Fig. 4: Evaluation of the ability to integrate heterologous genes of various lengths at a single site.
From: Efficient and markerless gene integration with SlugCas9-HF in Kluyveromyces marxianus
a Schematic illustration of CRISPR-mediated gene integration via homologous recombination. Four gene fragment lengths (1.5 k, 3 k, 6 k, and 12 k) were selected and constructed as Donors. b A representative image of diagnostic PCR confirming gene integration. The schematic of PCR design is shown above. Red arrows indicate primers. c Integration efficiency of various lengths of heterologous genes at the sg1 locus. Integration efficiency was assessed by diagnostic PCR. Three independent replicate experiments were conducted. Forty-eight colonies for Donor- 1.5 kb and thirty- two colonies for Donor- 3 kb were randomly selected separately. For Donor- 6 kb and Donor- 12 kb, all colonies were selected due to the limited number of colonies formed. Error bars represent the mean ± SD of biological triplicates.
