CRISPR/Cas9-mediated gene manipulation to create single-amino-acid-substituted and floxed mice with a cloning-free method

Xiaolong Ma,Xinmin Zhou,Jennifer Veevers,Ju Chen,Chao Chen,Wei Feng,Robert S Ross

doi:10.1038/srep42244

Abstract

Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology is a powerful tool to manipulate the genome with extraordinary simplicity and speed. To generate genetically modified animals, CRISPR/Cas9-mediated genome editing is typically accomplished by microinjection of a mixture of Cas9 DNA/mRNA and single-guide RNA (sgRNA) into zygotes. However, sgRNAs used for this approach require manipulation via molecular cloning as well as in vitro transcription. Beyond these complexities, most mutants obtained with this traditional approach are genetically mosaic, yielding several types of cells with different genetic mutations. Recently, a growing body of studies has utilized commercially available Cas9 protein together with sgRNA and a targeting construct to introduce desired mutations. Here, we report a cloning-free method to target the mouse genome by pronuclear injection of a commercial Cas9 protein:crRNA:tracrRNA:single-strand oligodeoxynucleotide (ssODN) complex into mouse zygotes. As illustration of this method, we report the successful generation of global gene-knockout, single-amino-acid-substituted, as well as floxed mice that can be used for conditional gene-targeting. These models were produced with high efficiency to generate non-mosaic mutant mice with a high germline transmission rate.

Highlights

The clustered regulatory interspaced short palindromic repeat (CRISPR)/Cas[9] system was initially described as an acquired prokaryotic immune defense system consisting of a Cas[9] nuclease and two small RNAs: CRISPR RNA, which acts as a guide for gene targeting, and trans-activating crRNA, which binds to crRNA and forms a ribonucleoprotein complex with Cas[9] to direct sequence-specific Cas[9] double-stranded DNA cleavage[4,5,6]
In our current study we report a method that capitalized on a cloning-free CRISPR/Cas[9] system using commercial Cas[9] protein combined with chemically synthesized crRNA, tracrRNA, and single-strand oligodeoxynucleotides
The CRISPR/Cas[9] system is proving to be a powerful yet simple tool to manipulate the genome for the generation of genetically modified animals

Summary

Introduction

The CRISPR/Cas[9] system was initially described as an acquired prokaryotic immune defense system consisting of a Cas[9] nuclease and two small RNAs: CRISPR RNA (crRNA), which acts as a guide for gene targeting, and trans-activating crRNA (tracrRNA), which binds to crRNA and forms a ribonucleoprotein complex with Cas[9] to direct sequence-specific Cas[9] double-stranded (ds) DNA cleavage[4,5,6]. To overcome the target vector construction and in vitro RNA transcription required for sgRNA, a recent study used crRNA and tracrRNA in combination with commercially available Cas[9] protein and a regular gene targeting vector, to generate knock-in mice[2]. In our current study we report a method that capitalized on a cloning-free CRISPR/Cas[9] system using commercial Cas[9] protein combined with chemically synthesized crRNA, tracrRNA, and single-strand oligodeoxynucleotides (ssODNs). This system allowed us to consistently obtain high-efficiency editing of multiple genes in the mouse, and successfully generate non-mosaic mutant mouse models with a variety of editing schemes, including, frame-shift indel mutations, single-amino-acid substitutions, and LoxP-inserted conditional alleles. Our method is a simple, cloning-free, and highly efficient technique, which accelerates our ability to produce rapid mouse genome editing in vivo

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Conclusion