Oligo pool design and library
We designed each the oligonucleotide to contain a guide RNA encoding sequence and its target sequence flanked by the correct PAM(TTTV) , in a total length of 140 bases(Fig1.a) 12,044 guide RNA sequences were generated targeting human genes, and 500 were generated targetingE. coli genome.
Each oligonucleotide included two constant 20-base sequences at either end for PCR amplification; A unique 8-base barcode sequence was inserted in each oligonucleotide.
Oligonucleotides were synthesized electrochemically on arrays (Custom Array, GenScript). These oligonucleotides (140 bases each) were amplified by PCR using Q5 Polymerase (NEB) and gel-purified by using a QIAquick Purification Kit (QIAGEN). Purified PCR products were assembled with the PUC19 vector using a NEBuidler HiFi DNA assembly kit (NEB). Assembly reaction products were transformed into Trans1-T1 competent cells (Transgene). Transformed cells were cultured onto Luria–Bertani (LB) agar plate with 100 μg/ml ampicillin. The resulting number of colonies yielded more than 60× library coverage. The total plasmids were extracted with a Plasmid Maxiprep Kit (Biomed).
Library sequences are given in TABLE.(excel)