CRISPR/Cas9-based genome editing can easily generate knockout mouse models by disrupting the gene sequence, but its efficiency for creating models that require either insertion of exogenous DNA (knock-in) or replacement of genomic segments is very poor. The majority of mouse models used in research involve knock-in (reporters or recombinases) or gene replacement (e.g., conditional knockout alleles containing exons flanked by LoxP sites). A few methods for creating such models have been reported that use double-stranded DNA as donors, but their efficiency is typically 1–10% and therefore not suitable for routine use. We recently demonstrated that long single-stranded DNAs (ssDNAs) serve as very efficient donors, both for insertion and for gene replacement. We call this method efficient additions with ssDNA inserts–CRISPR (Easi-CRISPR) because it is a highly efficient technology (efficiency is typically 30–60% and reaches as high as 100% in some cases). The protocol takes ∼2 months to generate the founder mice.
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This work was supported in part by a Grant-in-Aid for Young Scientists (B) (16K18821) from the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) to H.M.; and by 2014 Tokai University School of Medicine Research Aid, the MEXT-Supported Program for the Strategic Research Foundation at Private Universities 2015–2019 to Tokai University, the Research and Study Project of Tokai University General Research Organization, a Grant-in-Aid for Scientific Research (16H04685) from the MEXT, and funding from 2016–2017 Tokai University School of Medicine Project Research to M.O.; and by an Institutional Development Award (principal investigator: S. Smith) P20GM103471 (to C.B.G. and R.M.Q.). We thank A. Koesters, University of Nebraska Medical Center, for her editorial contribution and J.M. Miano, University of Rochester, for his helpful comments on the manuscript. We also gratefully acknowledge the contribution of the staff of the Support Center for Medical Research and Education, Tokai University, for sequencing and microinjection.
C.B.G., M.O., and H.M. have filed a patent application relating to the work described in this paper with international application number PCT/US2016/035660, filed June 3, 2016 (DNA editing using single-stranded DNA).
Integrated supplementary information
1μg DNA was used for RNA synthesis according to each manufacturer's protocol. The RNA samples in lanes 1 and 2 were synthesized using kits from two different vendors. 1% of the eluates, from purification using the MEGAclear columns, were loaded. This figure illustrates that kits can vary in their efficiencies. It should be noted that the identity of the products is not an important point: we have observed a little better performance of a different batch from vendor 1, and somewhat poorer performance of another batch of kit from vendor 2.
cDNAs (ssDNA) were synthesized using reverse transcriptases from three different vendors (1 to 3). 1.7μg RNA was used for cDNA synthesis according to each manufactures protocol and 50% of the reaction volumes were loaded. The cDNA yield from all three transcriptases are comparable.
1.7μg (lane 1 and 2) or 5μg (lanes 3 and 4) of RNA was used for cDNA synthesis and the reactions were incubated for 10 minutes (lanes 1 and 3) or 2 hours (lanes 2 and 4). 15% of the reaction volume was loaded. The band intensities suggest that 5μg RNA input produces optimal yields of cDNA.
(a) Typical smear-like appearance of the cDNA (ssDNA) preparations in an agarose gel showing a prominent band (box # 1) and a less prominent band (box # 2). (b and c) Gel slices excised for DNA extraction. (d) The purified ssDNAs loaded in another gel showing both bands migrate similarly. Either of the gel preparations (1 or 2) can be used for microinjection, or both preparations can be pooled before use.
About 150 ng of ~0.5kb cDNA was incubated with or without S1 nuclease at 37°C for 15 minutes.
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Miura, H., Quadros, R., Gurumurthy, C. et al. Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors. Nat Protoc 13, 195–215 (2018). https://doi.org/10.1038/nprot.2017.153
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