5′ modifications improve potency and efficacy of DNA donors for precision genome editing
Ghanta, Krishna et al. (2021), 5′ modifications improve potency and efficacy of DNA donors for precision genome editing, Dryad, Dataset, https://doi.org/10.5061/dryad.f7m0cfxwr
Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5′-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.
Regions of interest were amplified from genomic DNA and sequenced on an Illumina MiniSeq platform. PCR1 ((98° C- 2min, 24 cycles of (98° C- 15sec, 64° C- 20sec, 72° C- 15sec), 72° C- 5min) was performed using 200ng gDNA, 1.25uL of 10uM forward and reverse primers that contain Illumina adapter sequences, 12.5uL NEBNext UltraII Q5 Master Mix, and water to bring the total volume to 25uL. PCR2 (98° C- 2min, 10 cycles of (98° C- 15sec, 64° C- 20sec, 72° C- 15sec), 72° C- 5min) was done using 1uL of unpurified PCR1 reaction mixture, 1.25 uL of 10uM forward and reverse primers that contain unique barcode sequences, 12.5uL NEBNext UltraII Q5 Master Mix, and water to bring the total volume to 25uL. PCR2 products were first analyzed using 2% agarose gel electrophoresis, and then similar amounts were pooled based on the band intensities. Pooled PCR2 products were first purified by gel extraction (Qiagen) and purified again by PCR cleanup columns (Qiagen). Concentration of final purified library was determined by Qubit (High Sensitivity DNA assay). The integrity of library was confirmed by Agilent Tapestation using Agilent High Sensitivity D1000 ScreenTape kit. The library was then sequenced on an Illumina Miniseq platform according to the manufacturer’s instructions using MiniSeq Mid Output Kit (300-cycles). Sequencing reads were demultiplexed using bcl2fastq2 (Illumina) and CRISPResso2 was used to align the reads and quantify editing efficiencies. Quantification window size was set as 30 to ensure the stringent analysis. HDR efficiency was calculated as percentage of (precise HDR reads) / (total reads). For Guide-Seq Experiments, two phosphorothioate linkages were incorporated between the first three and the last three nucleotides in the dsODN tags. Unmodified dsODN does not contain any further modifications whereas modified dsODN contains 5′ TEG (SP9) modification (Integrated DNA Technologies). Sequencing data was processed and analyzed using the GUIDE-seq analysis software.