Figure 2. Combined use of Alt-R HDR modified donors and Alt-R HDR Enhancer have an additive effect on HDR improvement. RNP complex (2 μM) targeting 3 genomic loci along with 0.5 μM single-stranded HDR donor oligo were delivered
to HeLa cells by electroporation using the 4D-Nucleofector™ System (Lonza). The RNP complex comprised Alt-R S.p. HiFi Cas9 Nuclease V3 complexed with Alt-R CRISPR-Cas9 crRNA and tracrRNA. Unmodified, PS-modified, or Alt-R HDR modified donor
templates were used. Immediately after electroporation, cells were plated in media with or without 30 μM Alt-R HDR Enhancer. Genomic DNA was isolated 48 hours after electroporation, and HDR efficiency was measured by amplicon sequencing on an Illumina™
MiSeq™ system (v2 chemistry, 150-bp paired-end reads).