Fig. 4

Long-read amplicon sequencing to evaluate the knock-in accuracy of dsDNA and ssDNA donors. a Schematic overview of the analysis of knock-in outcomes. After electroporation of Cas12a-RNP and HDR donors for mNG tagging of HNRNPA1, RPE1 cells were expanded for two to three weeks. mNG-positive cells were then collected by FACS, and genomic DNA was isolated. Libraries for sequencing were prepared from the amplified target locus and subjected to long-read amplicon sequencing by PacBio. After analysis of sequencing outputs, including CCS generation, knock-knock categorized each read into a specific category of a knock-in outcome. b Representative plots generated by knock-knock showing the distribution frequency of amplicon lengths. The range of read lengths corresponding to WT and indels, perfect HDR, truncated integrations, and duplication of homology arm(s) are indicated. c Distribution of integration events across the donor types. For each category, the percentage within total integration events was calculated. Data from three biological replicates are shown. For each sample, 11,559–44,431 reads were categorized as the integration events from 43,697–91,850 total reads. d Data from (c), the frequencies of perfect HDR are highlighted. A two-tailed, unpaired Student’s t-test was used to obtain the P-value. *P < 0.05. s: sense strand, as: antisense strand. Data are presented as mean ± S.D