Academic journal article Genetics

A Mouse Geneticist's Practical Guide to CRISPR Applications

Academic journal article Genetics

A Mouse Geneticist's Practical Guide to CRISPR Applications

Article excerpt

PHENOTYPIC characterization of mutations is the most accurate and widely used method for elucidating in vivo gene functions and the genetics of diseases. Generation of human disease models is constrained by available genetic tools for a given model system. The laboratory mouse is the most widely used mammalian model due to its powerful genetics, embryonic stem (ES) cell technology, and routine transgenesis and mutagenesis. Traditional gene knockouts produced by gene targeting in ES cells usually produce null mutations; strategies to generate more subtle changes to proteins involve multiple rounds of manipulation in ES cells or forward genetic approaches such as ENU mutagenesis. The discoveries of sequence- specific nucleases have allowed researchers to precisely manipulate embryonic genomes in a wide range of experimental models (including mouse, rat, pig, fish, rabbit, fruit fly, frog, rhesus monkey, etc.), obviating the need for ES cells as an essential intermediate. This new genre of genome editing technologies involves generation of DNA double strand breaks (DSBs) in precise genomic locations by targetable nucleases and exploiting cellular repair machinery to produce mutations. The recently developed clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system is revolutionizing genetics not only in organisms in which gene targeting was not previously possible, but also in the laboratory mouse, where ES cell technology has enabled gene targeting and genome manipulation for nearly three decades.

The "CRISPR" system is a versatile prokaryotic antiviral defense mechanism providing adaptive immunity for a host bacterium against extrachromosomal genetic material (Horvath and Barrangou 2010). This RNA-guided bacterial innate immune system essentially involves three distinct steps: (1) acquisition of foreign DNA, (2) synthesis and maturation of CRISPR RNA (crRNA) followed by formation of RNA-Cas nuclease protein complexes, and (3) target recognition by crRNA and destruction of foreign DNA by Cas nuclease cleavage (Aida et al. 2014; Mashimo 2014; Sander and Joung 2014). Three different types of CRISPR-Cas systems have been described (Makarova et al. 2011). However, due to the simplicity, high efficiency, and multiplexing capability of the type II CRISPR/Cas system, it has been adopted as the genome editing technology of choice. The type II system utilizes a single Cas9 nuclease sufficient to cleave the target DNA specified by crRNA. The ability of targeting any genomic location opened new genome manipulation possibilities. In addition to genome editing, the system was quickly developed as a tool to regulate gene expression. Here we provide an overview of current advancements in this rapidly evolving technique to manipulate the mouse genome.

Mutagenic Capabilities of the CRISPR/Cas9 System

The versatility of CRISPR/Cas9 as a genome editing tool arises from its ability to recognize virtually any sequence in the genome and introduce a controlled break in the DNA. These breaks are repaired by error-prone or high-fidelity cellular mechanisms. The nuclease activity of the CRISPR/Cas9 system is guided by two noncoding RNA elements: (1) crRNA containing 20 bp of unique target sequence (spacer sequence) and (2) tracrRNA (trans-activating crRNA). The crRNA:tracrRNA duplex (also termed guiding RNA or gRNA) directs Cas9 nuclease to target DNA in the genome via complementary base pairing between the spacer on the crRNA and the complementary sequence (called protospacer) on the target DNA. Target specificity of Cas9 protein relies on the presence of specific nucleotides 39 to the protospacer sequence, termed the protospacer adjacent motif (PAM). The Cas9 RNA-guided endonuclease from Streptococcus pyogenes, spCas9, requires a 59-NGG-39 PAM, whereas Cas9 from S. thermophilus (stCas9) and Neisseria meningitidis (nmCas9) require 59-NNAGAAW-39 PAM(W= A or T) and 59-NNNNGATT-39 PAM motifs, respectively (Hou et al. …

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