CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos

Abstract

Conventional embryonic stem cell (ESC)–based gene targeting, zinc-finger nuclease (ZFN) and transcription activator–like effector nuclease (TALEN) technologies are powerful strategies for the generation of genetically modified animals. Recently, the CRISPR/Cas system has emerged as an efficient and convenient alternative to these approaches. We have used the CRISPR/Cas system to generate rat strains that carry mutations in multiple genes through direct injection of RNAs into one-cell embryos, demonstrating the high efficiency of Cas9-mediated gene editing in rats for simultaneous generation of compound gene mutant models. Here we describe a stepwise procedure for the generation of knockout and knock-in rats. This protocol provides guidelines for the selection of genomic targets, synthesis of guide RNAs, design and construction of homologous recombination (HR) template vectors, embryo microinjection, and detection of mutations and insertions in founders or their progeny. The procedure from target design to identification of founders can take as little as 6 weeks, of which <10 d is actual hands-on working time.

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Figure 1: Schematic of RNA-guided Cas9 nuclease.
Figure 2: DSB repair promotes gene editing.
Figure 3: Workflow for production of mutant rats using the CRISPR/Cas system.
Figure 4: PCR approach for sgRNA template production.
Figure 5: Schematic diagrams showing the strategy for constructing a rat gene-targeting vector.

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Acknowledgements

We thank F. Zhang of the Broad Institute of MIT and Harvard for kindly providing the Cas9 expression vector. We thank S. Siwko for comments and advice. This work was partially supported by grants from the State Key Development Programs of China (2010CB945403 to D.L. and 2012CB910400 to Mi.L.), grants from the National Natural Science Foundation of China (no. 31371455, 31171318 and 81330049) and a grant from the Science and Technology Commission of Shanghai Municipality (12XD1406100) and 14140900300.

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Authors

Contributions

Y.S., Y.G., L. Wang, Z.Q., Me.L., Y.C., L. Wu and Y.L. performed the experiments; Y.S., Y.G., X.M., Mi.L. and D.L. analyzed the data; and Y.S., Y.G., Mi.L. and D.L. wrote the manuscript.

Corresponding authors

Correspondence to Mingyao Liu or Dali Li.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 In vitro validation of sgRNA activity.

4 nM PCR product containing the target sequence was mixed with purified H6MBP-Cas9-4NLS recombinant protein (2 nM final concentration) in NEB buffer 3 with (lane 2) or without (lane 1) corresponding gRNAs for 1 hour. After the incubation, the protein was discarded and the DNA was analyzed by agarose gel electrophoresis.

Supplementary Figure 2 Design of PCR primers for genotyping of knock-in mutations in rat genome.

(a) A primer pair (F and R) is designed outside of the homology arms of the ssODN. (b) Two primer pairs (5'F/5'R and 3'F/3'R) are designed. For each pair, one primer is located in the GOI (the inserted fragment). The other primer is located in rat genome outside the homology arms (left arm or right arm). About 400 bp extension of both left and right arm of the donor plasmid can be used as the positive control for testing the primers (optional).

Supplementary Figure 3 Generation of double mutant rats by co-injection of Cas9/sgRNAs targeting the Fah and Il2rg loci.

Detection of mutations in F0 rats generated by injection of Cas9/sgRNAs targeting Fah (a) and Il2rg (b) after T7EI digestion using PCR products amplified from RNA-injected F0 rat tail genomic DNA. Double mutant rats are indicated by arrowheads. M, DNA marker. (c) DNA Sequence of Fah and Il2rg loci double mutant rats. The target sites are in blue with PAM in red.

Supplementary Figure 4 Anticipated results for precise knock-in by ssODNs.

(a) A diagram of Cas9 nickase induced precise gene editing. The sequence of the target site and the ssODNs are listed. (b) Typical sequences of the founders after RNA/DNA injection. The PCR products were amplified with the primers outside of the homology region indicated in (a). Sequences were determined by DNA sequencing of individual TA clones of PCR products. The sgRNA target is underlined, the EcoRI site is in red and the LoxP site is in italics. (c) An example of a DNA sequencing result for precise HR.

Supplementary information

Supplementary Figure 1

In vitro validation of sgRNA activity. (PDF 142 kb)

Supplementary Figure 2

Design of PCR primers for genotyping of knock-in mutations in rat genome. (PDF 243 kb)

Supplementary Figure 3

Generation of double mutant rats by co-injection of Cas9/sgRNAs targeting the Fah and Il2rg loci. (PDF 238 kb)

Supplementary Figure 4

Anticipated results for precise knock-in by ssODNs. (PDF 244 kb)

Supplementary Method 1

Preparation of recombinant Cas9 protein. (PDF 163 kb)

Supplementary Method 2

Functional validation of sgRNA and Cas9 protein in vitro. (PDF 98 kb)

Supplementary Table 1

Summary of injection statistics in rat embryos from published reports. (PDF 177 kb)

Supplementary Table 2

Primer or oligo sequences. (PDF 174 kb)

Pronuclear microinjection

This video demonstrates the microinjection of the CRISPR-Cas system into the pronucleus of a rat zygote fixed with a holding pipette on the left. An obvious pronuclear swelling is shown to demonstrate the successful pronuclear injection. (MOV 2515 kb)

Supplementary Data 1: Full DNA sequence of pGS3-T7-sgRNA vector for in vitro synthesis of gRNA.

The pair of reverse located BbsI restriction sites used for cloning is indicated in bold and underlined. The DraI restriction site used to linearize the plasmid for run-off transcription is shown in bold, italicized text. The “stuffer” sequence that will eventually be replaced with the target-coding oligonucleotides is highlighted in yellow and the “scaffold” portion of sgRNA is shown as italicized underlined text. The sequence of an M13 primer binding site is shown as underlined text. (PDF 208 kb)

Supplementary Data 2: Full DNA sequence of SP6-Cas9 vector for in vitro mRNA synthesis.

The SP6 promoter sequence (highlighted in green) is followed by a Kozak sequence (highlighted in yellow) is subcloned into the Cas9-containing plasmid pX260 (Addgene plasmid# 42229) using NcoI restriction site (in red), NLS-hCas9-NLS-coding cassette is underlined. (PDF 185 kb)

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Shao, Y., Guan, Y., Wang, L. et al. CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos. Nat Protoc 9, 2493–2512 (2014). https://doi.org/10.1038/nprot.2014.171

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