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?

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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 in positions 12 and 13 prior genetic manipulation of the target cell line is required, and of each repeat, which are referred to as repeat variable diresidue a simple siRNA transfection can result in a loss-of-function (RVD) (Joung and Sander, 2013). Since the DNA:protein binding phenotype. code of RVDs has been deciphered (Boch et al., 2009; Moscou Specificity and Off-Target Effects of RNAi and Bogdanove, 2009), it is possible to design TALEs that bind Since its discovery, off-target effects have been a growing any desired target DNA sequence by engineering an appropriate concern when using RNAi as a tool to study gene function. It DBD. Typically, the TALEs are designed to recognize 15 to 20 has been found that siRNAs can induce silencing of non-target DNA base-pairs, balancing specificity with potential off targeting mRNAs with limited sequence complementarity, often via inter- (Miller et al., 2011; Mussolino et al., 2011). This means that for actions with the 30 UTR (Carthew and Sontheimer, 2009). These every individual target site in the genome, it is necessary to design interactions occur when short regions of the mRNA 30 UTR a different DBD, each consisting of 500–700 amino acids, which contain imperfect matches to the small RNA, triggering transla- can be a lengthy process. tional repression and/or degradation (Jackson et al., 2006; Si- In recent years, the inherent programmability of TALEs has goillot and King, 2011). In this way, depending on the sequence, been used to execute a variety of functions, simply by fusing one siRNA can potentially repress hundreds of transcripts (Si- various effector domains to the DBD.

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A such as FokI are fused to the TALE DBD, resulting in a TALEN (Gaj et al., 2013). FokI is active only as a dimer; hence, pairs of TALENs are constructed to position the FokI nuclease domains to adjacent genomic target sites, where they introduce DNA double strand breaks (DSBs) (Figure 1C). Although this strategy requires the construction of two TALENs per target site, great specificity is achieved since a DSB occurs only after correct positioning and dimerization of FokI (Christian et al., 2010). Once the DSB is introduced, DNA repair can be achieved via B two different mechanisms: the high fidelity homologous recombination repair (HRR) or the error-prone non-homologous end joining (NHEJ) (Valerie and Povirk, 2003). Repair of DSBs via NHEJ frequently results in DNA target site deletions, insertions, or substitutions (Durai et al., 2005). In TALEN technology this is desired, and users typically target TALEN pairs to the most 50 exons of genes, promoting early frame shift mutations or prema- ture stop codons. Given the permanence of the genetic lesion, there is no need for maintained TALEN activity inside the target cell, once the mutation has been introduced. This is in contrast C to the reversible nature of RNAi, where loss of the small RNA equates to the loss of any evoked phenotype. In addition to the introduction of random reading frameshift mutations via NHEJ, TALENs can also be utilized to introduce non-random point mutations, targeted deletion, or addition of large DNA fragments via the aforementioned HRR. In the past, genome editing in mammalian cells was primarily achieved via the introduction of donor DNA that contained homologous sequences to the target locus. However, this process works very inefficiently in mammalian cells (Vasquez et al., 2001). More recently it was found that the introduction of DNA DSBs in- creases the efficiency of gene editing via homologous recombination, in the presence of suitable donor DNA (Moehle et al., 2007). Since targeted DSBs can be easily introduced into DNA using TALENs, the combination of such DSBs and the transfec- Figure 1. Mechanisms of Action of RNAi and TALE/N tion of a homologous donor DNA repair template has become a (A) RNAi degrades transcripts post-transcriptionally in the cytoplasm. popular approach to precisely engineer mammalian genomes, (B) TALE repression prevents transcription when targeted to the TSS. (C) Dimerized TALENs induce a site-specific DSB that is repaired via error- as reviewed in detail by Gaj et al. (2013). prone NHEJ, potentially leading to frameshift mutations when exons are Specificity and Off-Target Effects of TALEN targeted. Technologies Because of the requirement of FokI to dimerize in order to have TALE Transcriptional Repressor nuclease activity, no single TALEN can ever introduce a DSB into Gene silencing by means of transcriptional repression can a target site. As a consequence, two TALENs must off-target be achieved using KRAB-TALEs, where the transcriptional adjacent sites to introduce an unspecific DSB into the DNA. repressor Kru¨ ppel-associated box (KRAB) domain (Margolin Consequently, the off-target activity observed from TALENs is et al., 1994) is fused to DBDs that target the transcription start typically low (Guilinger et al., 2014a; Hockemeyer et al., 2011; site (TSS) (Cong et al., 2012)(Figure 1B). A crucial difference of Mussolino et al., 2011). Unlike TALENs, TALE transcriptional this approach when compared to RNAi is that TALE repression repressors function as monomers and can hence mediate off- prevents the transcription of targeted genes in the nucleus, while target effects autonomously. RNAi degrades them post-transcriptionally in the cytoplasm. This allows, for instance, the study of functional effects that Clustered Regularly Interspaced Short Palindromic derive from transcription per se, as recently described for Repeats (CRISPR) lncRNAs (Bassett et al., 2014). In another way though, TALE- Cas9: An RNA Guided DNA Endonuclease repression mimics the hypomorphic effect of RNAi, such that Similar to TALENs, the CRISPR-associated protein 9 (Cas9) is gene function is reduced but not shut off permanently, as it is also a programmable DNA nuclease. However, its mode of oper- the case for TALENs. ation differs in several ways: first of all, Cas9 possesses innate TALEN nuclease activity. The enzyme is active as a monomer and TALE DBDs have also been successfully used to mutate genes in catalyzes DSBs, (Hsu et al., 2014); hence, targeting a single mammalian cells. In this strategy, nuclease effector domains Cas9 molecule to the early exons of genes generates random

Molecular Cell 58, May 21, 2015 ª2015 Elsevier Inc. 577 Molecular Cell Review

A screen several clonal lines for homozygous gene disruption (Ran et al., 2013b). Another major difference between TALENs and Cas9 is the way both proteins recognize their genomic target sites. While TALENs encode the DNA target motif specificity in the amino acid sequence of their DBD, Cas9 requires a single chimeric guide RNA (sgRNA) to recognize its target site (Mali et al., 2013b)(Figure 2A). So, while for TALENs the amino acid sequence of their DBD has to be painstakingly re-designed for each target site, one invariant Cas9 nuclease can be tar- B geted to a large variety of DNA motifs, simply by co-expression of a target-site-specific sgRNA. It is this easy programmability that allowed the CRISPR/Cas9 platform to rapidly replace other genome editing platforms, including ZFNs and TALENs. In the Type II CRISPR/Cas9 system from Streptococcus pyogenes, which is currently the most popular system used for genome editing in mammalian cells, the sgRNA is composed of two separately expressed RNAs, a CRISPR RNA (crRNA), and a trans-activating crRNA (tracrRNA), which are processed C by the endogenous bacterial machinery to yield the mature gRNA (Chylinski et al., 2014; Deltcheva et al., 2011). Later it was shown that it is possible to express a single chimeric sgRNA, a fusion of crRNA, and tracrRNA, which is able to act as a mature sgRNA in vitro, without the necessity of further process- ing (Jinek et al., 2012). This chimeric sgRNA, similar to the fully processed native bacterial sgRNA, consists of a constant region that mediates interaction with Cas9 and a variable region that mediates interaction with the genomic target site. Chimeric sgRNAs currently used for genome engineering typically contain a 17- to 20-nt long variable region which is complementary to the genomic target sequence. More recently, a modified chimeric sgRNA design was developed in order to enhance its expression and interaction with Cas9. For this purpose, a potential PolIII terminator was removed from the constant region of the sgRNA and a hairpin was extended by 5 bp (Chen et al., 2013) to restore the full S. pyogenes hairpin sequence identified by (Deltcheva et al., 2011). Importantly, a major specificity determinant of Cas9 is the pro- Figure 2. Mechanisms of Action of CRISPR-Based Technologies 0 tospacer adjacent motif (PAM), a short region immediately 3 of (A) Cas9 nuclease monomer is guided to its target site via sgRNA and induces a site-specific DSB that is repaired via error-prone NHEJ, potentially leading to the target sequence (Sternberg et al., 2014). This PAM has the frameshift mutations when exons are targeted. sequence NGG for Cas9 from Streptococcus pyogenes and (B) CRISPRi prevents transcription when targeted to the TSS. varies for Cas proteins from different species (Horvath et al., (C) CRISPRa activates transcription when targeted to the TSS. 2008). Examples of orthogonal protospacer adjacent motifs include NGGNG (Horvath et al., 2008), NAAR (van der Ploeg, knockout phenotypes via DSBs and NHEJ repair, similar to tar- 2009), and NNAGAAW (Deveau et al., 2008). Although the de- geting pairs of TALENs to the same site. As with TALENs, to pendency of the PAM limits Cas9 target space, it offers another achieve a complete knockout of that gene, every functional level of specificity as discussed later, in the Off-Target Effects copy of a target gene needs to be disrupted. A maximum in section. the fraction of successfully edited cells is typically observed at CRISPR Interference around 5 to 8 days post-transduction (Wang et al., 2014; Zhou The ease with which Cas9 can be programmed to bind a specific et al., 2014). Thus far, current protocols yield editing efficiencies region of the genome prompted several groups to develop tran- of up to 50% when quantified via SURVEYOR assay (Hsu scriptional repressors. Similar to TALE transcriptional repres- et al., 2013; Mali et al., 2013c). Even in the near haploid cell sors, an enzymatically inactive version of Cas9 (deadCas9 or line KBM7, the maximum editing efficiency reported is 70% dCas9) is fused to a repressor domain (e.g., KRAB), triggering (Wang et al., 2014). Hence, to obtain homozygous knockout heterochromatin mediated gene silencing at the TSS (Qi et al., cell lines for a target gene of interest, it is usually necessary to 2013)(Figure 2B). This approach is termed CRISPRi (Gilbert

578 Molecular Cell 58, May 21, 2015 ª2015 Elsevier Inc. Molecular Cell Review

et al., 2013) and leads to transcriptional reduction of the target geting the same TSS (Cheng et al., 2013; Maeder et al., 2013; RNA, similar to TALE transcriptional repression. Mali et al., 2013a; Perez-Pinera et al., 2013). To overcome this In comparison to RNAi, CRISPR interference (CRISPRi) ap- limitation, several novel strategies were recently described pears to produce a more consistent and robust knockdown (Chavez et al., 2015; Konermann et al., 2015; Tanenbaum given the same number of effector RNAs. For example, six out et al., 2014). All approaches demonstrated efficient transcrip- of eight sgRNAs were found to repress GFP levels by at least tional activation of endogenous genes via expression of only 75% (Gilbert et al., 2013), and a direct comparison to gene inhi- one sgRNA per cell. bition via RNAi shows significantly stronger loss-of-function Similar to CRISPRi, the target site is crucial for CRISPR activa- phenotypes when using CRISPRi (Gilbert et al., 2014). tion (CRISPRa) to obtain maximal activation. The region up- Like any technology, CRISPRi comes with its own caveats. stream of the TSS (400 to 50 nt) is typically considered to Similar to RNAi, we still lack a solid understanding of the rules be the optimal target site for CRISPRa sgRNAs (Gilbert et al., by which a given sgRNA may engage and be active on a given 2014; Konermann et al., 2015). This is mutually exclusive target site. We do know that the accessibility and location of with the CRISPRi target space downstream from the TSS the TSS is critical: Cas9 needs to physically access its target (0 to +500 nt). The dependency of CRISPRa on the TSS of the tar- site. This process has been shown to be dependent on chro- geted transcript means that challenges are similar to the afore- matin accessibility (Kuscu et al., 2014; Wu et al., 2014). Hence, mentioned ones associated with CRISPRi. Most importantly, it genomic regions with closed chromatin states may prevent bind- is essential to know the location of the TSS to effectively activate ing and thus the function of Cas9. expression, and second, activation of multiple transcripts from To repress transcription with maximum efficiency, the KRAB one and the same TSS cannot be controlled individually. domain should be targeted to a window of 500 bp downstream RNA Polymerase Promoters for sgRNA Expression from the TSS (Gilbert et al., 2014). Hence, it is important to know The vast majority of studies have used polymerase III promoters the location of the TSS of targeted transcripts. Importantly, even (U6, H1, 7SK, and tRNA promoters) to transcribe the sgRNA, in fully sequenced genomes, like the human genome, some given its natural ability to transcribe small RNAs at high levels TSSs may be experimentally hard to annotate, such as miRNA (Kabadi et al., 2014). In some contexts, it may be desirable to TSSs (Georgakilas et al., 2014). Moreover, many genes have express sgRNAs using polymerase II promoters. For example, alternative transcripts, some with TSSs very distant from each tissue-specific expression could be achieved by the use of other. The usage of these TSSs can vary between cell types cell-type-specific promoters. However the use of polymerase II (Sandelin et al., 2007), so it is important to target the correct promoters complicates the ability to express functional sgRNAs, TSS for a given cell type. Moreover, different transcript variants given the natural path of capped polyadenylated RNAs to shuttle expressed simultaneously from alternative TSSs can act redun- through the ribosome as an mRNA. To overcome this issue, Nis- dantly (Davuluri et al., 2008). Consequently, if multiple transcript sim et al. (2014) developed several approaches that allow the variants are expressed in a cell, blocking the transcription of one expression of single and multiple sgRNAs from human RNA po- of them at a particular TSS may not be sufficient to produce a lymerase II promoters, permitting CRISPRi and CRISPRa in tran- loss-of-function phenotype. sient transfection assays. This was accomplishable by flanking A different problem arises when distinct transcripts share the the sgRNA with either ribozyme or Csy4 RNA cleavage sites. same TSS, as commonly is the case for intragenic non-coding Future studies should determine whether these approaches RNAs (He et al., 2012). Targeting dCas9-KRAB to such TSSs are transferable to single copy settings, such as genomically in- could potentially block the transcription of all associated tran- tegrated sgRNA expression cassettes. scripts. Attributing the observed knockdown phenotype to one transcript or another thus requires additional experiments. Specificity and Off-Target Effects of CRISPR CRISPR Activation Technologies Gain-of-function studies in mammalian cells have traditionally Specificity and Target Site Recognition been carried out by overexpressing transgenic open reading A number of studies have examined Cas9 for off-targeting and frames (ORFs or cDNAs) in target cells. This approach involves found that the PAM plays an essential role in DNA target site cloning an ORF behind a promoter and introducing it into target recognition. A random collision model was proposed—in which cells. More recently, CRISPR systems have been adapted to Cas9 dissociates much more rapidly from the DNA when no allow the expression of genes from their endogenous genomic PAM is present (Sternberg et al., 2014). Once Cas9 recognizes locus, bypassing the need to clone and express the oftentimes the PAM, it then interrogates the 50-flanking DNA for sgRNA large ORFs in cells. To date, several dCas9-based approaches complementarity. According to this model, target site specificity have successfully been used to activate transcription in is determined by the sequence adjacent to the PAM. In support mammalian cells (Cheng et al., 2013; Farzadfard et al., 2013; of this model, Hsu et al. found that Cas9 can only cut target sites Maeder et al., 2013; Mali et al., 2013a; Perez-Pinera et al., 2013). after recognizing the PAM (Hsu et al., 2013). They found that the All approaches rely on targeting transcriptional activation do- proximal 8–14 nucleotides immediately upstream of the PAM mains to the TSS of genes; most commonly, VP64 (Beerli et al., were most important for target site cleavage. Other studies pro- 1998) is fused to dCas9 (Figure 2C). Direct dCas9-VP64 fusion vided additional evidence for the existence of a seed region adja- proteins, however, have been shown in numerous studies to effi- cent to the PAM (Cong et al., 2013; Fu et al., 2013). Both studies ciently activate transcription of endogenous genes only when showed that mismatches in the seed region reduced Cas9 activ- they are co-expressed with multiple (three to five) sgRNAs tar- ity more than mismatches further away from the PAM.

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Off-Target Effects by the error-prone NHEJ. This approach has been shown to By means of dCas9-ChIP-seq, Wu et al. (2014) found that dCas9 reduce off-targeting by 50- to 1,500-fold (Cho et al., 2014; Ran binds to a large number of off-target sites. In fact, most dCas9- et al., 2013a). gRNA complexes were bound to sites other than the target site. A second approach very closely resembles TALENs and is Interestingly, all top ranking off-target motifs were 50 of a PAM based on the fusion of the FokI domain to a catalytically inactive (NGG) and contained the correct 5 bp directly adjacent to the Cas9. This approach makes use of the dependency of FokI on PAM. The degree of off-targeting appeared to be related to dimerization in order to cut the target DNA. It was shown that sgRNA levels. In a very similar study using dCas9-ChIP-seq, dCas9-FokI dimerization reduces off-target activity even further Kuscu et al. (2014) came to the same conclusions, namely that when compared to the Cas9 nickase approach (Guilinger et al., the PAM is the strongest determinant for off-target binding fol- 2014b; Tsai et al., 2014). In addition to modifications of Cas9, lowed by the nucleotides most proximal to the PAM. Both truncations of the sgRNA to 17 or 18 nt have been shown to studies found that the frequency of DNA cleavage in the identi- reduce off-targeting without reducing on-target efficiency (Fu fied off-target binding sites was much lower than the frequency et al., 2014). of binding. To further investigate off-target cleavage in an unbiased Design Rules and Tools for siRNAs, shRNAs, and sgRNAs manner, several groups have developed approaches to To maximize on-target and minimize off-target effects, careful directly analyze unwanted DSBs produced by catalytically attention should be paid to siRNA, shRNA, and sgRNA design. active Cas9 (Crosetto et al., 2013; Frock et al., 2015; Kim In this section we provide a very brief overview of the currently et al., 2015; Tsai et al., 2015). Using GUIDE-seq, Tsai et al. accepted design rules for each of those RNA reagents. (2015) found that the number of off-target cutting events varies Design of siRNAs and shRNAs widely between different sgRNAs (from 0 to 150 off-target Optimization of siRNA and shRNA design rules has been an events per sgRNA) and does not correlate with GC content active area of research for over a decade and various rules or genomic location of the target site. Moreover, these ChIP- have been established (Fellmann and Lowe, 2014; Tafer, 2014). seq-independent approaches confirm that the process of Typically, the duplex length ranges from 19 to 25 nt, and se- DNA cleavage by Cas9 is distinct and separable from simple quences are designed to target regions of the ORF that are DNA binding. common among transcript variants. It has generally been recom- While the large degree of non-specific binding of dCas9 ap- mended to avoid sequences with extremely high or low GC con- pears to be a concern, it is important to remember that most tents, miRNA seed matches, and internal complementarity. To dCas9 applications (such as CRISPRi or CRISPRa) are highly minimize off-targeting, sequences having perfect or near-perfect dependent on the genomic position of stable dCas9 binding complementarity to unintended target sites should be avoided (Gilbert et al., 2014). Inhibition as well as activation of gene (Birmingham et al., 2007). To assist researchers with si/shRNA expression appears to require dCas9-KRAB/-VP64 to be tar- sequence design, a large variety of computational tools have geted to a narrow window around the TSS of genes (Gilbert been developed. A listing of those web-based tools together et al., 2014). Hence, off-targeting of dCas9-KRAB/-VP64 to sites with their URLs can be found in Lagana et al. (2014). In addition, other than the TSS may not actually result in transcriptional numerous commercially available resources exist that claim repression or activation. to have validated sequences and those enriched for potent However, the KRAB domain’s ability to induce heterochromat- activity. in formation at any genomic locus could potentially lead to phe- While siRNA/shRNA design rules have substantially improved notypes resulting from displacement of regulatory factors from over previous years, de novo RNAi design still exhibits variation their target site. Data showing little to no anti-proliferative effects in efficiency and specificity between small RNAs. Hence, it is for thousands of negative control sgRNAs in a CRISPRi screen imperative to test every new reagent to measure knockdown ef- appear to support the hypothesis that off-target effects are not ficiency and to reproduce phenotypes with multiple si/shRNAs a major issue (Gilbert et al., 2014). This is in contrast to off-target- targeting the same gene. ing of siRNA, which can inhibit and/or degrade mRNA from a Design of sgRNAs large number of unspecific sites (Jackson et al., 2006), poten- To evoke gene knockout via early frameshift mutations, tially leading to a large variety of phenotypes (Jackson and Lins- sgRNAs are frequently designed to target Cas9 nuclease to ley, 2010). the N-terminal coding exons of protein-encoding genes. For Increasing Target Site Specificity of Cas9 CRISPRi/a on the other hand, sgRNAs are currently designed Certain applications, such as for instance human gene therapy, to target a region around the TSS. Hence, for every CRISPR/ require highly specific genome editing with minimized off-target- Cas9 platform, the design of sgRNAs must fit the specific ing. For this purpose, two separate Cas9-based systems have system. Nonetheless, common design rules for sgRNAs from been developed to increase target site specificity and decrease different platforms have been empirically determined. off-target effects. The first approach involves the mutation of one For all CRISPR systems described to date, the sgRNA should of the two Cas9 nuclease domains, turning Cas9 from a nuclease target a genomic DNA sequence that is adjacent to a PAM, into a nickase. Single DNA strand cuts, or nicks, are typically re- without actually containing the PAM itself (Sternberg et al., paired with high fidelity (Ran et al., 2013a). Consequently, two 2014). Sequences flanking the PAM have been described to Cas9 nickases need to target appropriate, adjacent sites in the impact the success of sgRNA targeting. Specifically, CGGH is genome in order to introduce DSBs, which are then repaired the recommended optimal PAM sequence for S. pyogenes

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Figure 3. User Guide Decision Tree for Help to Choose the Right Tool (1) Known sequence. For your targeted organism, what type of known sequence information is available? Transcriptome sequence alone allows the targeting of mRNA (si/shRNAs) and DNA exons (TALENs and Cas9 nuclease). If your organism’s genome has been sequenced and TSSs have been annotated, all technologies become available, since TALE repression and CRISPRi/a target promoter regions. (2) Loss-of-function-phenotype. What type of loss- of-function phenotype do you want? While RNAi, TALE-repression, and CRISPRi induce reversible gene knockdown, TALEN and Cas9 nuclease induce a permanent knockout. (3) Time to phenotype. How long will it take from the initial planning of the experiment to ﬁnally having a loss-of-function phenotype in cells? Transfection of siRNAs remains the most direct and quickest route. For RNAi using shRNAs as well as CRISPRi/a and Cas9 nuclease, appropriate shRNA/sgRNA plasmids need to be cloned. Moreover, it may be necessary to validate the functionality of Cas9 in target cells. The most time consuming way to induce loss-of-function is TALENs, since each target site requires the design of a speciﬁc amino acid sequence of the TALEN DBD. (4) Type of phenotype. RNAi, TALE repression, and CRISPRi/a are best suited to generate hypomorphic phenotypes that mimic for instance the inhibition of a target by a drug. TALEN and Cas9 nuclease on the other hand can generate complete knockout phenotypes (null allele) via genome editing. (5) Cost of experiment. How much will the production of required reagents cost? shRNAs and sgRNAs are generated by simple cloning or they can be purchased. Because TALEN-based technologies require a more complicated synthesis, cloning them or purchasing them generally costs more. Performing RNAi via siRNAs requires the costly synthesis of short RNAs. (6) Ease of experiment. How much relative effort does it take until one arrives at the desired loss-of-function phenotype? RNAi via siRNAs necessitates only the purchase or synthesis of short RNAs, followed by a simple transfection into target cells. RNAi via shRNAs and CRISPR-based approaches additionally involve the cloning of shRNAs or sgRNAs. The design and cloning of TALEN-based technologies are relatively more complicated, making it less easy to use. (7) Off-targeting. How severe are the known off-target effects? So far, the described off-target effects for CRISPR as well as TALEN technology appear to be less of an issue compared to RNAi. While off-targeting of an si/shRNA to a given RNA can result in an undesired phenotype, it seems likely that TALEN and CRISPR off- targeting to the genomic DNA may not often produce an undesired phenotype.

(Doench et al., 2014). The typical length of an sgRNA is 20 nt, ommended to test several sgRNA reagents and to confirm although slightly shorter sgRNAs (17 to 18 nt) have been found similar if not identical phenotypes in a given experiment with at to reduce off-targeting without compromising efficiency (Fu least two different sgRNAs. et al., 2014). Similar to si/shRNA rules, extremely high or low GC contents and homopolymeric nucleotide stretches should Choosing the Right Tool for the Job be avoided (Doench et al., 2014; Gilbert et al., 2014). Importantly, Do You Want to Generate a Knockout or Knockdown? targeting the sense or antisense strand appears to have no influ- Each loss-of-function technology has its own advantages and ence on efficiency of Cas9 nuclease (Doench et al., 2014)or limitations, so choosing the right tool for the job largely depends CRISPRi/a (Gilbert et al., 2014). Given that sgRNAs appear to on the specific experimental design. As a guide, Figure 3 gives an possess a seed region proximal to the PAM, sequences should overview of the most common points to be considered before be designed to avoid perfect or near-perfect matches to the beginning an experiment; it highlights the appropriate tools seed sequence outside the target site (Fu et al., 2013; Wu that can be used in everyday scenarios. et al., 2014). Local chromatin structure has been shown to It is probably fair to say that CRISPR/Cas9 has already leap- strongly impact Cas9 binding to its target DNA site (Kuscu frogged TALEN technology, mostly because Cas9 offers an et al., 2014; Wu et al., 2014). unprecedented simplicity to target a large variety of functional Several web-based software tools can assist researchers with domains to various genomic sites. Hence in the following section the design of sgRNAs for genome editing, each of them taking we will focus on contrasting RNAi, CRISPRi, and Cas9 nuclease into account empirically determined parameters for optimal for genome editing without specifically mentioning TALEN tech- sgRNA design, such as length and the GC contents of sgRNAs nology. Perhaps the most striking difference among these (Doench et al., 2014; Heigwer et al., 2014; Hsu et al., 2013). A various approaches is that RNAi and CRISPRi induce transient list of those tools together with their URLs can be found here (La- knockdown of gene expression, whereas genome editing via gana et al., 2014). While rules for si/shRNA sequence design Cas9 nuclease can induce permanent damage of the target have been developed over many years, the field of sgRNA design DNA, leading to a null phenotype in some cases. Therefore, a is still developing, and design rules will probably be improved major question is ‘‘do you want to generate a complete knockout with additional studies. Similar to si/shRNAs, it is currently rec- or a hypomorphic knockdown’’?

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Gene Knockout Another important issue is time: RNAi knockdown cell lines For many applications, a full knockout may be desirable, and can be obtained within a matter of days—simply via siRNA trans- perhaps even required. For instance, in cases of slow protein fection—and do not require time-consuming screening for ho- degradation, reduced transcript levels—as caused by knock- mozygous knockout clones. This may be especially important down approaches—may not necessarily result in low protein in primary cells with limited proliferative capacity or limited cell levels. Moreover, if a gene product is not rate limiting, even health in culture models. Moreover, the selection of homozygous strongly reduced protein levels may not produce a loss-of-func- knockout clones may not faithfully represent the original compo- tion phenotype. A knockout on the other hand would sooner or sition of cell populations. Hence, sub-populations that are un- later lead to full depletion of functional protein. derrepresented or for which the knockout was cell lethal may A significant advantage of Cas9 nuclease-mediated genome be excluded from subsequent analysis. editing compared to transcriptional repression via CRISPRi is Similar to RNAi, CRISPRi appears to generate a homogenous that it targets exonic regions rather than TSSs. Why is this impor- phenotype for an entire population of cells, providing all cells are tant? First of all, exons are generally very well annotated and can robustly expressing both the sgRNA and Cas9. As such, pheno- be deduced from transcriptome sequencing data. More impor- typic heterogeneity is minimized compared to Cas9 nuclease- tantly though, genome editing can disrupt exons that are shared mediated knockouts that depend on frameshift mutations or by all variants of a given gene, even if these are transcribed from loss of specific DNA sequence elements. separate TSSs. Like this, all transcript variants and even whole RNAi: A Phasing-Out Technology? gene families can be knocked out using a single sgRNA against With the rapid development of CRISPR technology, a reasonable exons that are conserved between all family members. question to ask is ‘‘is the era of RNAi based tools over?’’ After all, A true knockout—defined as the complete disruption of a RNAi often exhibits significant off-target effects and unpredict- target gene’s function—should cause a homogenous knockout able knockdown efficiencies. In contrast, CRISPRi appears phenotype. However, it is important to keep in mind that to be advantageous since knockdown efficiencies appear to genomic mutations introduced via NHEJ repair of DSBs are be superior to RNAi. Although the field is still exploring the extent somewhat random (Valerie and Povirk, 2003). Therefore, approx- of CRISPR-centric off-targeting, some results suggest that imately one third of the introduced mutations via Cas9 nuclease CRISPRi barely produces off-target phenotypes (Gilbert et al., would be expected to be in-frame and hence should not disrupt 2014). To answer the above question, one needs to consider the ORF. That said, depending on the location within a protein, a the strengths and weaknesses of each tool. few extra amino acids removed or added can alter activity, As discussed above, RNAi seems to be the simplest system to complicating the prediction of phenotypic outcome for each mu- create hypomorphic knockdowns. In contrast to CRISPRi, RNAi tation and each gene. Hence, genetic heterogeneity could yield does not require additional genes to be introduced into cells prior phenotypic heterogeneity. to the actual knockdown experiment, since most cells come with An additional and often overlooked consideration is ploidy. the full RNAi silencing machinery. Hence the execution of RNAi is Most common laboratory cancer cell lines exhibit changes in more rapid, saving investigators time and money. ploidy, with many lines carrying four or more copies of a chromo- Unlike CRISPRi, RNAi does not target TSSs. Therefore, some (Paulsson et al., 2013; Rondo´ n-Lagos et al., 2014). In these RNAi can be used in species for which transcriptome but not cases, true knockout phenotypes may only emerge once every genome sequencing data exists and whose TSSs have not yet functional copy of the target gene is mutated. In practice, this been annotated via additional sequencing approaches like may be difficult—although not impossible—to accomplish. De- CAGE (Kodzius et al., 2006). Similar to genome editing via pending on the experiment, it may be imperative to sequence Cas9 nuclease, RNAi can target sequences that are conserved the target loci to verify homozygous knockout when generating between transcript variants and gene family members. Hence, mutant clonal cell lines. one single si/shRNA can target all transcripts with conserved Gene Knockdown sequence—regardless of what TSS they originate from. This is While not always capable of disrupting gene function com- in contrast to CRISPRi and could put RNAi ahead of CRISPRi- pletely, hypomorphic knockdowns can offer a number of advan- based technologies. tages over knockouts. Since gene knockdown via RNAi or Finally, RNAi targets RNA transcripts in the cytoplasm and CRISPRi does not depend on frame shift mutations or ploidy, not genomic DNA in the nucleus. Hence, accessibility does not the aforementioned issues related to genetic heterogeneity are appear to be impaired by chromatin states, as observed for minimized. Moreover, gene knockdowns are reversible, a feature CRISPR-based approaches (Kuscu et al., 2014; Wu et al., that has proven very useful in the past (Xue et al., 2007). 2014). Taken together, despite the excitement surrounding Furthermore, partial gene knockdown allows researchers to CRISPR-based approaches, RNAi still retains a strong position study the function of essential genes whose knockout would in several experimental settings and should be expected to otherwise be cell lethal. Additionally, applications such as co-exist for some time. drug target discovery could benefit from phenotypic hypo- morphs, since an RNAi or CRISPRi knockdown—rather than a Future Perspectives knockout—may mimic the effects of an inhibitive drug more Clearly, the potential of CRISPR/Cas systems goes far beyond closely. However, in some cases, a full knockout may represent inducing loss-of-function and gain-of-function phenotypes. an idealized loss-of-function phenotype for the most potent In principle, dCas9 can be fused to virtually any functional inhibitive drugs. domain to target proteins to specific genomic sites, allowing

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