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. 2020 Aug 6;117(34):20803–20813. doi: 10.1073/pnas.2007837117

Table 1.

Primer sequences used to generate the sgRNAs and corresponding genomic fragments

Primer Orientation Nucleotide sequence
U6 promoter F GCC​GGT​ACC​TGA​GGG​CCT​ATT​TCC​C
R ACC​TCT​AGA​AAA​AAA​GCA​CCG​ACT​CGG​TGC​CAC​TTT​TTC​AAG​TTG​ATA​ACG​GAC​TAG​CCT​TAT​TTT​AAC​TTG​CTA​TTT​CTA​GCT​CTA​AAA​CNN​NNN​NNN​NNN​NNN​NNN​NNN​CGG​TGT​TTC​GTC​CTT​TCC​ACA​AG
Target sequence Rab5c R GAC​CCG​CCA​TTG​CCC​GTC​CA
Target sequence Rab7a R TCA​AAC​TAA​AGG​GGG​AAA​AG
Target sequence NPC1 R TAA​ATT​TCT​AGC​CCT​CTC​GC
Target sequence EEA1 R GGTGGTGGTTAAACCATG
Primers Rab5c F1up gaa​ttc​gag​ctc​ggt​acc​cGA​GAG​AAC​TAG​GGA​AGA​AGG​ATC​AG
R1up TGC​CCG​TCC​AGC​TGT​AGT​G
F2 RFP CCA​CTA​CAG​CTG​GAC​GGG​CAa​tgg​tgt​cta​agg​gcg​aag​agc
R2 RFP GGA​ACC​ACC​AGA​ACC​ACC​AGA​A
F3 down GGT​TCT​GGT​GGT​TCT​GGT​GGT​TCC​CTG​GCG​GGT​CGG​GGA​GGC​GCA
R3 down gtc​gac​tct​aga​gga​tcc​ccC​CTC​CTA​CCA​AGA​GAG​TAG​AGA​AAG
Primers Rab7a F1up gaa​ttc​gag​ctc​ggt​acc​cAC​TGC​TGT​CAG​CCT​TGC​CTT​CA
R1up CCT​TCA​AAC​TAA​AGG​GGG​AAA​AGG
F2 RFP CCT​TTT​CCC​CCT​TTA​GTT​TGA​AGG​atg​gtg​tct​aag​ggc​gaa​g
R2 RFP GGA​ACC​ACC​AGA​ACC​ACC​AGA​A
F3 down TTC​TGG​TGG​TTC​TGG​TGG​TTC​CAC​CTC​TAG​GAA​GAA​AGT​GTT​GCT​G
R3 down gtc​gac​tct​aga​gga​tcc​ccC​CTC​ACC​CAA​CCT​ACC​ACA​GAA​T
Primers NPC1 F1up gaa​ttc​gag​ctc​ggt​acc​cCC​ACT​GAG​ATG​AAG​GAG​TCC​AT
R1up GAA​ATT​TAG​AAG​CCG​TTC​GCG​C
F2 Halo CGC​GAA​CGG​CTT​CTA​AAT​TTC​gga​ggt​tct​ggt​ggt​tct​ggt​ggt​tcc​GCA​GAA​ATC​GGT​ACT​GGC​TTT​CCA
R2 Halo GCC​GGA​AAT​CTC​GAG​CGT​CGA​CAG
F3 down CTG​TCG​ACG​CTC​GAG​ATT​TCC​GGC​tag​ccc​tct​cgc​agg​gca​tcc
R3 down gtc​gac​tct​aga​gga​tcc​ccG​CTG​TCT​AAT​GAA​ACT​TCT​AGG​TC
Primers EEA1 F1 up gaa​ttc​gag​ctc​ggt​acc​cCT​CTT​TGG​CTG​AAA​TTA​GAA​GCA​GG
R1 up CAT​GGT​TTA​ACC​ACC​ACC​CGG​CG
F2 mScarlet CGC​CGG​GTG​GTG​GTT​AAA​CCA​TGg​tga​gca​agg​gcg​agg​cag​tga​t
R2 mScarlet ctt​gta​cag​ctc​gtc​cat​gcc​gc
F3 down GCG​GCA​TGG​ACG​AGC​TGT​ACA​AGg​gag​gtt​ctg​gtg​gtt​ctg​gtg​gtt​ccT​TAA​GGA​GGA​TTT​TAC​AGA​GGG​TAA​GAG
R3 down gtc​gac​tct​aga​gga​tcc​ccG​CTC​TAA​TCT​TTC​TAT​CCT​CAA​GGT​TTT​C

The sgRNAs with target sequences specific for Rab5c, Rab7a, NPC1, or EEA1 were generated using a free PCR strategy using a U6 promoter-containing primer and reverse primers including Rab5c, Rab7a, NPC1, or EEA1 specific target nucleotide sequences (at the positions indicated with N).