Fig. 5: Assessing the specificity of 24 Cas12a variants using an enhanced vision of GUIDE-seq.

a A schematic illustration showing turn-over dsDNA cleavage and NHEJ cycles for the Cas12a/crRNA complex. Cleavage sites are marked with triangles and ash-gray line indicate changes in the dsODN tag sequences. b Two rounds of nested anchored GUIDE-seq tag specific PCR. The primers Nuclease_off_+/−_GSP1 were used to the first PCR; The primers Nuclease_off_+/−_GSP2 were used to the second PCR; P5 means the Illumina P5 adapter primers. c Incomplete dsODN tag sequences induced by Cas12a/crRNA complex in HEK293T cells at IL12A and PRKCH targets. The bar graph shows the percentage of different lengths of dsODN tag that can be detected after 48 h transfection of AsCas12a and crRNA expression plasmid as well as 34 nt dsODN tag. The primers Target F/Tag-R4 and Target F/Tag-F4 were used for the PCR amplification of the sites integrated with the dsODN tag. The percentage of different lengths of the dsODN tag was determined by deep sequencing. Data are presented as mean values +/− SEM. ****P < 0.0001 by Two-tailed paired t-test. d Two rounds of nested anchored enhanced vision of the GUIDE-seq tag specific PCR. Notably, the primers Tag-F5 (Nuclease_off_+_GSP1) and Tag-R5 (Nuclease_off_-_GSP1) were used to the first PCR; The primers Tag-F7 (Nuclease_off_+_GSP2) and Tag-R7 (Nuclease_off_-_GSP2) were used to the second PCR; P5 means the Illumina P5 adapter primers. e Comparison of the general activities and specificities of the Cas12a variants. The general activity score of each Cas12a was calculated as the average indel frequency at the target sequences containing the same protospacers 7 days after transduction of Cas12a variant lentiviruses into library integrated HEK293T cells. The general specificity score of each Cas12a calculated from the enhanced vision of GUIDE-seq identified off-targets sites. f Total translocations induced by Cas12a variants. Data are presented as mean values +/− SEM. g Comparison of the general activities and translocations of the Cas12a variants. h Comparison of the general specificities and translocations of the Cas12a variants. Source data are provided as a Source Data file.