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Efficient CRISPR/Cas9-Mediated Genome Editing in Mice by Zygote Electroporation of Nuclease.

Qin W, Dion SL, Kutny PM, Zhang Y, Cheng AW, Jillette NL, Malhotra A, Geurts AM, Chen YG, Wang H - Genetics (2015)

Bottom Line: The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is an adaptive immune system in bacteria and archaea that has recently been exploited for genome engineering.Although the technology is robust, delivery remains a bottleneck, as it involves manual injection of the components into the pronuclei or the cytoplasm of mouse zygotes, which is technically demanding and inherently low throughput.Our results demonstrate that mice carrying CRISPR/Cas9-mediated targeted mutations can be obtained with high efficiency by zygote electroporation.

View Article: PubMed Central - PubMed

Affiliation: The Jackson Laboratory, Bar Harbor, Maine 04609.

No MeSH data available.


ZEN enables high throughput genome editing in mice. (A) Flow chart of CRISPR/Cas9-mediated genome editing delivered by ZEN. (B) ZEN compared to microinjection.
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fig3: ZEN enables high throughput genome editing in mice. (A) Flow chart of CRISPR/Cas9-mediated genome editing delivered by ZEN. (B) ZEN compared to microinjection.

Mentions: We demonstrated electroporation is an effective method for delivering CRISPR/Cas9 material into the mouse zygotes. Using our zygote electroporation of nuclease (ZEN) protocol (Figure 3A), in one experimental setting, we were able to generate mice carrying NHEJ mutations at 100% efficiency (11/11) and HDR-mediated modifications at 27% efficiency (3/11) at the Tet2 locus. To our surprise, for hybrid embryos, electroporation appears to be less invasive, as compared to the microinjection method. The live birth rate of ZEN-treated embryos is 59% (Table 2), a level approaching that of untreated embryos, and significantly higher than the 30% live birth rate from microinjected embryos in our hands. In addition, an obvious advantage is that a large number of embryos can be processed with the ZEN protocol (20–50 embryos were loaded into a single cuvette in our study) in one single run and multiple projects in parallel (Figure 3B).


Efficient CRISPR/Cas9-Mediated Genome Editing in Mice by Zygote Electroporation of Nuclease.

Qin W, Dion SL, Kutny PM, Zhang Y, Cheng AW, Jillette NL, Malhotra A, Geurts AM, Chen YG, Wang H - Genetics (2015)

ZEN enables high throughput genome editing in mice. (A) Flow chart of CRISPR/Cas9-mediated genome editing delivered by ZEN. (B) ZEN compared to microinjection.
© Copyright Policy - open-access
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4492369&req=5

fig3: ZEN enables high throughput genome editing in mice. (A) Flow chart of CRISPR/Cas9-mediated genome editing delivered by ZEN. (B) ZEN compared to microinjection.
Mentions: We demonstrated electroporation is an effective method for delivering CRISPR/Cas9 material into the mouse zygotes. Using our zygote electroporation of nuclease (ZEN) protocol (Figure 3A), in one experimental setting, we were able to generate mice carrying NHEJ mutations at 100% efficiency (11/11) and HDR-mediated modifications at 27% efficiency (3/11) at the Tet2 locus. To our surprise, for hybrid embryos, electroporation appears to be less invasive, as compared to the microinjection method. The live birth rate of ZEN-treated embryos is 59% (Table 2), a level approaching that of untreated embryos, and significantly higher than the 30% live birth rate from microinjected embryos in our hands. In addition, an obvious advantage is that a large number of embryos can be processed with the ZEN protocol (20–50 embryos were loaded into a single cuvette in our study) in one single run and multiple projects in parallel (Figure 3B).

Bottom Line: The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is an adaptive immune system in bacteria and archaea that has recently been exploited for genome engineering.Although the technology is robust, delivery remains a bottleneck, as it involves manual injection of the components into the pronuclei or the cytoplasm of mouse zygotes, which is technically demanding and inherently low throughput.Our results demonstrate that mice carrying CRISPR/Cas9-mediated targeted mutations can be obtained with high efficiency by zygote electroporation.

View Article: PubMed Central - PubMed

Affiliation: The Jackson Laboratory, Bar Harbor, Maine 04609.

No MeSH data available.