Published online 23 March 2015
`
`Nucleic Acids Research, 2015, Vol. 43, No. 7 3407–3419
`doi: 10.1093/nar/gkv226
`
`SURVEY AND SUMMARY
`Advances in CRISPR-Cas9 genome engineering:
`lessons learned from RNA interference
`Rodolphe Barrangou1,†, Amanda Birmingham2,†, Stefan Wiemann3, Roderick
`L. Beijersbergen4, Veit Hornung5 and Anja van Brabant Smith2,*
`
`1Department of Food, Bioprocessing and Nutrition Sciences, North Carolina State University, Raleigh, NC 27695,
`USA, 2Dharmacon, part of GE Healthcare, Lafayette, CO 80026, USA, 3Division of Molecular Genome Analysis, and
`Genomic & Proteomics Core Facility, German Cancer Research Center, 69120 Heidelberg, Germany, 4The
`Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands and 5Institute of Molecular Medicine, University
`Hospital, University of Bonn, 53128 Bonn, Germany
`
`Received January 15, 2015; Revised March 4, 2015; Accepted March 5, 2015
`
`ABSTRACT
`The discovery that the machinery of the Clustered
`Regularly Interspaced Short Palindromic Repeats
`(CRISPR)-Cas9 bacterial immune system can be re-
`purposed to easily create deletions, insertions and
`replacements in the mammalian genome has revo-
`lutionized the field of genome engineering and re-
`invigorated the field of gene therapy. Many paral-
`lels have been drawn between the newly discov-
`ered CRISPR-Cas9 system and the RNA interfer-
`ence (RNAi) pathway in terms of their utility for
`understanding and interrogating gene function in
`mammalian cells. Given this similarity, the CRISPR-
`Cas9 field stands to benefit immensely from lessons
`learned during the development of RNAi technology.
`We examine how the history of RNAi can inform to-
`day’s challenges in CRISPR-Cas9 genome engineer-
`ing such as efficiency, specificity, high-throughput
`screening and delivery for in vivo and therapeutic
`applications.
`
`INTRODUCTION
`From early classical genetic studies to present-day molec-
`ular ones, the ability to modulate gene content and ex-
`pression has been essential to understanding the function
`of genes within biological pathways and their correlation
`with disease phenotypes. The discovery of RNAi and its
`reduction to practice in mammalian cells in the early to
`mid 2000’s made reverse genetics approaches feasible on a
`
`genome scale in higher eukaryotes (1). In the last 24 months,
`another gene modulation technique, Clustered Regularly
`Interspaced Short Palindromic Repeats (CRISPR)-Cas9
`genome engineering (referred to as CRISPR-Cas9), has
`emerged; in that remarkably brief window, this approach
`has proven to be a powerful tool for studying individual
`gene function, performing genome-wide screens, creating
`disease models and perhaps developing therapeutic agents
`(2). These lightning advances have largely followed the path
`blazed by RNAi studies and we argue that further leverage
`is to be gained by examining relevant successes and failures
`in the last 14 years of RNAi.
`RNAi and CRISPR-Cas9 have many clear similarities.
`Indeed, the mechanisms of both use small RNAs with an
`on-target specificity of ∼18–20 nt. Both methods have been
`extensively reviewed recently (3–5) so we only highlight their
`main features here. RNAi operates by piggybacking on
`the endogenous eukaryotic pathway for microRNA-based
`gene regulation (Figure 1A). microRNAs (miRNAs) are
`small, ∼22-nt-long molecules that cause cleavage, degra-
`dation and/or translational repression of RNAs with ad-
`equate complementarity to them (6). RNAi reagents for re-
`search aim to exploit the cleavage pathway using perfect
`complementarity to their targets to produce robust down-
`regulation of only the intended target gene. The CRISPR-
`Cas9 system, on the other hand, originates from the bac-
`terial CRISPR-Cas system, which provides adaptive im-
`munity against invading genetic elements (7). Generally,
`CRISPR-Cas systems provide DNA-encoded (7), RNA-
`mediated (8), DNA- (9) or RNA-targeting(10) sequence-
`specific targeting. Cas9 is the signature protein for Type
`II CRISPR-Cas systems (11), in which gene editing is me-
`
`*To whom correspondence should be addressed. Tel: +1 720 890 5188; Fax: +1 303 604 3286; Email: anja.smith@ge.com
`†
`These authors contributed equally to this work.
`C(cid:3) The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
`This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which
`permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
`
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`3408 Nucleic Acids Research, 2015, Vol. 43, No. 7
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`Figure 1. The RNAi and CRISPR-Cas9 pathways in mammalian cells. (A) miRNA genes code for primary miRNAs that are processed by the
`Drosha/DGCR8 complex to generate pre-miRNAs with a hairpin structure. These molecules are exported from the nucleus to the cytoplasm, where
`they are further processed by Dicer to generate ∼22-nt-long double-stranded mature miRNAs. The RNA duplex associates with an Argonaute (Ago)
`protein and is then unwound; the strand with a more unstable 5(cid:4) end (known as the guide strand) is loaded into Ago to create the RNA-induced silencing
`complex (RISC) while the unloaded strand is discarded. Depending on the degree of complementarity to their targets, miRNAs cause either transcript
`cleavage and/or translational repression and mRNA degradation. siRNAs directly mimic mature miRNA duplexes, while shRNAs enter the miRNA path-
`way at the pre-miRNA hairpin stage and are processed into such duplexes. (B) CRISPR-Cas9-mediated genome engineering in mammalian cells requires
`crRNA, tracrRNA and Cas9. crRNA and tracrRNA can be provided exogenously through a plasmid for expression of a sgRNA, or chemically synthesized
`crRNA and tracrRNA molecules can be transfected along with a Cas9 expression plasmid. The crRNA and tracrRNA are loaded into Cas9 to form an
`RNP complex which targets complementary DNA adjacent to the PAM. Using the RuvC and HNH nickases, Cas9 generates a double-stranded break
`(DSB) that can be either repaired precisely (resulting in no genetic change) or imperfectly repaired to create a mutation (indel) in the targeted gene. There
`are a myriad of mutations that can be generated; some mutations will have no effect on protein function while others will result in truncations or loss of
`protein function. Shown are mutations that will induce a frameshift in the coding region of the mRNA (indicated by red X’s), resulting in either a truncated,
`non-functional protein or loss of protein expression due to nonsense-mediated decay of the mRNA.
`
`

`
`diated by a ribonucleoprotein (RNP) complex consisting
`of a CRISPR RNA (crRNA) (8) in combination with
`a trans-activating CRISPR RNA (tracrRNA) (12) and a
`Cas9 nuclease (13–16) that targets complementary DNA
`flanked by a protospacer-adjacent motif (PAM) (17–19).
`The molecular machinery from the CRISPR-Cas9 bacte-
`rial immune system can be repurposed for genome edit-
`ing in mammalian cells by introduction of exogenous cr-
`RNAs and tracrRNAs or a single guide RNA chimeric
`molecule (sgRNA) which combines crRNA and tracrRNA
`sequences, together with the Cas9 endonuclease to create a
`double-strand break (DSB) in the targeted DNA (16,20–22)
`(Figure 1B). The DSB is repaired either by non-homologous
`end joining (NHEJ) or homology-directed repair (HDR)
`(23). The error-prone NHEJ pathway typically generates
`small insertions or deletions (indels) that are unpredictable
`in nature, but frequently cause impactful and inactivating
`mutations in the targeted sequence; conversely, the HDR
`pathway is useful for precise insertion of donor DNA into
`the targeted site.
`Both RNAi and CRISPR-Cas9 have experienced signifi-
`cant milestones in their technological development, as high-
`lighted in Figure 2 (7–14,16–22,24–51) (highlighted top-
`ics have been detailed in recent reviews (2,4,52–58)). The
`CRISPR-Cas9 milestones to date have mimicked a com-
`pressed version of those for RNAi, underlining the prac-
`tical benefit of leveraging similarities to this well-trodden
`research path. While RNAi has already influenced many
`advances in the CRISPR-Cas9 field, other applications of
`CRISPR-Cas9 have not yet been attained but will likely
`continue to be inspired by the corresponding advances in
`the RNAi field (Table 1). Of particular interest are the po-
`tential parallels in efficiency, specificity, screening and in
`vivo/therapeutic applications, which we discuss further be-
`low.
`
`EFFICIENCY
`Work performed during the first few years of intensive
`RNAi investigations demonstrated that, when taking 70–
`75% reduction in RNA levels as a heuristic threshold for ef-
`ficiency (59), only a small majority of siRNAs and shRNAs
`function efficiently (24,60) when guide strand sequences are
`chosen randomly. This observation led to the development
`in 2004 of rational design algorithms for siRNA molecules
`(Figure 2), followed later by similar algorithms for shRNAs.
`These methods have been able to achieve ∼75% correlation
`and >80% positive predictive power in identifying func-
`tional siRNAs (61) but have been somewhat less effective
`for shRNAs (62) (perhaps because in most cases, shRNAs
`produce less knockdown than do siRNAs, likely due to a
`smaller number of active molecules in each cell). crRNAs
`also vary widely in efficiency: reports have demonstrated
`indel (insertion and deletion) creation rates between 5 and
`65% (20,25), though the average appears to be between 10
`and 40% in un-enriched cell populations. Indeed, a growing
`amount of evidence suggests a wide range of crRNA effi-
`ciency between genes and even between exons of the same
`gene, yielding some ‘super’ crRNAs that are more func-
`tional (26,27). However, such high-functioning crRNAs are
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`Nucleic Acids Research, 2015, Vol. 43, No. 7 3409
`
`likely to make up only a small percentage of those randomly
`selected for any given gene (28).
`Following the RNAi playbook, efforts to develop rational
`design algorithms for crRNA have already begun: Doench
`et al. (28) assayed thousands of sgRNAs in a functional
`assay and identified sequence features that predict sgRNA
`activity. In addition, the CRISPR-Cas9 field has moved
`quickly to determine relevant structures and details on the
`separate binding and cleavage processes (29–30,63), thus
`avoiding a deficit of mechanistic information that impeded
`early efforts to predict RNAi efficiency. From these early
`CRISPR-Cas9 studies, it is already understood that the ef-
`ficiency of the crRNA:tracrRNA:Cas9 RNP complex is de-
`termined by PAM-dependent, crRNA-driven Cas9 binding
`to target DNA as well as crRNA sequence complementarity
`to the target DNA (particularly in the first PAM-proximal
`8–10 nt, the ‘seed’ region of the crRNA). Nonetheless, many
`details of CRISPR-Cas9 activity remain to be learned. Be-
`cause an efficient sgRNA or crRNA must not only create a
`DSB in the sequence of interest, but also create a mutation
`that results in functional disruption of the resulting protein
`or non-coding structure, the type of indel as well as its posi-
`tion along the length of the coding sequence are likely to be
`important; thus, effective rational design efforts for func-
`tional knockouts may need to incorporate attributes gov-
`erning these characteristics as well. Furthermore, more than
`a decade of efforts in the RNAi field demonstrates that even
`a well-characterized system is not necessarily easy to pre-
`dict: crRNA design algorithms, like those for siRNAs and
`shRNAs, may plateau in their predictive power at a level
`that still necessitates testing of multiple reagents per target
`in order to guarantee selection of a functional one.
`It is worth noting that efficient gene silencing by an RNAi
`reagent requires a process of ongoing, active repression; this
`mechanism may be impaired by gene-specific factors that
`increase mRNA turnover and/or decrease RNA-induced
`silencing complex (RISC) turnover. As such, there are some
`genes for which no RNAi reagents can be found that qual-
`ify as effective. In contrast, efficiency of a crRNA depends
`upon the probability of a one-time event––the editing of a
`DNA site. Because of this difference, the success of rational
`design efforts may be less critical to the CRISPR field on an
`individual gene basis, as it will be widely possible to find an
`effective crRNA if one is willing to evaluate enough treated
`cells; highly effective crRNA designs, however, will still be
`necessary for genome-scale studies, as discussed further be-
`low.
`
`SPECIFICITY AND OFF-TARGET EFFECTS
`Perhaps in no other area are the lessons of RNAi as obvious
`as in that of specificity. While RNAi was originally hailed
`as exquisitely specific (64), subsequent research has shown
`that in some circumstances it can trigger non-specific ef-
`fects and/or sequence-specific off-target effects (65). Many
`non-specific effects seen with this approach are mediated by
`the inadvertent activation of pattern recognition receptors
`(PRRs) of the innate immune system that have evolved to
`sense the presence of nucleic acids in certain sub-cellular
`compartments. siRNA length, certain sequence motifs, the
`absence of 2-nt 3(cid:4) overhangs and cell type are important fac-
`
`

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`3410 Nucleic Acids Research, 2015, Vol. 43, No. 7
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`Figure 2. Timeline of milestones for RNAi and CRISPR-Cas9. Milestones in the RNAi field are noted above the line and milestones in the CRISPR-Cas9
`field are noted below the line. These milestones have been covered in depth in recent reviews (2,4,52–29).
`
`Table 1. Summary of improvements in the CRISPR-Cas9 field that can be anticipated by corresponding RNAi advances
`
`Milestone
`
`IND application
`
`Off-target driver
`identification
`
`Off-target-reducing
`modifications
`
`Large-scale arrayed
`screening
`
`in vivo use (human)
`
`Phase III entry
`
`RNAi
`
`2004
`
`2006
`
`2006
`
`2007
`
`2010
`
`2014
`
`CRISPR
`
`This step is undoubtedly imminent. The drug that was the subject of the first RNAi IND
`failed clinical trials when its effect was shown to be due to non-RNAi-related
`mechanisms; especially since CRISPR therapeutics require the delivery not only of a
`targeting RNA but of exogenous Cas9 (delivered as DNA, mRNA or protein),
`pharmaceutical developers must avoid allowing history to repeat itself.
`Current work is characterizing the nature and extent of the PAM-proximal crRNA
`‘seed’. Until it is complete, novel outcomes must be demonstrated using multiple reagents
`to the same target, as is routinely done for RNAi. Once the crRNA seed is understood,
`researchers should determine whether it could be leveraged to develop sequence-specific
`off-target controls such as RNAi’s C911 controls.
`While effective specificity-enhancing chemical modifications for CRISPR may have to
`wait until off-target drivers are more fully understood, synthetic crRNAs should be
`modifiable by precisely the same methods as synthetic siRNAs.
`Genome-wide arrayed screens using CRISPR are likely to be more challenging because
`the percentage of edited cells is typically lower than for RNAi. Nonetheless, CRISPR
`screening and analysis practices will build on and extend those designed for RNAi
`screening, just as the latter did with those for small-molecule screening.
`As CRISPR-driven editing in adult human cells has already been achieved, in vivo
`human use seems inevitable. Efficacious delivery, including that of the exogenous Cas9
`protein (or Cas9 mRNA) necessary to make integration-less DNA modifications, is likely
`to present a significant hurdle. Novel delivery formulations developed in pursuit of
`RNAi therapeutics will undoubtedly be among those tried first.
`CRISPRa and other dCas9-based approaches raise the hope of addressing conditions
`untreatable purely via RNAi-like down-regulation while retaining the reversible nature of
`RNAi. The two modalities might profitably be used in parallel.
`
`tors for induction of the mammalian interferon response
`(66–68). Additionally, the general perturbation of cellular
`or tissue homeostasis by the delivery process itself can also
`trigger unwanted responses (most likely secondary to innate
`immune damage-sensing pathways) such as the wide-spread
`alteration of gene expression caused by cationic lipids, es-
`pecially when used at high concentrations (69). Such non-
`
`specific effects associated with delivery will still exist for
`CRISPR-Cas9 but can likely be overcome by minimizing
`lipid concentration as is now routinely done in RNAi stud-
`ies. Similarly, the introduction of chemical modifications
`into the backbone of an siRNA duplex (e.g. 2(cid:4)-O-methyl
`ribosyl) can block the recognition of RNA molecules by
`PRRs (66,70–71), so such modifications may also address
`
`

`
`innate immune system recognition caused by synthetic cr-
`RNAs. Researchers would do well to investigate whether
`additional effects may result, potentially in a cell-line or
`cell-type dependent manner, as a response to creation of
`DSBs or the abundant expression of Cas9 or an sgRNA
`molecule (such as that seen when strong shRNA expression
`outcompetes that of endogenous miRNAs, leading to the
`breakdown of cellular regulation (72)). These types of non-
`specific off-target effects have already been reported with
`other genome engineering techniques (e.g. zinc finger nucle-
`ases (73)) but the ease-of-use and simplicity of the CRISPR-
`Cas9 system should allow researchers to address these types
`of questions fully in the near future.
`RNAi can also produce sequence-specific off-target ef-
`fects, which were initially described in early 2003 (31),
`but whose potential impact was not fully appreciated un-
`til well after the method had become a widely used re-
`search and screening technique (e.g. (74)). Cleavage-based
`off-targeting, which occurs when RISC encounters an
`unintended transcript target with perfect or near-perfect
`complementarity to its guide strand, can induce knock-
`down equivalent to that of intended target down-regulation
`and was originally hypothesized to be the main cause of
`sequence-specific off-target effects. It took several years to
`determine that these effects were in fact primarily caused
`by RNAi reagents acting in a ‘miRNA-like’ fashion, down-
`regulating unintended targets by small (usually <2-fold)
`amounts primarily through seed-based interactions with
`the 3(cid:4) UTR of those unintended targets. Because miRNA-
`like off-targeting is generally seed-based and all transcripts
`contain matches to a variety of 6–8-base motifs, such off-
`targeting can affect tens to hundreds of transcripts. Further-
`more, if the RNAi reagent contains a seed mimicking that
`of an endogenous miRNA, the off-targeting may affect the
`pathway or family of targets evolutionarily selected for reg-
`ulation by that miRNA. It is not possible to design RNAi
`reagents that do not contain seed regions found in the tran-
`scriptome’s 3(cid:4) UTRs and the non-seed factors that conclu-
`sively determine whether or not a seed-matched transcript is
`in fact off-targeted have not yet been identified. Both ratio-
`nal design and chemical modifications such as 2(cid:4) O-methyl
`ribosyl substitutions can mitigate seed-based off-target ef-
`fects (32), but without a full solution, specificity remains a
`well-known pain point for RNAi users.
`Inspired by these concerns, an initial evaluation of the
`off-target potential of CRISPR-Cas9 was published within
`months of the technique’s debut and work to refine these
`early findings has continued apace. Studies have revealed
`some sequence flexibility, and tolerance for mismatches
`and bulges, that have generated concerns about specificity
`and sequence-directed off-target cleavage (25,75–79). Sev-
`eral variations of CRISPR-Cas9 have been developed to ad-
`dress specificity including paired nickases (77), short sgR-
`NAs (76) and Cas9 fused to FokI (80), and the rapid ad-
`vances in understanding CRISPR-Cas9 mechanism and
`structure are likely to further fuel such developments. Re-
`cent papers, however, have uncovered very few to no off-
`target mutations that can be attributed to CRISPR-Cas9
`(33,34) and conclude that clonal artifacts that derive from
`isolating CRISPR-Cas9-edited cells may be a larger con-
`cern. This apparent discrepancy between prediction and re-
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`Nucleic Acids Research, 2015, Vol. 43, No. 7 3411
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`ality, while encouraging, highlights a treacherous pitfall in
`studying off-target gene editing: to date the primary ap-
`proach has been to predict putative off-target sites and then
`search for editing at those sites, but this approach risks
`falling prey to the ‘streetlight effect’, in which one searches
`only where it is easy to look. The RNAi field learned this
`lesson painfully: early off-target prediction efforts focused
`on strong overall complementarity as a determinant and
`thus largely failed to identify genes that were actually off-
`targeted due to short, seed-based complementary (35). Un-
`less the CRISPR-Cas9 field learns from RNAi’s mistakes,
`it is in danger of repeating the same very one, especially as
`CRISPR-Cas9 specificity has recently also been shown to
`depend on an as-yet-not-fully-defined seed region (20,81).
`At this stage, computational predictions of putative off-
`target gene-editing sites are at best questionable guesses and
`thus cannot be depended upon in assessing the full effect of
`off-targeting. Unfortunately, while RNAi specificity stud-
`ies are limited to the transcriptome, analysis of CRISPR-
`Cas9 specificity requires identifying effects that may occur
`anywhere in the genome, generally through next-generation
`sequencing. This process is costly and depends upon non-
`trivial data analysis and processing, so it remains unclear
`whether the field has the will to commit to this work. Until
`such time as a less biased understanding of CRISPR-Cas9
`off-targeting emerges, researchers are advised to emulate the
`best-practice of RNAi by using multiple crRNAs or sgR-
`NAs in order to show redundancy of phenotype by multi-
`ple reagents targeting the same gene, thus ensuring that the
`phenotype is due to on-target effects rather than off-target
`effects.
`
`GENOME-SCALE SCREENING TOOLS
`Interest in genome-scale CRISPR-Cas9-based screening
`has blossomed, with some pooled screening resources al-
`ready available (26,27) and arrayed ones likely to emerge in
`the near future. Because genome editing screens will also be
`affected by a large number of the factors that make RNAi
`screens more challenging than small-molecule ones (82),
`practitioners would do well to study the hard-won victories
`in this field since the first published whole-genome synthetic
`lethal screen for sensitization to paclitaxel (36) (Figure 2)
`before diving into these costly experiments.
`Of particular importance is evaluating whether the lower
`efficiencies seen using CRISPR-Cas9 are sufficient to gen-
`erate a desired phenotype in the screening assay––that is,
`determining whether the phenotype is detectable in the tar-
`geted cell population. In this regard, two factors are of spe-
`cial concern: the ploidy of the gene locus of interest (as
`tumor cell lines are often aneuploid) and the likelihood
`of disrupting the reading frame by the induced mutation
`(since +3 or −3 indels would not serve this purpose). Tak-
`ing these factors into account, the chance of obtaining a
`high percentage of cells that have a functional knockout
`in a bulk cell culture is relatively low under typical screen-
`ing conditions. Consequently, it is unlikely that traditional
`arrayed loss-of-signal screens such as those common in
`RNAi will be widely feasible in bulk-transfected cells using
`CRISPR-Cas9. Nevertheless, the CRISPR-Cas9 technol-
`ogy may have an advantage in screens for which a complete
`
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`3412 Nucleic Acids Research, 2015, Vol. 43, No. 7
`
`knockout is required to uncover a phenotype (for exam-
`ple, when targeting kinases where a residual expression of
`10% is sufficient for activity). The use of analysis techniques
`that examine effects at the single-cell level (using, for exam-
`ple, high-content microscopy or fluorescence-activated cell
`sorting-based read-outs) could also be informative, as it has
`been for gene silencing screens. A more novel possibility
`is leveraging HDR to develop systems in which CRISPR-
`Cas9-mediated DSBs are repaired using an exogenous tem-
`plate harboring a marker or resistance gene that can later be
`used to select cells with a functional, inactivating, recombi-
`nation event (83).
`Homologous recombination could similarly be used to
`validate RNAi screening hits by introducing variants in
`the transcript that do not alter the protein but create mis-
`matches with the siRNA or shRNA reagents such that tar-
`get knockdown no longer occurs, providing a fast method
`to distinguish on-target and off-target effects. More simply,
`gene editing can be used to knock out a putative hit to deter-
`mine if the knockout results in a similar phenotype to that
`obtained with knockdown during the RNAi screen (84). Al-
`though (as discussed further below) knockout phenotypes
`may differ from knockdown phenotypes (so that a negative
`result may not necessarily indicate a false positive), confir-
`mation of the knockdown phenotype with a CRISPR-Cas9
`knockout would be a strong indication of a true positive.
`Conversely, RNAi reagents may be an effective validation
`strategy for CRISPR-Cas9-based screens and the numer-
`ous other effective approaches optimized for RNAi screens,
`such as confirmation of assay phenotype by multiple inde-
`pendent reagents and the use of additional related assays
`(58), will prove directly applicable to genome editing vali-
`dation.
`The development and application of RNAi-based pooled
`screening approaches have greatly enhanced the field of
`functional genomics screening in mammalian cells: the
`ability to quantify in large populations the relative abun-
`dance of individual cells, each carrying a gene-specific gene-
`modifying reagent, allows for different screening models
`such as identification of genotype-specific essential genes,
`synthetic lethal genes or genes involved in resistance to spe-
`cific drugs (85). Crucial requirements for this approach are
`the availability of reagents with high efficiency and speci-
`ficity, the presence of tractable markers for integrated con-
`structs and the availability of large collections of gene-
`perturbing reagents in retroviral or lentiviral vectors. In
`contrast to the current lack of arrayed screening resources
`for CRISPR-Cas9, large, genome-scale collections of sgR-
`NAs that fulfill these criteria are available alongside anal-
`ogous shRNA collections. Numerous examples of both
`pooled RNAi screens and pooled CRISPR-Cas9 screens
`have been published (26–27,86–90,37).
`CRISPR-Cas9 pooled screens share with their RNAi-
`based cousins the necessity of inactivating gene activity at
`single-copy integration of the shRNA- or sgRNA-encoding
`expression cassette. This proves a difficulty for both tech-
`nologies, as efficiency of shRNA-mediated knockdown
`varies for different platforms but never reaches complete
`knockdown for all vectors targeting a specific gene (91,92)
`and the observed frequency of CRISPR-Cas9-based gene
`inactivation using single-copy sgRNAs shows great vari-
`
`ability among the different studies, on average not reaching
`frequencies higher than 50% (20,25). RNAi screeners have
`demonstrated that such limited efficiency is more challeng-
`ing for screening models aimed at the identification of lethal
`genes, synthetic lethal genes or response enhancers than for
`enrichment experiments like resistance screens or positive
`selection of a phenotype such as expression of a cell surface
`marker. To combat this limitation, libraries for CRISPR-
`Cas9 pooled screening are already including multiple inde-
`pendent reagents for each gene. As shown by shRNA-based
`pooled screens, this approach insures against false nega-
`tives due to individual inefficient reagents and provides a
`means of corroborating each reagent’s result, thereby reduc-
`ing false positives.
`False-positive off-target effects in both RNAi and
`CRISPR-Cas9 pooled libraries will also be mitigated by ra-
`tional design, as discussed above. Further, recently devel-
`oped C911 seed match controls (93) can be implemented
`in large-scale shRNA screening collections as internal off-
`target matched controls; such a technique would be highly
`desirable for CRISPR-Cas9 screening but its feasibility will
`depend on further understanding of the relevant specificity
`mechanisms. So far, the levels of off-target effects in pooled
`screens using CRISPR-Cas9-based gene editing remains
`unclear, although results from the comparison of shRNA
`and CRISPR-Cas9 screens in the same screening model (for
`genes whose loss confer resistance to vemurafenib, a BRAF
`protein kinase inhibitor) in A375 melanoma cells support
`a low frequency of off-target effects (26) in the CRISPR-
`Cas9 system. With regards to false negatives, one would
`expect that a considerable advantage of the CRISPR-Cas9
`technology over shRNA-mediated knockdown would be in-
`creased strength of phenotype due to the ability to com-
`pletely abolish expression of the targeted gene. As a re-
`sult, one would predict improved recovery of genes involved
`in the phenotype of interest, and indeed, it has been re-
`ported that the recovery rate for essential genes is higher for
`CRISPR-Cas9 than for shRNA (94). However, the afore-
`mentioned genome-wide CRISPR-Cas9 screen for cellu-
`lar resistance to vemurafenib identified a limited number
`of hits compared to a similar screen with a genome-wide
`shRNA collection (26). It may be that CRISPR-Cas9-based
`screens are unable to identify genes that are lethal upon
`complete loss, but are associated with the desired pheno-
`type when knocked down by 70–90%. An example of such
`a gene is SOX10, which causes a slow-growth phenotype
`upon knockdown; this phenotype is associated with resis-
`tance to vemurafenib (95). CRISPR-Cas9 screeners should
`take this potential bias into account when analyzing their
`screening results.
`
`INVIVOSTUDIES
`Following the footsteps of RNAi, CRISPR-Cas9 has
`quickly advanced beyond studies in cell lines and primary
`cell cultures to in vivo studies aimed at everything from
`examination of the biology of particular genes and dis-
`ease phenotypes to development of potential therapeutic
`agents. Notably, however, this technology provides signifi-
`cant advances in the creation of animal models for mecha-
`nistic studies that RNAi, given its transient and partial na-
`
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`ture, cannot offer. Focusing on in vivo studies in the mouse,
`Wang et al. (96) demonstrated that CRISPR-Cas9 can be
`introduced into embryonic stem cells in a multiplex fash-
`ion to create animals carrying multiple specific mutations
`in several genes in a manner requiring only one genera-
`tion, thereby dramatically decreasing the time required to
`generate transgenic animal models. Additional studies have
`used CRISPR-Cas9 to create mouse models of various can-
`cers by mutating a combination of tumor suppressor genes
`and oncogenes in the livers of wild-type mice (97) or in
`mouse hematopoietic stem cells (98), eliminating the need
`for time-consuming creation and crossbreeding of genet-
`ically engineered mouse strains. The modification of the
`mouse genome using CRISPR-Cas9 technology is not lim-
`ited to gene mutations: large chromosomal deletions, inver-
`sions and translocations can be produced by using multiple
`sgRNAs (99–101).
`The final goal of much in vivo work is the development
`of therapeutic tools. In spite of challenges regarding deliv-
`ery and non-specific effects (including those that caused the
`first RNAi-based therapeutic candidate by OPKO Health
`to fail phase III clinical trials in 2009), considerable efforts
`and investments continue in the pursuit of RNA-targeting
`therapeutics. More than 30 clinical trials are currently in
`progress or completed on indications from pachyonychia
`congenita to high cholesterol (102,103). Recently, advances
`in non-viral delivery systems have been made with the de-
`velopment of lipopeptide nanoparticles that offer the op-
`portunity to treat disease via in vivo delivery to endothe-
`lial cells or hepatocytes (104,105). Given this enduring in-
`terest in gene-modulation-based drugs, it seems certain that
`CRISPR-Cas9-based treatments will shortly enter the ther-
`apeutics pipeline; recent proof-of-principle studies (Table 2)
`point to likely indications (106–115). Gene-editing thera-
`peutics may enjoy a smoother road than gene-silencing-
`based ones since they have no requirement for continuous
`delivery of siRNAs or continuous expression of integrated
`shRNAs. As a consequence, gene editing can be done with-
`out leaving a footprint in the genome other than the cor-
`rected DNA sequence. While gene-editing therapeutics may
`have the advantage of not requiring continuous delivery
`or expression of RNAs, RNAi has the advantage of