Journal: eLife

Article Title: Genome-wide base editor screen identifies regulators of protein abundance in yeast

doi: 10.7554/eLife.79525

Figure Lengend Snippet: ( A ) The low-copy plasmid pGal-BE3 encodes the base editor v3 (BE3; ) under a galactose-inducible promoter ( GALL ). This plasmid also encodes LEU2 for selection. ( B ) The high-copy pgRNA-backbone plasmid encodes a 2 kb placeholder flanked by the SNR52 promoter (a Polymerase-III promoter) and SUP4 terminator. The placeholder can be removed by Esp3I/BsmBI restriction digest and then a gRNA can be cloned in either by Golden Gate Assembly or by Gibson Assembly. This plasmid also encodes the URA3 gene for selection. ( C ) This plasmid is identical to the one shown in (B) but with a barcode cassette added. The barcode cassette contains a Kanamycin resistance gene. ( D ) Sequences at the gRNA cloning site of the pgRNA-backbone plasmid to facilitate gRNA plasmid cloning (individually by Golden Gate Assembly or as a library by Gibson Assembly). Rows 3 and 4 show the two types of DNA fragments containing the gRNA that can be cloned into the digested backbone plasmid.

Article Snippet: The resulting double-stranded DNA fragment with 4-nucleotide overhangs was then inserted into the pgRNA-backbone plasmid by Golden Gate Assembly ( ) using the Esp3I/BsmBI restriction enzyme (NEB #E1602S).

Techniques: Plasmid Preparation, Selection, Clone Assay