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Fig. 1 | Journal of Biological Engineering

Fig. 1

From: Seamless site-directed mutagenesis of the Saccharomyces cerevisiae genome using CRISPR-Cas9

Fig. 1

Outline of the two-step, stuffer-assisted genome site-directed mutagenesis strategy. Two variations of the strategy were applied. In the stuffer strategy a protospacer target sequence located near the site to mutagenize is replaced by a heterologous 20-nucleotide sequence (the stuffer) by CRISPR-Cas9 assisted homologous recombination, leaving the PAM site intact. The stuffer may be a standard, randomly generated sequence (the standard stuffer, left box) or a degenerate sequence bearing at least seven mismatches with the original protospacer (a silent stuffer, middle box). The second CRISPR step uses the stuffer as a protospacer, restoring the original protospacer sequence and introducing the desired mutation in a single homologous recombination event. A second variation on the strategy replaces the entire target ORF – or nearby ORF if an intergenic region is the target of mutagenesis – by a heterologous stuffer ORF (e.g. GFP), which is targeted by one or more gRNAs in the second step (right box). Homologous recombination restores the original ORF with mutations. Successful integration is easily assessed by PCR. The strategies were tested at the positions indicated in the boxes. Positions are identified by the coordinate of the first nucleotide of the PAM site (NGG) with respect to the nearest ORF. For each position, stuffer insertion was successful in either all strains tested (green check), two out of three strains (yellow check), in all strains but led to a slow growth phenotype (yellow-x), or in none of the strains (red-x). Once a stuffer was inserted, its removal and replacement by the point mutant sequence was successful in all cases

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