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Choosing the Right Tool for the Job: Rnai, TALEN, Or CRISPR

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Choosing the Right Tool for the Job: Rnai, TALEN, Or CRISPR Molecular Cell Review Choosing the Right Tool for the Job: RNAi, TALEN, or CRISPR Michael Boettcher1 and Michael T. McManus1,* 1Department of Microbiology and Immunology, UCSF Diabetes Center, Keck Center for Noncoding RNA, University of California, San Francisco, San Francisco, CA 94143, USA *Correspondence: [email protected] http://dx.doi.org/10.1016/j.molcel.2015.04.028 The most widely used approach for defining gene function is to reduce or completely disrupt its normal expression. For over a decade, RNAi has ruled the lab, offering a magic bullet to disrupt gene expression in many organisms. However, new biotechnological tools—specifically CRISPR-based technologies—have become available and are squeezing out RNAi dominance in mammalian cell studies. These seemingly competing technologies leave research investigators with the question: ‘‘Which technology should I use in my experiment?’’ This review offers a practical resource to compare and contrast these technologies, guiding the investigator when and where to use this fantastic array of powerful tools. Introduction by showing that simple injection of double-stranded RNA into The triumphal sequencing of the Human Genome Project C. elegans could potently silence any gene sequence and pro- (Lander et al., 2001) offered new insights into the fundamental duce phenotypes that revealed gene function (Fire et al., 1998). inner workings of humans, promising a big step toward curing As the mechanisms for RNAi were established, it soon found humankind of most diseases. More than 15 years after its use in human cells to inhibit specific genes (Elbashir et al., completion, biologists continue to struggle with this vast amount 2001). Its ease of use made RNAi the method of choice for deci- of genomic sequence encoding tens of thousands of genes of phering gene function. However, RNAi produces hypomorphic unknown function (Birney et al., 2007). Sequencing the genome phenotypes, which do not always mirror the complete loss-of- was an incredible challenge but, in broader perspective, was function that can occur with genetic mutation. This and other only the first small step. The most difficult challenge lies ahead; practical caveats have encouraged scientists to develop new deciphering the cryptic meaning of the 3.3 billion base pairs of tools for reverse genetics. DNA, by assigning functions to the tens of thousands of genes, Complete loss-of-function reverse genetics approaches and determining how they work together to make us human. became available with the discovery of zinc-finger nucleases This is the grand biological promise yet to be fulfilled, and with (ZFNs) and later, transcription activator-like effector nucleases the recent development of new biotechnological tools, the (TALENs) (Gaj et al., 2013). These approaches utilize customiz- biggest discoveries are yet to come. able DNA-binding domains (DBDs) that are engineered to recog- The gold standard for deciphering gene function is to disrupt nize specific target DNA sequences. Fused to nucleases, DBDs normal gene expression and study the resulting phenotypes. can be used to introduce double-strand breaks (DSBs) and sub- Such loss-of-function experiments have been performed for sequent frameshift mutations into genes, which can lead to their more than 100 years, beginning with the work of Thomas Morgan knockout (Sung et al., 2013). who discovered that genes are on chromosomes and carry mu- A more recent and incredibly powerful addition to the genome tations responsible for phenotypes. With this knowledge, gener- editing toolbox is the CRISPR/Cas system. Its involvement in ations of scientists have worked hard to comprehensively bacterial resistance against viral infections was initially described mutate genes, using chemicals, radiation, and random virus in Streptococcus thermophilus (Barrangou et al., 2007). integration to painstakingly map each phenotype to a specific Currently, the Type II CRISPR/Cas9 system from Streptococcus mutant gene. This forward genetics approach has taken de- pyogenes is the most widely used CRISPR system and was suc- cades, as artisan techniques fraught with numerous technical cessfully applied to edit human genomes (Cho et al., 2013; Cong challenges complicate the attempts to map random and seem- et al., 2013; Jinek et al., 2013; Mali et al., 2013c). The full history of ingly minute lesions in a sea of genomic DNA. The sequencing the discovery and development of the CRISPR/Cas system has of the human genome offered a map by which gene function been excellently reviewed previously (Doudna and Charpentier, could be deciphered but lacked the means to selectively disrupt 2014). Together, these powerful tools offer a new promise to a specific gene in a nonrandom manner. rapidly and efficiently decipher any gene’s function. The discovery of RNAi by Fire and Mello promised a magic bul- With the increasing variety of molecular tools available for loss- let to target any gene, provided the investigator knows the DNA of-function experiments, many researchers have difficulty with sequence (reverse genetics). The timing of the discovery could selecting the most appropriate system. This review compares not have been more perfect, as the method was published and contrasts the fantastic new tools summarized in Table 1, shortly after the human genome sequence became freely avail- and offers researchers a ‘‘practical guidebook’’ for determining able. The work of Fire and Mello astounded the scientific world how to uncover gene function in cells. Which one should you use? Molecular Cell 58, May 21, 2015 ª2015 Elsevier Inc. 575 Molecular Cell Review Table 1. Comparisons between RNAi, TALE, and CRISPR Centric Technologies RNAi TALE Repression TALEN Cas9 Nuclease CRISPRi CRISPRa Loss-of-function Post-transcriptional Repression of Frame shift DNA Frame shift DNA Repression of Activation of mechanism RNA degradation transcription mutation mutation transcription transcription Result Reversible Reversible Permanent Permanent Reversible Reversible knockdown knockdown knockout knockout knockdown activation Transgenes si/shRNA TALE-KRAB TALEN Cas9 nuclease dCas9-KRAB dCas9-VP64 sgRNA sgRNA sgRNA Guiding sequence si/shRNA DBD DBD sgRNA sgRNA sgRNA Required sequence Transcriptome Annotated TSS Transcriptome Transcriptome Annotated TSS Annotated TSS information Off-target space Transcriptome Window around Genome; requires Genome; cuts Window around Window around TSS FokI dimerization as monomer TSS TSS Transcript variants All variants via Only variants from All variants via All variants via Only variants from Only variants from conserved region the same TSS conserved region conserved region the same TSS the same TSS RNAi goillot and King, 2011). These off-target effects appear to be RNAi is currently the most extensively used reverse genetics dosage dependent (Wang et al., 2009), and phenotypes derived approach to study gene function in mammalian cells. Its success from them can be dominant over the intended on-target pheno- can be attributed to an evolutionarily conserved endogenous types (Franceschini et al., 2014). pathway that regulates gene expression via small RNAs (Wilson In addition to sequence specific off-target effects, the arti- and Doudna, 2013). The endogenous RNAi machinery can be ficial introduction of siRNAs or shRNAs into a target cell ‘‘hijacked’’ by introducing synthetic small RNAs into cells; this line can cause non-sequence-specific off-target effects. As is commonly achieved using short interfering RNAs (siRNAs) or mentioned above, RNAi uses a natural pathway that regulates short hairpin RNAs (shRNAs) (Mohr et al., 2014). The result cellular gene expression levels, where the system is governed from either approach is similar, such that the introduced RNA by endogenous microRNAs. Flooding the system with exoge- is loaded into the RNA-induced silencing complex (RISC), which nous sequences can displace microRNAs from RISC. This in turn promotes the degradation of perfectly complementary can impair the functions of endogenous microRNAs, leading target mRNA (Figure 1A) (Carthew and Sontheimer, 2009). Using to alterations in gene transcript levels and consequently this approach, target mRNA and subsequently target protein to off-target phenotypes (Khan et al., 2009). These observa- levels can be reduced postranscriptionally. tions fueled the effort to develop alternative reverse genetics However, given that the RNAi machinery appears to be mostly approaches. active in the cytoplasm, nuclear transcripts—for example long non-coding RNAs (lncRNAs)—may be more difficult to target Transcription Activator-Like Effector (Derrien et al., 2012; Fatica and Bozzoni, 2014). Moreover, it A fundamentally different approach to modulate gene function was recently shown that the transcription of lncRNA itself can became possible with the development of programmable DNA- have functional consequences (Kornienko et al., 2013). Hence, binding proteins: transcription activator-like effectors (TALEs). reducing lncRNAs at a post-transcriptional level via RNAi may This approach makes use of synthetic transcription factor DBDs not always reveal their full loss-of-function phenotype (Bassett that can be programmed to recognize specific DNA motifs. et al., 2014). TALE DBDs contain 7–34 highly homologous direct repeats, A major advantage of RNAi is that the silencing machinery is each consisting of 33–35 amino acids. The key to specificity is present in practically every mammalian somatic cell. Hence, no contained in the two amino acid residues
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