Basto / Oegema Centrosome and Centriole

1. Auflage 2015
ISBN: 978-0-12-802638-0
Verlag: Elsevier Science & Techn.
Format: EPUB
Kopierschutz: 6 - ePub Watermark

E-Book, Englisch, Band Volume 129, 434 Seiten

Reihe: Methods in Cell Biology

This new volume of Methods in Cell Biology looks at methods for analyzing centrosomes and centrioles. Chapters cover such topics as methods to analyze centrosomes, centriole biogenesis and function in multi-ciliated cells, laser manipulation of centrosomes or CLEM, analysis of centrosomes in human cancers and tissues, proximity interaction techniques to study centrosomes, and genome engineering for creating conditional alleles in human cells. - Covers sections on model systems and functional studies, imaging-based approaches and emerging studies - Chapters are written by experts in the field - Cutting-edge material

Autoren/Hrsg.

Basto, Renata

Oegema, Karen

Leseproben

Chapter 2

Generation of a conditional analog-sensitive kinase in human cells using CRISPR/Cas9-mediated genome engineering

Tyler C. Moyer and Andrew J. Holland1     Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA
1 Corresponding author: E-mail: [e-mail] 

Abstract

The ability to rapidly and specifically modify the genome of mammalian cells has been a long-term goal of biomedical researchers. Recently, the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system from bacteria has been exploited for genome engineering in human cells. The CRISPR system directs the RNA-guided Cas9 nuclease to a specific genomic locus to induce a DNA double-strand break that may be subsequently repaired by homology-directed repair using an exogenous DNA repair template. Here we describe a protocol using CRISPR/Cas9 to achieve bi-allelic insertion of a point mutation in human cells. Using this method, homozygous clonal cell lines can be constructed in 5–6 weeks. This method can also be adapted to insert larger DNA elements, such as fluorescent proteins and degrons, at defined genomic locations. CRISPR/Cas9 genome engineering offers exciting applications in both basic science and translational research.

Keywords

Analog-sensitive kinase; Centrosome; Chemical genetics; CRISPR; Genome editing; Plk4

Introduction

Genome engineering is a term used to describe the process of making specific, targeted alterations in the genome of a living organism. Genome engineering exploits the repair of a DNA double-strand break (DSB) through the endogenous pathway of homologous recombination (HR). By providing an exogenous DNA repair template that contains homology to the targeted site, it is possible to exploit the HR machinery to create defined alterations close to the site of a DSB. However, mammalian genomes comprise billions of base pairs and there is a low probability of a spontaneous DSB occurring close to the region to be targeted; as a consequence, desired recombination events occur extremely infrequently (Capecchi, 1989). A major breakthrough came with the demonstration that targeted DSBs greatly increase the frequency of homology-directed repair (HDR) at a specific locus (Choulika, Perrin, Dujon, & Nicolas, 1995; Plessis, Perrin, Haber, & Dujon, 1992; Rouet, Smih, & Jasin, 1994; Rudin, Sugarman, & Haber, 1989). This discovery has spurred the development of programmable endonucleases that can be exploited to promote site-specific cleavage of the genome.

Zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) are artificial restriction enzymes produced by fusing customizable DNA binding domains to the sequence-independent nuclease domain of the restriction enzyme Fok1 (Boch et al., 2009; Christian et al., 2010; Miller et al., 2007, 2011; Moscou & Bogdanove, 2009; Urnov et al., 2005). Fok1 requires dimerization for its activity, and thus a pair of ZFNs or TALENs is required to bind to opposite strands of DNA on either side of a target site to allow Fok1 dimerization and DNA cleavage. While ZFNs and TALENs have been shown to be capable of creating targeted DNA breaks and introducing genomic sequence changes through HDR, difficulties in protein design and synthesis proved to be a barrier to their widespread use (Hsu, Lander, & Zhang, 2014).

1. CRISPR/Cas System

Recently, a new tool based on clustered, regularly interspaced, short palindromic repeats (CRISPR) systems from bacteria have been exploited for genome engineering in human cells and have generated considerable excitement (Hsu et al., 2014). CRISPR systems have the distinct advantage of using RNA-guided nuclease activity to target cleavage of DNA and thereby eliminate the need for protein engineering and optimization.

CRISPR/Cas modules were identified in bacteria as part of an adaptive immune system that enables hosts to recognize and cleave foreign invading DNA (Horvath & Barrangou, 2010; Marraffini & Sontheimer, 2010). CRISPR modules comprise arrays of short nucleotide repeats interspersed with unique spacers that share homology with foreign phage or plasmid DNA. Of the three CRISPR/Cas systems that have evolved in bacteria, the type II system is the simplest and involves only three components: a processed RNA that is complementary to the spacers, known as a CRISPR-RNA (crRNA), a trans-activating tracrRNA that hybridizes to the crRNA, and the Cas9 nuclease. The crRNA and the tracrRNA form an RNA double-strand structure that directs Cas9 to generate DSBs at a site complementary to the targeting region of the crRNA (Brouns et al., 2008; Deltcheva et al., 2011; Garneau et al., 2010). The RNA components of the CRISPR/Cas9 system (the crRNA and the tracrRNA) can be combined into a singular guide RNA (gRNA) (Jinek et al., 2012). The gRNA directs Cas9 to induce DSBs in the genome of cells at sites complementary to a ~20 base pair targeting sequence in the gRNA. The simplicity of these RNA-guided nucleases has allowed scientists to repurpose the CRISPR/Cas9 system to create site-specific DNA breaks in a variety of eukaryotic cells (Cong et al., 2013; Mali et al., 2013).

2. Analog-Sensitive Kinases

Nearly one-third of the proteome is subject to phosphorylation by protein kinases. Adenosine triphosphate (ATP)-competitive small molecule inhibitors are powerful tools for probing the function of kinases in living cells. However, many kinases possess a similar catalytic core, and thus achieving specificity in inhibiting kinase activity in cells is a major challenge. One method to overcome this limitation is to exploit a chemical genetic strategy in which a kinase is engineered to accept ATP analogs that are not efficiently utilized by wild-type kinases. These engineered kinases are referred to as analog-sensitive (AS) kinases (Bishop et al., 2000). This is achieved through the mutation of a bulky hydrophobic ‘gatekeeper’ amino acid in the ATP binding pocket to a smaller amino acid (alanine or glycine) (to identify the gatekeeper residue for a kinase see http://sequoia.ucsf.edu/ksd) (Liu et al., 1999). An AS kinase can be specifically inhibited with generic nonhydrolyzable bulky ATP analogs, allowing rapid and reversible control of kinase activity in cells. Despite broad utility, the use of the AS kinase approach in mammalian cells has been hampered by the difficulty of functionally replacing an endogenous kinase with appropriate levels of an AS kinase. The development of CRISPR/Cas9 offers a facile method for introducing AS mutations into endogenous mammalian kinases.

Here, we outline a method for using CRISPR/Cas9 genome engineering to introduce an AS point mutation in a single step into both alleles of Polo-like kinase 4 (Plk4). This protocol can be used to introduce point mutations into any target gene of choice and could also be adapted to insert larger DNA elements, such as fluorescent proteins and degrons, at defined genomic locations.

3. Methods

3.1. Designing a Guide RNA for Sequence-Specific DNA Cleavage by SpCas9

The CRISPR/Cas9 system is capable of generating targeted DSBs in the genome of mammalian cells. The Streptococcus pyogenes (Sp) CRISPR/Cas9 system is the most widely used system and will be the focus of this protocol. The specificity of SpCas9 targeting is determined by a 20-nucleotide (nt) targeting sequence within the gRNA that is complementary to the genomic target sequence. The genomic target sequence must precede an “NGG” sequence known as the protospacer adjacent motif (PAM), which is necessary for target cleavage, but is not encoded within the gRNA (Figure 1) (Mojica, Diez-Villasenor, Garcia-Martinez, & Almendros, 2009; Shah, Erdmann, Mojica, & Garrett, 2013). The PAM sequence has evolved to ensure that the CRISPR/Cas9 system does not self-target the CRISPR modules in the bacterial genome (Shah et al., 2013). SpCas9 usually cleaves the DNA 3-nt upstream of (i.e., 5' to) the PAM to produce a blunt-ended DSB (Figure 1). Breaks can either be repaired by HDR or through error-prone nonhomologous end-joining (NHEJ) pathway, which usually introduces insertions and deletions (InDels) of bases at the cut site.

Several plasmid constructs are available for SpCas9/gRNA expression in mammalian cells. In our experiments we have used the PX459 vector (available from Addgene, vector #62988), which enables expression of a gRNA, SpCas9, and a puromycin resistance gene from a single vector. In this section we describe how to design a gRNA to direct cleavage at a specific genomic site.

Figure 1 Genome Editing Using the CRISPR/Cas9 System.

SpCas9 nuclease is directed to a specific locus through base-pairing of the targeting sequence (underlined) of its associated guide RNA (gRNA) with a genomic target sequence. The genomic target sequence is followed by a protospacer-adjacent motif (PAM, red (light gray in print versions)) that is required for SpCas9 recognition and cleavage. Double-stranded cleavage usually occurs three base pairs upstream of the PAM. The double-strand break (DSB) may be repaired through homology directed repair (HDR) using an exogenously supplied repair template that contains a mutation of interest (green (dark gray in print versions)). Inserting a second mutation...