CRISPR-READI: Efficient Generation of Knockin Mice by CRISPR RNP Electroporation and AAV Donor Infection

Sean Chen; Sabrina Sun; Dewi Moonen; Clancy Lee; Angus Yiu-Fai Lee; David V Schaffer; Lin He

doi:10.1016/j.celrep.2019.05.103

CRISPR-READI: Efficient Generation of Knockin Mice by CRISPR RNP Electroporation and AAV Donor Infection

Cell Rep. 2019 Jun 25;27(13):3780-3789.e4. doi: 10.1016/j.celrep.2019.05.103.

Authors

Sean Chen¹, Sabrina Sun², Dewi Moonen¹, Clancy Lee³, Angus Yiu-Fai Lee⁴, David V Schaffer⁵, Lin He⁶

Affiliations

¹ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA.
² Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, USA.
³ Department of Environmental Science and Policy Management, University of California, Berkeley, Berkeley, CA, USA.
⁴ Cancer Research Laboratory, University of California, Berkeley, Berkeley, CA, USA.
⁵ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA; Department of Chemical and Biomolecular Engineering, University of California, Berkeley, Berkeley, CA, USA; Department of Bioengineering, University of California, Berkeley, Berkeley, CA, USA; The Helen Wills Neuroscience Institute, University of California, Berkeley, Berkeley, CA, USA. Electronic address: schaffer@berkeley.edu.
⁶ Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, CA, USA. Electronic address: lhe@berkeley.edu.

Abstract

Genetically engineered mouse models harboring large sequence insertions or modifications are critical for a wide range of applications including endogenous gene tagging, conditional knockout, site-specific transgene insertion, and gene replacement; however, existing methods to generate such animals remain laborious and costly. To address this, we developed an approach called CRISPR-READI (CRISPR RNP electroporation and AAV donor infection), combining adeno-associated virus (AAV)-mediated HDR donor delivery with Cas9/sgRNA RNP electroporation to engineer large site-specific modifications in the mouse genome with high efficiency and throughput. We successfully targeted a 774 bp fluorescent reporter, a 2.1 kb CreERT2 driver, and a 3.3 kb expression cassette into endogenous loci in both embryos and live mice. CRISPR-READI is applicable to most widely used knockin schemes requiring donor lengths within the 4.9 kb AAV packaging capacity. Altogether, CRISPR-READI is an efficient, high-throughput, microinjection-free approach for sophisticated mouse genome engineering with potential applications in other mammalian species.

Keywords: AAV; CRISPR; CRISPR-EZ; HDR editing; electroporation; genome editing; knockin; mouse models.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Animals
Clustered Regularly Interspaced Short Palindromic Repeats*
Dependovirus* / genetics
Dependovirus* / metabolism
Electroporation*
Female
Gene Knock-In Techniques*
Mice
Mice, Transgenic
Parvoviridae Infections* / genetics
Parvoviridae Infections* / metabolism
Ribonucleoproteins* / genetics
Ribonucleoproteins* / metabolism

Substances

Ribonucleoproteins

Abstract

Publication types

MeSH terms

Substances

Grants and funding