CRISPR RNP & ssDNA For Transgenic Mice Generation

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Get The Correct Pup in The First Round

How to save on your transgenic mice project? Use ssDNA as CRISPR HDR donor template for one-step, high efficiency knockin to decrease the number of mice and embryos you have to manage.

Using Long ssDNA for the Generation of Foxi1-rtTA Transgenic Mice

In collaboration with Dr. Brian Lin at Massachusetts General Hospital, USA

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HDR Donor Options

Plasmids DNA – Editing Efficiency : 10-30%, but requires very long homology arms^1,2
dsDNA – Editing efficiency: 1- 10%^3-8
Two ssODN for LoxP insertion – Editing Efficiency < 10%^9-11
ssDNA – Editing Efficiency ~ 30-60%^6-9

"We found that insertion efficiency was 2.5 times higher, and the viability of injected embryos was 2.4 times higher using an ssDNA donor than a dsDNA donor." - Miura, H., Quadros, et al. Easi-CRISPR for creating knock-in and conditional knockout mouse models using long ssDNA donors. Nat Protoc 13, 195–215 (2018).

"lssDNA generally provides more efficient KIs of approximately 10–50% in zygotes compared with double stranded DNA that normally provides less than 10%." - Miyasaka, Y., et al. CLICK: one-step generation of conditional knockout mice. BMC Genomics 19, 318 (2018).

"We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We find that the one-donor methods are 10- to 20-fold more efficient than the two-donor approach." -Gurumurthy, C.B., et al. Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation. Genome Biol 20, 171 (2019).

What Does High Editing Efficiency Mean to Your Projects

2-3 fold increase in KI efficiency with ssDNA compared to plasmids, dsDNA, and short ssODNs.
2 X Editing Efficiency = 50% Less Mice, 50% Less Injection, 50% Less Sequencing.
Save 6-8 weeks with one less breeding round using one step knockin with long ssDNA vs. two ssODNs.

In general, ssDNA have been accepted as the preferred donor formats to maximize on-target integration for conditional KO/KI and larger KI animal model generation. The increased availability of long ssDNA in recent years has certainly made these methods more feasible.

References

1. Aida, T., Nakade, S., Sakuma, T. et al. Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ. BMC Genomics 17, 979 (2016).

2. Yang, H., Wang, H., Shivalila, C. S., Cheng, A. W., Shi, L. & Jaenisch, R. 2013. One‐Step generation of mice carrying reporter and conditional alleles by CRISPR/Cas‐mediated genome engineering. Cell 154, 1370–1379.

3. Inui M, et al. Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system. Sci Rep. 2014;4.

4. Ma Y, et al. Generating rats with conditional alleles using CRISPR/Cas9. Cell Res. 2014;24:122–5.

5. Horii T, Hatada I. Challenges to increasing targeting efficiency in genome engineering. J Reprod Dev. 2016;62:7–9.

6. Quadros, R.M., Miura, H., Harms, D.W. et al. Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins. Genome Biol 18, 92 (2017).

7. 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).

8. Miyasaka, Y., Uno, Y., Yoshimi, K. et al. CLICK: one-step generation of conditional knockout mice. BMC Genomics 19, 318 (2018).

9. Gurumurthy, C.B., O'Brien, A.R., Quadros, R.M. et al. Reproducibility of CRISPR-Cas9 methods for generation of conditional mouse alleles: a multi-center evaluation. Genome Biol 20, 171 (2019).

10. Bishop KA, et al. CRISPR/Cas9-mediated insertion of loxP sites in the mouse Dock7 gene provides an effective alternative to use of targeted embryonic stem cells. G3 Bethesda Md. 2016;6:2051–61.

11. Miano JM, Zhu QM, Lowenstein CJ. A CRISPR path to engineering new genetic mouse models for cardiovascular research. Arterioscler Thromb Vasc Biol. 2016;36:1058–75.