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, 2016 (2), pdb.top087536

Editing the Mouse Genome Using the CRISPR-Cas9 System

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Editing the Mouse Genome Using the CRISPR-Cas9 System

Adam Williams et al. Cold Spring Harb Protoc.

Abstract

The ability to modify the murine genome is perhaps one of the most important developments in modern biology. However, traditional methods of genomic engineering are costly and relatively clumsy in their approach. The use of programmable nucleases such as zinc finger nucleases and transcription activator-like effector nucleases significantly improved the precision of genome-editing technology, but the design and use of these nucleases remains cumbersome and prohibitively expensive. The CRISPR-Cas9 system is the next installment in the line of programmable nucleases; it provides highly efficient and precise genome-editing capabilities using reagents that are simple to design and inexpensive to generate. Furthermore, with the CRISPR-Cas9 system, it is possible to move from a hypothesis to an in vivo mouse model in less than a month. The simplicity, cost effectiveness, and speed of the CRISPR-Cas9 system allows researchers to tackle questions that otherwise would not be technically or financially viable. In this introduction, we discuss practical considerations for the use of Cas9 in genome engineering in mice.

Figures

Figure 1
Figure 1. Basic components of the Cas9 system
The Cas9 nuclease generates double strand breaks by using its two catalytic domains (HNH and RuvCI) to cleave each strand of a DNA target site next to a PAM sequence (red) and matching the 20-nucleotide sequence of the single guide RNA (sgRNA). The sgRNA includes a fused RNA sequence derived from CRISPR RNA (crRNA) and the trans-activating crRNA (tracrRNA) that binds and stabilizes the Cas9 nuclease.
Figure 2
Figure 2. Procedures to generate genetically modified mice using the CRISPR/Cas9 genome editing system
Isolated zygotes are co-injected with Cas9 mRNA and sgRNAs to generate mice carrying indel mutations or targeted chromosomal deletions. Alternatively, the Cas9 mRNA and sgRNAs are co-injected in combination with donor ssDNAs or circular plasmids to generate mice harboring point mutations, tags, loxP sites or large DNA fragments such as a fluorescent protein.

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