Expanding the Biologist’s Toolkit with CRISPR-Cas9

Few discoveries transform a discipline overnight, but biologists today can manipulate cells in ways never possible before, thanks to a peculiar form of prokaryotic adaptive immunity mediated by clustered regularly interspaced short palindromic repeats (CRISPR). From elegant studies that deciphered how these immune systems function in bacteria, researchers quickly uncovered the technological potential of Cas9, an RNA-guided DNA cleaving enzyme, for genome engineering. Here we highlight the recent explosion in visionary applications of CRISPR-Cas9 that promises to usher in a new era of biological understanding and control. Few discoveries transform a discipline overnight, but biologists today can manipulate cells in ways never possible before, thanks to a peculiar form of prokaryotic adaptive immunity mediated by clustered regularly interspaced short palindromic repeats (CRISPR). From elegant studies that deciphered how these immune systems function in bacteria, researchers quickly uncovered the technological potential of Cas9, an RNA-guided DNA cleaving enzyme, for genome engineering. Here we highlight the recent explosion in visionary applications of CRISPR-Cas9 that promises to usher in a new era of biological understanding and control. It was only 7 years ago that a fledgling group of international scientists met at the University of California, Berkeley, for the first annual meeting on CRISPRs. A diverse range of expertise was represented—microbiology, biochemistry, metagenomics, food science—allowing the mystery of CRISPR immune system function to be unraveled collectively from multiple lines of experimentation. Each subsequent conference boasted more breakthrough discoveries, and the increasing rate of CRISPR-related publications reflected an intensifying interest in the topic. A description of the molecular function of Cas9 and suggestion of its use for genome engineering, presented at the 2012 meeting, foreshadowed an explosion of research using CRISPR-Cas9 that was soon to come. Beginning in January 2013, a flurry of studies demonstrated that site-specific DNA editing in eukaryotic cells could be achieved through the heterologous expression of Cas9 together with a guide RNA. Two years and >1,000 publications later (Figure 1A), the technology has gone viral. The genomes of virtually all model plants and animals have been modified with CRISPR-Cas9, and creative new tools continue to expand the capabilities of this system. While CRISPR biology remains an active area of study, the memorable acronym is now more commonly associated with genome engineering than it is with prokaryotic adaptive immunity. In this perspective, we provide a concise summary of how the CRISPR-Cas9 technology emerged and is enabling remarkable innovations in the biological sciences. We encourage the reader to consult the recent literature for more comprehensive reviews (Doudna and Charpentier, 2014Doudna J.A. Charpentier E. Genome editing. The new frontier of genome engineering with CRISPR-Cas9.Science. 2014; 346: 1258096Crossref PubMed Scopus (3617) Google Scholar, Hsu et al., 2014Hsu P.D. Lander E.S. Zhang F. Development and applications of CRISPR-Cas9 for genome engineering.Cell. 2014; 157: 1262-1278Abstract Full Text Full Text PDF PubMed Scopus (3638) Google Scholar, Mali et al., 2013bMali P. Esvelt K.M. Church G.M. Cas9 as a versatile tool for engineering biology.Nat. Methods. 2013; 10: 957-963Crossref PubMed Scopus (892) Google Scholar). Detailed protocols for the numerous applications involving CRISPR-Cas9 can be found in a recent volume of Methods in Enzymology (Doudna and Sontheimer, 2014Doudna J.A. Sontheimer E.J. Methods in Enzymology. The use of CRISPR/Cas9, ZFNs, and TALENs in generating site-specific genome alterations. Preface.Methods Enzymol. 2014; 546: xix-xxCrossref PubMed Scopus (15) Google Scholar). Finally, we apologize to our many colleagues whose work we could not discuss due to length constraints, or because the work was published after completion of this article. So concluded a study published in 1987, in which the authors inadvertently discovered the first genomic CRISPR locus in Escherichia coli while sequencing the iap gene (Ishino et al., 1987Ishino Y. Shinagawa H. Makino K. Amemura M. Nakata A. Nucleotide sequence of the iap gene, responsible for alkaline phosphatase isozyme conversion in Escherichia coli, and identification of the gene product.J. Bacteriol. 1987; 169: 5429-5433Crossref PubMed Google Scholar). CRISPRs have since been found in roughly 40% and 90% of all bacterial and archaeal species, respectively (Grissa et al., 2007Grissa I. Vergnaud G. Pourcel C. The CRISPRdb database and tools to display CRISPRs and to generate dictionaries of spacers and repeats.BMC Bioinformatics. 2007; 8: 172Crossref PubMed Scopus (738) Google Scholar), and are characterized by short direct repeats interrupted at regular intervals by unique spacer sequences. Yet it wasn't until 2005 that a potential connection between CRISPRs and antiviral immune defense was established, when multiple laboratories reported that spacers derive from foreign genetic elements (Bolotin et al., 2005Bolotin A. Quinquis B. Sorokin A. Ehrlich S.D. Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin.Microbiology. 2005; 151: 2551-2561Crossref PubMed Scopus (1130) Google Scholar, Mojica et al., 2005Mojica F.J.M. Díez-Villaseñor C. García-Martínez J. Soria E. Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements.J. Mol. 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CRISPR provides acquired resistance against viruses in prokaryotes.Science. 2007; 315: 1709-1712Crossref PubMed Scopus (3857) Google Scholar). The central role of noncoding CRISPR RNA (crRNA) in this pathway was revealed shortly thereafter by pioneering work from the van der Oost laboratory (Brouns et al., 2008Brouns S.J.J. Jore M.M. Lundgren M. Westra E.R. Slijkhuis R.J.H. Snijders A.P.L. Dickman M.J. Makarova K.S. Koonin E.V. van der Oost J. Small CRISPR RNAs guide antiviral defense in prokaryotes.Science. 2008; 321: 960-964Crossref PubMed Scopus (1680) Google Scholar). CRISPRs function together with CRISPR-associated (cas) genes that typically flank CRISPR loci in the genome, and the entire pathway is consequently referred to as CRISPR-Cas. Adaptive immunity proceeds in three stages: acquisition (or adaptation), CRISPR RNA biogenesis, and interference (for recent reviews, see Barrangou and Marraffini, 2014Barrangou R. Marraffini L.A. CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity.Mol. Cell. 2014; 54: 234-244Abstract Full Text Full Text PDF PubMed Scopus (496) Google Scholar, van der Oost et al., 2014van der Oost J. Westra E.R. Jackson R.N. Wiedenheft B. Unravelling the structural and mechanistic basis of CRISPR-Cas systems.Nat. Rev. Microbiol. 2014; 12: 479-492Crossref PubMed Scopus (485) Google Scholar). During acquisition, new spacers are selected from foreign nucleic acids and integrated at one end of the CRISPR locus. RNA precursors are then transcribed from the CRISPR locus and enzymatically processed into mature crRNAs, which are bound by one or more Cas proteins to form ribonucleoprotein targeting complexes that each contain a single spacer (guide) sequence. Finally, Cas nucleases cleave target nucleic acids that are marked for degradation via complementary base pairing to the crRNA. CRISPR-Cas immune systems have been classified into three types and numerous subtypes based primarily on cas gene phylogeny (Makarova et al., 2011Makarova K.S. Haft D.H. Barrangou R. Brouns S.J.J. Charpentier E. Horvath P. Moineau S. Mojica F.J.M. Wolf Y.I. Yakunin A.F. et al.Evolution and classification of the CRISPR-Cas systems.Nat. Rev. Microbiol. 2011; 9: 467-477Crossref PubMed Scopus (1671) Google Scholar), and while many mechanistic features are widely conserved, significant differences exist. For example, Type I and III systems require only crRNA for targeting, while Type II systems also use trans-activating CRISPR RNA (tracrRNA) (Deltcheva et al., 2011Deltcheva E. Chylinski K. Sharma C.M. Gonzales K. Chao Y. Pirzada Z.A. Eckert M.R. Vogel J. Charpentier E. CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.Nature. 2011; 471: 602-607Crossref PubMed Scopus (1675) Google Scholar). In addition, the protein composition of crRNA-Cas targeting complexes is highly variable, with complexes in Type I and III systems typically comprising greater than eight subunits (Brouns et al., 2008Brouns S.J.J. Jore M.M. Lundgren M. Westra E.R. Slijkhuis R.J.H. Snijders A.P.L. Dickman M.J. Makarova K.S. Koonin E.V. van der Oost J. Small CRISPR RNAs guide antiviral defense in prokaryotes.Science. 2008; 321: 960-964Crossref PubMed Scopus (1680) Google Scholar, Hale et al., 2009Hale C.R. Zhao P. Olson S. Duff M.O. Graveley B.R. Wells L. Terns R.M. Terns M.P. RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex.Cell. 2009; 139: 945-956Abstract Full Text Full Text PDF PubMed Scopus (774) Google Scholar). In contrast, Type II systems require only a single polypeptide: Cas9 (Sapranauskas et al., 2011Sapranauskas R. Gasiunas G. Fremaux C. Barrangou R. Horvath P. Siksnys V. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli.Nucleic Acids Res. 2011; 39: 9275-9282Crossref PubMed Scopus (549) Google Scholar). Cas9 is a DNA endonuclease that generates double-stranded breaks (DSBs) in DNA target sequences identified through base pairing to the guide RNA (Figure 1B) (Gasiunas et al., 2012Gasiunas G. Barrangou R. Horvath P. Siksnys V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria.Proc. Natl. Acad. Sci. USA. 2012; 109: E2579-E2586Crossref PubMed Scopus (1683) Google Scholar, Jinek et al., 2012Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (9424) Google Scholar). Cas9 functions naturally with a dual-guide RNA composed of crRNA and tracrRNA (Jinek et al., 2012Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (9424) Google Scholar); the 5′ end of the crRNA base-pairs with target DNA, whereas the 3′ end forms a double-stranded stem with the tracrRNA to facilitate Cas9 recruitment. Because DNA targets are recognized via RNA-DNA base pairing, changing the sequence of the guide RNA easily alters DNA specificity. Efficient targeting also requires the presence of a short sequence motif proximal to the DNA target sequence, known as the protospacer adjacent motif (PAM) (Mojica et al., 2009Mojica F.J.M. Díez-Villaseñor C. García-Martínez J. Almendros C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system.Microbiology. 2009; 155: 733-740Crossref PubMed Scopus (1014) Google Scholar). By specifically selecting spacers at PAM sites during acquisition (Yosef et al., 2012Yosef I. Goren M.G. Qimron U. Proteins and DNA elements essential for the CRISPR adaptation process in Escherichia coli.Nucleic Acids Res. 2012; 40: 5569-5576Crossref PubMed Scopus (481) Google Scholar), CRISPR-Cas immune systems can discriminate between self and non-self sequences during interference via PAM recognition. Targets found in foreign DNA contain a PAM and are targeted, whereas matching targets in the CRISPR locus itself, from which the crRNA is transcribed, do not contain a PAM and are avoided. Cas9 from Streptococcus pyogenes, which has been the focus of most studies to date, recognizes a 5′-NGG-3′ PAM sequence (Jinek et al., 2012Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (9424) Google Scholar, Mojica et al., 2009Mojica F.J.M. Díez-Villaseñor C. García-Martínez J. Almendros C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system.Microbiology. 2009; 155: 733-740Crossref PubMed Scopus (1014) Google Scholar). The ability to edit genomic DNA inside cells has been limited by the dearth of effective tools to introduce site-specific DSBs (Carroll, 2014Carroll D. Genome engineering with targetable nucleases.Annu. Rev. Biochem. 2014; 83: 409-439Crossref PubMed Scopus (382) Google Scholar). Earlier methods relied on protein-only systems such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), but the feasibility of engineering these designer enzymes to recognize new sequences was limited. In contrast, the CRISPR-Cas system has the distinct advantage of relying on RNA for specificity. And while Cas9 shares molecular capabilities with other CRISPR-Cas systems, its compositional simplicity has been paramount to its successful application. Not only does it encompass only a single polypeptide, but remarkably, it retains full activity with a chimeric single-guide RNA (sgRNA), generated by connecting the 3′ end of the crRNA to the 5′ end of the tracrRNA (Jinek et al., 2012Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (9424) Google Scholar). Six months after the first description of the molecular function of Cas9 (Jinek et al., 2012Jinek M. Chylinski K. Fonfara I. Hauer M. Doudna J.A. Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.Science. 2012; 337: 816-821Crossref PubMed Scopus (9424) Google Scholar), six studies demonstrated that Cas9 together with sgRNA can be used to specifically edit the genomes of human cells (Cho et al., 2013Cho S.W. Kim S. Kim J.M. Kim J.-S. 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Efficient genome editing in zebrafish using a CRISPR-Cas system.Nat. Biotechnol. 2013; 31: 227-229Crossref PubMed Scopus (2150) Google Scholar), and bacteria (Jiang et al., 2013Jiang W. Bikard D. Cox D. Zhang F. Marraffini L.A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems.Nat. Biotechnol. 2013; 31: 233-239Crossref PubMed Scopus (1680) Google Scholar). The technology was rapidly extended to other model organisms, and by May of 2013, the Jaenisch laboratory reported the one-step generation of mice mutated at multiple alleles via zygotic injection of Cas9 mRNA and sgRNAs (Wang et al., 2013Wang H. Yang H. Shivalila C.S. Dawlaty M.M. Cheng A.W. Zhang F. Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering.Cell. 2013; 153: 910-918Abstract Full Text Full Text PDF PubMed Scopus (2597) Google Scholar). What had once been laborious and time-consuming was now facile and rapidly achievable. In the simplest embodiment of the CRISPR-Cas9 technology (Figure 1C), the sgRNA guide sequence is designed to target a complementary 20-bp site flanked by NGG within the gene of interest. Heterologous expression of Cas9 together with sgRNA may be accomplished by stable lentiviral transfection, transient plasmid transfection, direct DNA or RNA injection, or transfection with purified ribonucleprotein complex, with the optimal strategy depending on the desired application and the cell or organism being edited. After trafficking to the nucleus through an appended nuclear localization signal, Cas9 targets the locus of interest and induces a DSB that is repaired by the cell's endogenous machinery via non-homologous end-joining (NHEJ). Because NHEJ is an error-prone repair pathway that results in small insertions or deletions (indels), the open reading frame is disrupted and the gene becomes inactivated. While the editing efficiency with CRISPR-Cas9 can be as high 80% (Kim et al., 2014Kim S. Kim D. Cho S.W. Kim J. Kim J.-S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins.Genome Res. 2014; 24: 1012-1019Crossref PubMed Scopus (1136) Google Scholar, Zuris et al., 2015Zuris J.A. Thompson D.B. Shu Y. Guilinger J.P. Bessen J.L. Hu J.H. Maeder M.L. Joung J.K. Chen Z.-Y. Liu D.R. Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.Nat. Biotechnol. 2015; 33: 73-80Crossref PubMed Scopus (953) Google Scholar), it is cell and site specific and depends on the delivery method. In addition, the resulting cell population will be inherently heterogeneous, both in the percentage of cells that were edited and in the specific genotype of the edited cells. The genome can also be edited in a more precise manner using homology-directed repair (HDR). By combining Cas9-sgRNA delivery with a donor DNA that bears homology to sequences flanking the targeted site, DSBs are repaired using the donor DNA as a template. Importantly, this strategy enables new sequences to be introduced into the gene of interest, such as epitope tags, and specifically defined mutations to be installed, such as those that might mimic or correct disease-causing alleles. However, efficiencies of HDR are significantly lower than NHEJ, and more work is needed to develop strategies that bias the cell's natural DNA repair machinery toward the desired outcome. Genome editing with CRISPR-Cas9 is now a routine procedure for virtually all model plants and animals, and recent progress has pushed the technology into ever more interesting directions. A unique advantage of CRISPR-Cas9 over earlier genome editing methods is that multiplexable targeting is easily achieved by co-expressing Cas9 with multiple sgRNAs simultaneously. In addition to editing multiple chromosomal loci in a single experiment with this approach, large chromosomal deletions can be achieved by using two sgRNAs to induce DSBs at sites that flank the region of interest (Xiao et al., 2013Xiao A. Wang Z. Hu Y. Wu Y. Luo Z. Yang Z. Zu Y. Li W. Huang P. Tong X. et al.Chromosomal deletions and inversions mediated by TALENs and CRISPR/Cas in zebrafish.Nucleic Acids Res. 2013; 41: e141Crossref PubMed Scopus (310) Google Scholar). Furthermore, large-scale chromosomal rearrangements resembling those found in specific tumors can be introduced (Choi and Meyerson, 2014Choi P.S. Meyerson M. Targeted genomic rearrangements using CRISPR/Cas technology.Nat. Commun. 2014; 5https://doi.org/10.1038/ncomms4728Crossref Scopus (222) Google Scholar, Torres et al., 2014Torres R. Martin M.C. Garcia A. Cigudosa J.C. Ramirez J.C. Rodriguez-Perales S. Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR-Cas9 system.Nat. 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When expressed in bacteria, dCas9 together with sgRNA can sterically occlude RNA polymerase from binding promoter sequences and thereby downregulate the expression of specific transcripts (Bikard et al., 2013Bikard D. Jiang W. Samai P. Hochschild A. Zhang F. Marraffini L.A. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system.Nucleic Acids Res. 2013; 41: 7429-7437Crossref PubMed Scopus (736) Google Scholar, Qi et al., 2013Qi L.S. Larson M.H. Gilbert L.A. Doudna J.A. Weissman J.S. Arkin A.P. Lim W.A. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.Cell. 2013; 152: 1173-1183Abstract Full Text Full Text PDF PubMed Scopus (3031) Google Scholar). 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For example, DNA loci can be imaged in live cells using dCas9-GFP fusions (Chen et al., 2013Chen B. Gilbert L.A. Cimini B.A. Schnitzbauer J. Zhang W. Li G.-W. Park J. Blackburn E.H. Weissman J.S. Qi L.S. Huang B. Dynamic imaging of genomic loci in living human cells by an optimized CRISPR/Cas system.Cell. 2013; 155: 1479-1491Abstract Full Text Full Text PDF PubMed Scopus (1273) Google Scholar), offering new insights into the dynamics and conformation of genomic loci. Robust genetic circuits can be constructed and implemented using dCas9-mediated transcriptional control (Kiani et al., 2014Kiani S. Beal J. Ebrahimkhani M.R. Huh J. Hall R.N. Xie Z. Li Y. Weiss R. CRISPR transcriptional repression devices and layered circuits in mammalian cells.Nat. Methods. 2014; 11: 723-726Crossref PubMed Scopus (157) Google Scholar, Liu et al., 2014Liu Y. Zeng Y. Liu L. Zhuang C. Fu X. Huang W. Cai Z. 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Programmable RNA recognition and cleavage by CRISPR/Cas9.Nature. 2014; 516: 263-266Crossref PubMed Scopus (435) Google Scholar), suggesting that programmable manipulation of cellular RNA transcripts may become possible in the near future. CRISPR-Cas9 enables facile, targeted perturbation of specific genes in the cell, either through permanent genome editing or temporary gene regulation. A systematic investigation of gene function, however, requires this level of control to be extended across the genome. A number of recent studies have harnessed the programmable nature of CRISPR-Cas9 to conduct powerful genome-scale screens. Importantly, this approach enables the hypothesis-free discovery of novel pathways that underlie a given biological process. Using lentiviral sgRNA libraries and catalytically active Cas9, loss-of-function gene knockout screens were performed in both human and mouse cells (Koike-Yusa et al., 2014Koike-Yusa H. Li Y. Tan E.-P. Velasco-Herrera Mdel.C. Yusa K. 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Deep sequencing of the sgRNA pool after either positive or negative selection revealed genes essential for cell viability, as well as genes involved in resistance to specific small-molecule drugs. While focused libraries will prove useful for targeted screens in which the candidate genes are selected by the researcher, genome-wide libraries that query all protein-coding genes will