Fig.3

RNP screening process and prioritization steps for paired guide selection. The process for screening endonucleases to identify a Cas9, single dual-guide pair for CD163 allele modification is outlined. Left panel: Initially many guides are tested as RNPs in porcine fibroblast cell lines to measure on-target INDEL frequency short read Illumina DNA sequencing. Graph displays the frequency and range of on-target cutting by each RNP. This step eliminates no or low activity guideRNAs. Middle panel: Next a few high-activity RNPs (less than 10) are used to digest porcine genomic DNA in vitro to identify concentration dependent off-target sites by SITE-Seq biochemical screen. The sequences captured by SITE-Seq are aligned to the on-target sequence and mismatched nucleotides are highlighted in color. Right panel: A few RNP candidates having high frequency of desired repair outcomes and reduced mismatch off-target cutting sites are selected and injected as combinations (A + B, A + D, C + D, C + D) of dual-guide and screened in blastocysts to measure the on-target cut site to cut site repair and off-target INDELs at a few SITE-Seq identified mismatch sequences. Based on this criterion, one dual-guide pair is graduated to the commercial editing process (Fig. 4)