A Protocol for Production of Mutant Mice Using Chemically Synthesized crRNA/tracrRNA with Cas9 Nickase and FokI-dCas9

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is the most widely used genome editing tool. A common CRISPR/Cas9 system consists of two components: a single-guide RNA (sgRNA) and Cas9. Both components are required for the introduction of a double-strand break (DSB) at a specific target sequence. One drawback of this system is that the production of sgRNA in the laboratory is laborious since it requires cloning of an sgRNA sequence, in vitro transcription reaction and sgRNA purification. An alternative to targeting Cas9 activity by sgRNA is to target it with two small RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Both of these small RNAs can be chemically synthesized which makes the production of these RNAs less difficult when compared to sgRNA. Another downside of the CRISPR/Cas9 systems is that off-target effects have been reported. However, modified forms of Cas9 have been developed to minimize off-target effects. For example, nickase-type Cas9 (nCas9) and FokI domain-fused catalytically-inactive Cas9 (FokI-dCas9; fCas9) induce DSBs only when two guide RNAs bind opposite strands within a defined distance. In this protocol, we describe our experimental system for the production of mutant mice using a CRISPR/Cas9 system that combines crRNA, tracrRNA, and modified forms of Cas9. This method not only facilitates the preparation of reagents for the genome editing system but it can also reduce the risk of off-target effects.