Fig. 4: Activity-attenuated crRNA enhances transformation and editing efficiency for CRISPR-mediated homologous recombination.

a Schematic of the experimental setup for CRIPSR-mediated homologous recombination aimed at fragment knockouts and knock-ins. RT, repair template. b,c Genome editing assay in E. coli NEB 10-beta-gfp to knock out 500 bp within gfp (b) or knock in 1200 bp mcherry-expressing fragment (c). Each dot for the transformations represents a single biological replicate. NT indicates non-targeting gRNAs. Each dot for the editing efficiencies represents the average of 16 colonies screened from one biological replicate. Data are shown as mean ± s.d. for n  =  3 biologically independent samples. d Phenotypic characterization of knockout and knock-in strains. Confirmed knockout and knock-in strains were cultured, resuspended in PBS buffer, and imaged using fluorescence microscopy. The left panel presents a schematic showing the effects of knockout and knock-in, while the right panel shows fluorescence microscopy images, confirming that the phenotypes of the knockout and knock-in strains were as expected. Scale bar: 10  μm. Representative micrographs of wild type (WT) strains were derived from a single experiment without replication. KO indicates knockout. KIN indicates knock-in. Source data are provided as a Source Data file.