4. 1Â TAE buffer for electrophoresis: 40 mM Tris base, 20 mM
acetic acid, 1 mM EDTA sodium salt dihydrate (prepared by
diluting 50Â TAE stock in nuclease-free water).
5. 1% Agarose gel: 1% (w/v) Agarose diluted in 1Â TAE buffer.
6. Gel extraction and PCR cleanup kits.
7. AgeI, BamHI, and AseI restriction enzymes.
8. Gibson assembly reaction mixture [30] (see Note 4).
2.3 Primer Pairs
Used for Cloning
1. dCas9-Forward: 5
0 -GCTACCGGTCGCCACCATGGTGCC
CAAAAAGAAGAGGAAAGTGG-3
0 ; dCas9-Reverse: 5
0 -ACT
GCTGGATCCGTGGAACT ACCTACCTTGCGCTTTTTC
TTGGGA-3
0 .
2. U6-sgTelo-MTSa-Forward: 5
0 -ATTAATAGTTATTAGAGGG
CCTATTTC CC-3
0 ; U6-sgTelo-MTSa-Reverse: 5
0 -ATGACC
CCGTAATTGATTACTATTAATCGGTTGGCAGTGACTC
CGTCTC-3
0 .
3. EGFP-Forward: 5
0 -ATTACCGCCATGCATTAGTTATTACG
CGTTACATAACTTACGGTAAATG-3
0 ; EGFP-Reverse: 5
0 -
AATAGGCCCTCTA ATAACTATTAATAAGATACATTGATGAGTTTGGAC-3
0 .
4. BFP-Forward: 5
0 -ATTACCGCCATGCATTAGTTATTACGC
GTTACATAACTTACGGTAAATG-3
0 ; BFP-Reverse: 5
0 -AAT
AGGCCCTCTAATAACTATTAATAAGATACATTGATGAG
TTTGGAC-3
0 .
5. U6-sgSat-MTSb-Forward: 5
0 -ATTAATAGTTATTAGAGGG
CCTATTTCCC-3
0 ; U6-sgSat-MTSb-Reverse: 5
0 -ATGACC
CCGTAATTGATTACTATTACTCGTCGGTCCCGGCATC
CGAT-3
0 .
2.4 MBs
MTS-targeting MBs with a backbone composed of 2
0 -O-methyl
RNA (2Me) and a fully phosphorothioate (PS)-modified loop
domain can be synthesized by an oligonucleotide synthesis service
provider (see Note 5). Sequences of MBs used in our experiments
are listed below (see Note 6) (m represents 2Me modification, ∗
represents PS linkage modification):
1. AMTSa-targeting MB labeled with an ATTO647N fluorophore at the 5
0 end and an Iowa Black RQ quencher at the 3
0
end (see Note 7) has the sequence: 5
0 -mCmUmUmCmG∗
mU∗mC∗mC∗mA∗mC∗mA∗mA∗mA∗mC∗mA∗mC∗
mA∗mA∗mC∗mU∗mC∗mC∗mU∗mGmAmAmG-3
0 .
2. MTSb-targeting MB labeled with an Iowa Black FQ quencher
at the 5
0 end and an ATTO488 fluorophore at the 3
0 end (see
Note 7) has the sequence: 5
0 -mCmUmCmAmG∗mC∗
mG∗mU∗mA∗mA∗mG∗mU∗mG∗mA∗mU∗mG∗mU
∗mC∗mG∗mU∗mG∗mA∗mCmUmGmAmG-3
0 .
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Xiaotian Wu et al.