTCCGGG
ADH1T.R
AAGCTTGCATGCCGGTAG
KanMX4.F
CGCGAATTCATAACTTCGTATAATGTATG
KanMX4.R
CGCACTAGTGGATCGATCCATAACTTCG
identification of
SSA-mediated Gal4 reporter
gene repair in yeast assay
HO-L.F
CGGGGTACCctaGCTAGCAATTATCCTGGGCACGAGTG
HO-L.R
GGGGTCGACCGCCATTTTAAGTCCAAAG
HO-R.F
TCCCCGCGGCTGGGGGAACAACTTCAC
HO-R.R
CCCGAGCTCCTGTAAGATTCCGCCACAT
P.ADH1.F
GGACTAGTCATCCTTTTGTTGTTTCCGG
T.ADH1.R
TCCCCGCGGAAGCTTGCATGCCGGTAG
PCR primers for
construction of the yeast
integrating reporter vectors
PCR primers for
5'AD
TACCACTACAATGGATGATG
3'HO
GTTACCACAACTCTTATGAG
identification of the
integration of the reporter
constructs
For mammalian cell assay system
Cloning bStCas9 for
GCTACCGGTCGCCACCATGACTAAGCCATACTCAATTGG
M.bStCas9.F
mammalian expression
(Paired with Y.bStCas9.R)
22
vector construction
PCR primers for
CMV.F1
CCCAAGCTTTAGTTATTAATAGTAATCAATTAC
generating the eGFP gene
eGFPL.R1
CCGGAATTCGCGGCCGCTGCGCTCCTGGACGTAG
reporter construct with SSA
eGFPR.F2
CCGGAATTCGGGGATCCTAAGTGACTAACAAGTTCAGCGTGTCCG
arms of 215 bp
ployA.R2
PCR primers for cloning
CMV.F
CCGCTCGAGTAAGATACATTGATGAGTTTG
CGCTCTAGAGCATTAGTTATTAATAGTAATCAATT
the DsRed expression
cassette
DsRed.R
CGCGGATCCTTACAGGAACAGGTGGTGGCG
eGFP.F
GTAAACGGCCACAAGTTCAG
eGFP.R
GTCGTCCTTGAAGAAGATGG
PCR primers for
identification of the repair of
the eGFP reporter construct
in mammalian cell assay
GACggtctcGGATCCACATTGCATGGATTATACAGTTTT
R26A.sgRopti.F1
AGAGCTGTAGGATCCAAC
PCR primers for
GACggtctcGGATCCGTCTTTCTAGAAGATGGGCGTTTT
generating sgRNA.Opti
R26B.sgRopti.F1
AGAGCTGTAGGATCCAAC
designs for mouse ROSA26
GACggtctcGGATCCTGGGCCTATTCTCAGTCCAGTTTT
locus targeting
R26C.sgRopti.F1
AGAGCTGTAGGATCCAAC
M.sgRopti.R
CAGCTCGAGAAAAAAAACACCGAATCGGTGCCA
GACggtctcGGATCCGGGGCCACTAGGGACAGGATGTTTT
PCR primers for
AAVS1.sgRopti.F
AGAGCTGTAGGATCCAAC
generating sgRNA.Opti
GACggtctcGGATCCCACACTTGTCACCACCCCAAGTTTT
designs for human
CCR5a.sgRopti.F
AGAGCTGTAGGATCCAAC
endogenous gene targeting
(Paired with M.sgRopti.R)
GACggtctcGGATCCGACAAGTGTGATCACTTGGGGTTTT
CCR5b.sgRopti.F
AGAGCTGTAGGATCCAAC
AAVS1.TS.F
GGCCGCGGGGCCACTAGGGACAGGATTGGTGAG
AAVS1.TS.R
GATCCTCACCAATCCTGTCCCTAGTGGCCCCGC
CCR5a.TS.F
GGCCGCCACACTTGTCACCACCCCAAAGGTGAG
fragments for construction of
CCR5a.TS.R
GATCCTCACCTTTGGGGTGGTGACAAGTGTGGC
the reporter vectors for the
CCR5b.TS.F
GGCCGCGACAAGTGTGATCACTTGGGTGGTGAG
human endogenous gene
CCR5b.TS.R
GATCCTCACCACCCAAGTGATCACACTTGTCGC
Annealing primers for
generating
target sequence
The designed restriction enzyme sites within the PCR primers or the sticky ends flanking the
annealing primers were underlined.
23