Fig. 2

Relationship of DNA cleavage efficiency with gene expression and target specificity. a Impact of gene expression on editing efficiency. We divided the target sites into those that occur in lowly expressed genes (FPKM < 25, blue boxplots) and those that occur in highly expressed genes (FPKM ≥ 25, red bloxplots) using our RNA-seq data. The FPKM value of 25 was chosen to divide the target sites into two groups of roughly equal sizes for the five Cas nucleases. Here, all sgRNAs were considered in the analysis. Overall, we found from our deep sequencing experiments that SpCas9, AsCpf1, and LbCpf1 were able to edit highly expressed genes more efficiently than lowly expressed genes (P < 0.05, Wilcoxon rank sum test). In contrast, the two smaller nucleases, SaCas9 and NmCas9, were less affected by gene expression. b Similar analysis to a, except that only sgRNAs of optimal lengths were considered. In the current study, we set the optimal lengths of SpCas9 as 17–22 nt inclusive, SaCas9 as ≥ 21 nt, NmCas9 as ≥ 19 nt (based on our results in Fig. 1b and Additional file 1: Figure S5b as well as a previous report [22]), AsCpf1 as ≥ 19 nt, and LbCpf1 as ≥ 19 nt. c Comparison of AsCpf1 with either SpCas9 (left boxplot) or LbCpf1 (right boxplot). Only sgRNAs of the optimal lengths for SpCas9 and the Cpf1 nucleases (19–22 nt inclusive) were considered. From deep sequencing analysis, we found that the editing activity of AsCpf1 was significantly lower than that of both SpCas9 and LbCpf1 (P < 0.001, Wilcoxon rank sum test). d To assess the specificities of SpCas9, AsCpf1, and LbCpf1, we examined the tolerance of these enzymes to single mismatches along the spacer targeting the A17 site in the NF1 gene. Red letters indicate the mutated bases. e Using the spacers indicated in d, we determined the editing activities of SpCas9, AsCpf1, and LbCpf1 by deep sequencing. The cells were harvested 24 h after transfection. For all three nucleases, we observed an increased tolerance to mismatches around the middle of the spacer. Importantly, while SpCas9 and LbCpf1 exhibited higher cleavage efficiencies than AsCpf1 with a perfect matched (PM) spacer, they also showed an overall higher tolerance to mismatches between the spacer and the target DNA. Data represent mean ± standard error of the mean (n ≥ 4)