Genomic DNAs from LE exposed to the RNPs were isolated as described [1 (link)]. The target omega-1 locus in the replicates of gDNAs was amplified using a control primer pair (Figure 2 a). The amplicon of 790 bp in size from control and experimental groups, both KO and KI focused manipulations, was purified by NucleoSpin® Gel and PCR clean-up (Macherey-Nagel, Bethlehem, PA, USA) after which the nucleotide sequence was determined by the Sanger method (Genewiz, South Plainland, NJ, USA). Sanger sequence traces from the twelve independent replicates of the experimental groups for both SpCas9- or AsCas12a- [27 (link)]-catalyzed gene editing manipulations were analyzed and compared with reads from the control (mock) group using the tracking of indels by decomposition (TIDE) algorithm [71 (link),72 (link),73 (link)]. TIDE provides a rapid and informative assay and also informs decisions on whether proceed to more detailed high-throughput, next-generation sequencing (NGS) of targeting amplicons [74 (link)].
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