Integrated evaluation of off-target and developmental effect of FNLS-YE1 in human 2PN embryos and derived stem cells. (A) Schematic of sgRNA-dependent DNA off-targeting and sgRNA-independent RNA off-targeting analyses using targeted deep sequencing and RNA sequencing of FNLS-YE1-treated embryos undergoing whole-genome/whole-transcriptome amplification at the single cell level. FNLS-YE1 mRNA/FNLS-BE3 mRNA, PCSK9 sgRNA, and GFP mRNA or only GFP mRNA were co-injected into four blastomeres of 8-cell embryos whereas the other four blastomeres were left un-injected. When embryos developed to the early morula stage, GFP-positive and negative blastomeres were separated and analyzed by targeted deep sequencing or RNA-seq. (B) Genome-wide analysis of sgRNA-dependent off-target events with PEM-seq and targeted deep sequencing in human blastomeres with or without base editing. A schematic diagram of the PEM-seq workflow is shown in Fig. S2A. (C) Number of off-target SNVs identified by RNA-seq in human blastomeres injected with FNLS-YE1/PCSK9/GFP or FNLS-BE3/PCSK9/GFP compared with those injected with GFP only. (D) Proportion of C·G to U·A mutations identified by RNA-seq for GFP, FNLS-YE1/PCSK9/GFP, and FNLS-BE3/PCSK9/GFP groups. (E) Schematic of sgRNA-independent off-target analysis with WGS of hESCs derived from FNLS-YE1-treated or untreated embryos. FNLS-YE1 mRNA and PCSK9 sgRNA were co-injected into 8-cell stage embryos. Subsequently, hESCs were derived until injected embryos developed into blastocysts after trophectoderm biopsy and isolation of inner cell mass into a 4-well plate. Ultimately, genomic DNA of hESCs was extracted for WGS. In addition, peripheral blood genomes of both parents were used as a background control. (F) Percentage of on-target efficiency for one control and four independent hESCs derived from FNLS-YE1-treated and untreated embryos analyzed by WGS. (G and H) Number of SNVs (G) and indels (H) identified in one control and four independent hESCs by WGS. (I) Proportion of C·G to T·A mutations detected in hESCs derived from control and PCSK9-edited embryos using FNLS-YE1. (J) Distribution frequency of SNV mutation types. The number in each cell indicates the proportion of a certain type of mutation among all SNV mutations. (K) Representative morphology of the control embryos and the FNLS-YE1-injected embryos at different cleavage stages. (L) Developmental rate from MI-stage oocyte to blastocyst for un-injected and injected embryos with FNLS-YE1 targeting PCSK9. Data are presented as the mean ± SEM. P values were evaluated with unpaired Student’s t-test. Each dot in Fig. 2C–D and 2G–I represents a single blastomere and an embryonic stem cell line, respectively.