Extended Data Fig. 8: Assessing the contribution of the hGem tag on Xyl2:URA3 LINEAR editing efficiency with 50-bp homology arms.
From: A repackaged CRISPR platform increases homology-directed repair for yeast engineering
a, Equimolar quantities of each LINEAR fragment were transformed to S. stipitis UC7. Plate pictures were taken at 48 h post-transformation. Ten colonies selected at random (circled) for downstream analysis. Note that GFP cassette was placed in between sgRNA cassette and the donor DNA as a negative reporter to assess aberrant recombination. The colonies with strong GFP expression levels were false positives. b, The expected edit outcome on Xyl2 locus and the intact locus. c, PCR products obtained from the primer pair in b using the genomic DNA isolated from the colonies circled in a as templates. Individual PCR reactions for each biological sample were performed once. d, Sequencing of the amplicons associated with Cas9-hGem #10 and Cas9 #6 confirmed the presence of the intact wild type Xyl2. e, Sequencing of the amplicon associated with Cas9 #2 revealed an erroneous insertion of the donor template along with a portion of the upstream region of the LINEAR fragment.
