Table 2
Primer pairs for Golden Gate cloning up to 7 additional sgRNAs into the CRISPRi backbone.The 3
0 end
of each primer (green sequences) anneals to the CRISPRi plasmid backbone to PCR amplify the
[promoter-sgRNA-terminator] cassette. The blue sequences are the SapI recognition sites and the
5
0 -bolded black sequence is extra “filler” sequence added to improve SapI cleavage close to the end
of the DNA fragment. The bolded multicolored sequences are the SapI digestion overhangs that allow
assembly into the SapI-digested CRISPRi backbone. By adjusting the sequences of the overhangs,
multiple [promoter-sgRNA-terminator] cassettes (each with unique sticky ends) can be assembled in
tandem. Overhangs should be used such that the first sgRNA cassette’s forward primer has the
5
0 -GGA-3
0 overhang (amplified using oligo GG_vector_Forward) and the last sgRNA’s reverse primer
has the 5
0 -CTG-3
0 overhang (amplified using oligo GG_vector_Reverse) to ligate into the CRISPRi
plasmid. Furthermore, each consecutive sgRNA’s forward primer should have overhangs reverse
complementary to the previous sgRNA’s reverse primer (for example, in this primer set the overhang
5
0 -GAT-3
0 in GG_Reverse_1 is reverse complementary to the overhang 5
0 -ATC-3
0 in GG_Forward_2).
For optimal Golden Gate assembly, ensure the selected overhang sequences do not contain two
consecutive identical bases in the same positions (e.g., when using 5
0 -GGA-3
0 as an overhang
sequence, 5
0 -GTA-3
0 is permissible, but 5
0 -GGT-3
0 and 5
0 -TGA-3
0 would not be). A combination of
overhang sequences compatible with cloning seven additional sgRNA cassettes is provided in this
table. To clone an intermediate number of sgRNA cassettes, use the corresponding number of Golden
Gate primer pairs in the order provided, ensuring the first cassette uses the GG_vector_Forward oligo
and the last cassette uses the GG_vector_Reverse oligo. Lastly, ensure each sgRNA target sequence
is free of SapI digestion sites
358
Andrew I. Wong and Jeremy M. Rock
Primer pairs for Golden Gate cloning up to 7 additional sgRNAs into the CRISPRi backbone.The 3
0 end
of each primer (green sequences) anneals to the CRISPRi plasmid backbone to PCR amplify the
[promoter-sgRNA-terminator] cassette. The blue sequences are the SapI recognition sites and the
5
0 -bolded black sequence is extra “filler” sequence added to improve SapI cleavage close to the end
of the DNA fragment. The bolded multicolored sequences are the SapI digestion overhangs that allow
assembly into the SapI-digested CRISPRi backbone. By adjusting the sequences of the overhangs,
multiple [promoter-sgRNA-terminator] cassettes (each with unique sticky ends) can be assembled in
tandem. Overhangs should be used such that the first sgRNA cassette’s forward primer has the
5
0 -GGA-3
0 overhang (amplified using oligo GG_vector_Forward) and the last sgRNA’s reverse primer
has the 5
0 -CTG-3
0 overhang (amplified using oligo GG_vector_Reverse) to ligate into the CRISPRi
plasmid. Furthermore, each consecutive sgRNA’s forward primer should have overhangs reverse
complementary to the previous sgRNA’s reverse primer (for example, in this primer set the overhang
5
0 -GAT-3
0 in GG_Reverse_1 is reverse complementary to the overhang 5
0 -ATC-3
0 in GG_Forward_2).
For optimal Golden Gate assembly, ensure the selected overhang sequences do not contain two
consecutive identical bases in the same positions (e.g., when using 5
0 -GGA-3
0 as an overhang
sequence, 5
0 -GTA-3
0 is permissible, but 5
0 -GGT-3
0 and 5
0 -TGA-3
0 would not be). A combination of
overhang sequences compatible with cloning seven additional sgRNA cassettes is provided in this
table. To clone an intermediate number of sgRNA cassettes, use the corresponding number of Golden
Gate primer pairs in the order provided, ensuring the first cassette uses the GG_vector_Forward oligo
and the last cassette uses the GG_vector_Reverse oligo. Lastly, ensure each sgRNA target sequence
is free of SapI digestion sites
358
Andrew I. Wong and Jeremy M. Rock