Fig. 1: Construction and screening of crRNA DR library.

a Schematic of the secondary structure of canonical crRNA. The DR sequence is divided into three parts: flanking sequence, loop and stem, with each region mutated separately. b Fluorescence intensity derived from flanking sequence-mutated crRNAs guiding dCas12a to knock down gfp. c Screening of the crRNA DR library. crRNA mutants were used to guide dCas12a to knock down gfp and ranked based on the level of expression repression. d Relative expression levels of gfp derived from single-part-mutated crRNAs. The eight mutants in the black box are derived from Fig. 1b.e Relative expression levels of gfp derived from the multi-part-mutated crRNAs. The flanking sequences of the multi-part mutants were CAAUG or GAAUG, with the loop contained the mutation library. f Typical crRNA mutant sequences determined through Sanger sequencing and their corresponding designations. The left column indicates their position in (e) and (f), while the right column contains the designations used for referencing these crRNAs. g Diversity of gfp relative expression levels derived from the diversified crRNA mutants. The relative expression was calculated based on the specific fluorescence level (RFU/OD₆₀₀) and normalized by setting the value of the non-targeted control as 100%. Source data are provided as a Source Data file.