C.elegansis ideally suited for germline-editing, the syncytial germline allows each injection to deliver editing reagents to hundreds of germ nuclei. We recently described methodologies involving "asymmetric donors" (Dokshin et al, 2018), that yield one or more precision edits, such as GFP knock-ins per injected animal. Asymmetric donors are made by annealing a PCR product containing homology arms with an armless PCR product (containing, for example, gfp sequence only). We prefer the DNA marker Rol-6 as a metric and control for each of the many variables that influence editing efficiency. The presence of Rollers identifies a cohort of progeny inheriting long-dsDNA donors and edits. This cohort is not limited to the Rollers but also includes siblings of similar age to the Rollers. Scoring non-Rolling siblings is sometimes helpful when making a difficult edit. We do not prefer "co-editing" strategies such as "co-CRISPR" and "co-Conversion" when using CRISPR RNPs as these markers, likely due to a broader distribution of RNPs, do not necessarily identify animals inheriting longer precision DNA edits. After adjusting RNP and donor DNA levels such that the recovery of F1 Rollers is achieved at approximately 25% of the efficiency obtained when injecting
rol-6 DNA alone, we find that typically 10% of Rollers assayed have GFP insertions while shorter edits, such as Degron or Flag insertions are found in greater than 50% of Rollers assayed. Recently, we have begun exploring how editing efficiencies depend on other donor DNA properties including chemical modifications added to the 5? ends. These studies began with attempts to improve donor DNA nuclear uptake by adding a nuclear localization signal (NLS). These NLS donors increased the frequency of GFP knock-ins dramatically, similar to the over-all efficiencies observed with asymmetric donors. Strikingly, however, high knock-in efficiencies were achieved at very low concentrations of just 10 ng/ l, for 1kb long donors, suggesting that the end modifications greatly enhance donor potency. Upon further investigation we found that the NLS was not required for high efficiency editing. Instead, our findings suggest that increased potency is driven by both a Tetra-Ethylene-Glycol (TEG) linker and a 2?-O-Methyl RNA (used to attach the NLS). Unfortunately, short donor molecules prepared using commercially sourced TEG primers have not (as yet) shown any increased editing efficiency. We are now exploring the specific chemistries and mechanisms through which end-modified donors influence the efficiency and accuracy of homology driven repair, and whether combining end-modifications and asymmetry, can further stimulate efficiencies.