A CRISPR Path To Engineering New Genetic Mouse Models For .
Cutting-Edge ReviewA CRISPR Path to Engineering New Genetic Mouse Modelsfor Cardiovascular ResearchJoseph M. Miano, Qiuyu Martin Zhu, Charles J. LowensteinAbstract—Previous efforts to target the mouse genome for the addition, subtraction, or substitution of biologicallyinformative sequences required complex vector design and a series of arduous steps only a handful of laboratoriescould master. The facile and inexpensive clustered regularly interspaced short palindromic repeats (CRISPR) methodhas now superseded traditional means of genome modification such that virtually any laboratory can quickly assemblereagents for developing new mouse models for cardiovascular research. Here, we briefly review the history of CRISPRin prokaryotes, highlighting major discoveries leading to its formulation for genome modification in the animal kingdom.Core components of CRISPR technology are reviewed and updated. Practical pointers for 2-component and 3-componentCRISPR editing are summarized with many applications in mice including frameshift mutations, deletion of enhancersand noncoding genes, nucleotide substitution of protein-coding and gene regulatory sequences, incorporation of loxP sitesfor conditional gene inactivation, and epitope tag integration. Genotyping strategies are presented and topics of geneticmosaicism and inadvertent targeting discussed. Finally, clinical applications and ethical considerations are addressedas the biomedical community eagerly embraces this astonishing innovation in genome editing to tackle previouslyintractable questions. (Arterioscler Thromb Vasc Biol. 2016;36:1058-1075. DOI: 10.1161/ATVBAHA.116.304790.)Key Words: epitope frameshift mutation genetics genome editing genotype nucleotideThe 2007 Nobel Prize in Physiology or Medicine wasawarded for pioneering work in embryonic stem cells(ESC), which developed techniques to modify the mousegenome leading to the creation of thousands of knockoutmouse strains, including hundreds that exhibit cardiovascular phenotypes (ftp://ftp.informatics.jax.org/pub/reports/MGI Knockout Full.html). Traditionally, the generation ofknockin or knockout mouse models for cardiovascular studyrequired intricate design and construction of a targeting vector, electroporation of the vector into a limited number ofESC lines, selection and enrichment of targeted ESC clones,screening of clones with validated probes by Southern blotting, and expansion of correctly targeted clones for injectioninto the blastocyst of a mouse embryo.1 If ensuing chimericmice were able to pass the targeted allele through the germline, and if phenotypic haploinsufficiency leading to premature debilitating disease or death did not occur, heterozygousoffspring could be intercrossed to create mice homozygous forthe targeted allele. The time and effort involved in creating amouse in this manner is considerable (typically more than 1year) with no guarantee of success. The advent of nucleasedirected genome editing (NDGE) in animals with zinc fingernucleases2 and transcription activator–like effector nucleases3simplified the labor-intensive task of generating geneticallymodified mice while reducing the time to completion. Thesetechnologies also augmented the range of animals that couldsuccessfully undergo genome modifications.4 However, themore recent clustered regularly interspaced short palindromicrepeats (CRISPR) system has rapidly ascended as the méthodede choix for genome editing (Figure 1A) because of its incredible simplicity and high efficiency. Indeed, more than half ofthe entire literature pertaining to CRISPR through 2015 wasreported in that year (Figure 1B).Although many reviews have been published on the subject of CRISPR,5–7 the field has moved at such a ferociouspace, with many pearls of knowledge only recently sharedand debated through a discussion group started in early2013 (https://groups.google.com/forum/#!forum/crispr), thatan update is timely, particularly in cardiovascular biologywhere only a few papers have been published using CRISPRgenome editing in mice. Here, we briefly summarize the historical roots of CRISPR and the key elements of the technology as they apply to the generation of new mouse models. Weprovide an overview of 2-component (2c) and 3-component(3c) CRISPR, highlighting the strengths and weaknesses ofeach with specific applications. We address important issuesrelated to the design and testing of crucial components ofCRISPR as well as genetic mosaicism, robust genotyping,Received on: March 14, 2016; final version accepted on: April 6, 2016.From the Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, Rochester, NY (J.M.M., Q.M.Z., C.J.L.);and Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA (Q.M.Z.).The online-only Data Supplement is available with this article at 61/ATVBAHA.116.304790/-/DC1.Correspondence to Joseph M. Miano, PhD, Aab Cardiovascular Research Institute, University of Rochester School of Medicine and Dentistry, 601Elmwood Ave, Box CVRI, Rochester, NY 14642, E-mail j.m.miano@rochester.edu 2016 American Heart Association, Inc.Arterioscler Thromb Vasc Biol is available at http://atvb.ahajournals.orgDOI: 10.1161/ATVBAHA.116.304790Downloaded from http://atvb.ahajournals.org/1058at University of Rochester on July 5, 2016
Miano et al CRISPR-izing Mice 1059Nonstandard Abbreviations and Acronyms2-component CRISPR3-component CRISPRclustered regularly interspaced short palindromic repeatsCRISPR-associated protein 9CRISPR RNAdouble-strand breakdouble-strand DNAembryonic stem cellhomology-directed repairinsertion or deletionlong noncoding RNAnuclease-directed genome editingnonhomologous end joiningprotospacer adjacent motifpremature termination codonsingle-guide RNAsingle-nucleotide polymorphismsingle-strand oligonucleotidetransactivating crRNAtranscription factor–binding siteand inadvertent (off) targeting, all of which must be carefully considered for unambiguous evaluation of CRISPRderived mice. Finally, we provide some clinical perspectivefor future applications and challenges that must be overcomefor CRISPR to have tangible impact in the treatment of cardiovascular disease.Origins of CRISPR and Its Transition to theAnimal KingdomIn 1987, a peculiar arrangement of homologous direct repeatshaving dyad symmetry and unknown function was discovered in Escherichia coli.8 Similarly arrayed sequences weresubsequently found in archaea and other species of bacteria.9These repeat sequences were named CRISPR.10 No CRISPRsequences were identified in eukaryotes or viruses, suggesting aunique function of CRISPR to species of prokaryotes.10 The firstAB# PubMed Papers15001200CRISPRTALENZFN90060030002002 2004 2006 2008 2010 2012 2014# PubMed CRISPR Papers2c CRISPR3c EJPAMPTCsgRNASNPSSOtracrRNATFBSclue as to the function of CRISPR sequences emerged with thediscovery of similar sized CRISPR spacers between CRISPRsas well as flanking CRISPR-associated (Cas) protein-codinggenes, implying inter-related functional gene loci.10 An important breakthrough occurred in 2005 when the CRISPR spacers were found to be of bacteriophage and plasmid origin.11,12These findings sparked the prescient hypothesis that CRISPRsendow bacteria with genetic immunity against foreign DNA.12Then, in 2007, studies proved CRISPR and adjacent Cas operons confer acquired resistance in bacteria to invading viruses.13The mechanism underlying such adaptive immunity occurs viaendonuclease-mediated cleavage of pre-CRISPR transcripts byCas proteins and subsequent processing into smaller CRISPRRNA (crRNA) that are complementary to viral DNA, leading tosilencing of the alien invasion.14 Extended CRISPR sequences,reflecting sequential infections of bacteria, imparted greaterresistance to bacteriophage assault.12,13 Thus, many prokaryotes acquire immunity that is hard wired into the host genome.Importantly, in the type II CRISPR-Cas system, the procurement of CRISPR spacers (or protospacers) from the initialinvasion of a bacteriophage or plasmid requires a protospaceradjacent motif (PAM) immediately 3′ of the CRISPR spacersequence in invading DNA.15 This orientation with respect toCRISPR spacer and PAM is also critical for CRISPR-mediatedNDGE in the mouse genome. Although the CRISPR acronymis more aptly applied to prokaryotic immunity, it has becomeengrained in the lexicon of eukaryotic biologists and the publicas a powerful means of altering genomes. For a more in depthstudy of CRISPR in prokaryotic biology, the reader is referredto several outstanding reviews from pioneers in the field.16–18Two key breakthroughs precipitated the “CRISPR Craze.”19First, a transactivating crRNA (tracrRNA) was established asessential for crRNA processing in Streptococcus pyogenes.20The second important discovery occurred 1 year later whencrRNA, tracrRNA, and Cas9 were combined in vitro andshown to cleave the complementary and noncomplementary strands of target plasmid DNA 3 base pairs upstreamof a PAM.21,22 Importantly, the crRNA and tracrRNA couldbe combined into 1 hybrid transcript (so-called singleguide RNA [sgRNA]) and still direct Cas9 cutting of targetDNA.21 Over the ensuing months, an avalanche of papers15001200900600300100806040200Figure 1. Explosive rise in clustered regularly interspaced short palindromic repeats (CRISPR)–related publications. A, Comparative publication track record for the 3 major methods of nuclease-directed genome editing during the past 14 years. B, Annual number of CRISPRpublications based on PubMed search. After a relatively inactive period (2002–2006), a first wave of papers was published based on theprokaryote adaptive immune function of CRISPR (2007–2011) followed by a second wave relating to CRISPR genome editing (2012–present). Data for both graphs obtained on December 30, 2015.Downloaded from http://atvb.ahajournals.org/ at University of Rochester on July 5, 2016
1060 Arterioscler Thromb Vasc Biol June 2016demonstrated NDGE in human and mouse cell lines23–25 aswell as various animal models26–34 using the main components comprising CRISPR genome editing, each of which isconsidered next.Components of CRISPR Genome EditingComponent 1: Cas9 EndonucleaseThe most common endonuclease used in CRISPR genomeediting is the class II effector protein, Cas9, from S pyogenes(Spycas9).35 The Spycas9 gene is transcribed as a 4.2-kbtranscript encoding a 1368 amino acid protein of 160 kDa.The transcript has been codon optimized for efficient translation in mammals and engineered to carry nuclear localizationsignals for proper targeting to the nucleus as well as epitopetags for easy detection.23–25 The SpyCas9 cDNA is found inseveral plasmids, most notably pX330 from addgene (https://www.addgene.org/42230/).23 The pX330 plasmid serves as atemplate for in vitro transcription of Spycas9. However, it ismuch easier to purchase ready-made SpyCas9 protein (PNABio, http://pnabio.com) or more frequently, Spycas9 mRNA(TriLink BioTechnologies, http://www.trilinkbiotech.com)for immediate microinjection in mouse zygotes. The longer 4.5-kb Spycas9 mRNA from TriLink includes a capped,5′ synthetic untranslated region harboring a strong Kozaksequence for enhanced translation, an α-globin 3′ untranslated region, and a 120-bp polyadenylated tail to augmentstability (Tiffany Teng of TriLink BioTechnologies, personalcommunication).It is possible to inject mouse zygotes directly with a plasmid containing both Spycas9 and sgRNA.36 However, thisapproach is complicated by the reduced chance of transmitting an edited allele through the germline because of embryonic cell divisions preceding Cas9/sgRNA transcription andCas9 translation. Furthermore, there are unpredictable effectsof a randomly integrated plasmid in the mouse genome andunknown consequences of continuous expression of Cas9 protein over the life of the mouse. Nevertheless, a recent studyshowed that mice carrying a Cas9 transgene under control ofthe cardiac-specific Myh6 promoter exhibited no observabletoxicity. These mice allow for rapid assessment of gene function in the adult heart after a single injection of adeno-associated virus carrying an sgRNA of interest, thus circumventingpotential embryonic lethality that otherwise occurs with conventional gene targeting strategies.37 Additional mouse models will likely emerge that further limit expression of Cas9to heart or blood vessels using drug-responsive, cell-specificpromoters driving Cas9 directly or indirectly via Cre recombinase in the context of the Rosa26 floxed stop Cas9 transgenicmouse (https://www.jax.org/strain/024857).38The SpyCas9 protein has a unique bilobed structure.39,40The α-helical target recognition lobe makes important contacts with the sgRNA bound to its target genomic DNA. Thenuclease lobe contains the HNH and RuvC-like nucleasedomains, each of which mediates an individual nick in thecomplementary (via HNH) or noncomplementary (via RuvClike) strand of the double helix, thus creating a blunt doublestrand break (DSB). In addition, a PAM-interacting domainwithin the nuclease lobe binds PAM sequences (NGG orNAG) that are necessary for igniting SpyCas9 nuclease activity, leading to target DNA cleavage41 3 base pairs upstream ofthe PAM sequence.42 The PAM-interacting domain may alsofunction to facilitate unwinding of the double helix in a 3′ to5′ direction from the PAM site to allow for strand invasion ofthe sgRNA, Watson–Crick base pairing between the sgRNAand its complementary target DNA sequence, and formationof a so-called R loop structure.40,43 PAM sequences may bediversified through alterations in SpyCas944 or the utilizationof other RNA-guided endonucleases.45–47 This increases thesequence space that may be efficiently targeted such that virtually every nucleotide in the mouse genome may undergogenome editing.Additional derivations of SpyCas9 have been engineered that expand its functionality. For example, either ofthe nuclease domains may be mutated (D10A or H840A) tocreate a nickase (Cas9n), which increases the specificity ofgenome editing.21,48 We do not advocate the use of Cas9nfor generating cardiovascular mouse models because of theinherent complexity in experimental design as well as thelower efficiency of SpyCas9n versus wild-type SpyCas9in mediating genome edits,49 although editing genomesis highly locus dependent and there may be instanceswhere Cas9n is desirable.50 However, newer generations ofSpyCas9 carrying mutations that do not alter the 2 nuclease domains have been developed that also reduce off-targeting events. These include enhanced specificity SpyCas9(https://www.addgene.org/71814/)51 and a high fidelitySpyCas9 (https://www.addgene.org/72247/).52 It will beimportant to see whether these newer versions of SpyCas9are offered as ready-made mRNA or protein and how wellthey perform in editing the mouse genome when comparedwith wild-type SpyCas9.Another modification of SpyCas9 involves mutationof both nuclease domains that effectively yields a dead ordeactivated version of the enzyme (dCas9). This alteration in SpyCas9 preserves its sgRNA-directed binding totarget DNA; however, dCas9 is unable to cut DNA rendering the protein a suitable platform for many applications,most notably activation or repression of genes through theconjugation of transcriptional activators or repressors.53This approach offers a potentially powerful means of rescuing mouse phenotypes resulting from CRISPR-mediatedNDGE of distal enhancer sequences by directly activatingthe endogenous promoter. Another application of dCas9involves joining the protein with a fluorophore for purposesof tracking Cas9 binding across the genome.54 A recentstudy utilizing such an approach revealed Cas9 targetingboth euchromatic and heterochromatic sequences througha diffusion-like mechanism, suggesting most of the mousegenome may be targeted for editing regardless of epigenetic state.55 This finding is consistent with Cas9-mediatedcleavage of methylated DNA.56 Recently, however, an invitro study showed that nucleosomes represent a barrier forCas9 binding and cleavage, suggesting the state of chromatin around a gene of interest in the mouse zygote may bean important determinant of CRISPR-mediated NDGE.57The latter point underscores the need to test each sgRNA inadvance of experimentation (below).Downloaded from http://atvb.ahajournals.org/ at University of Rochester on July 5, 2016
Miano et al CRISPR-izing Mice 1061Endonuclease activity of SpyCas9 requires a conformational change in its structure, which is mediated by thesgRNA/target DNA sequence.40 We know little, however,about endonuclease-independent activity of this protein inthe mouse. Preliminary physiological data suggest no overtphenotypes arising from constitutive expression of Cas9 inmice,37,38 and RNA-seq studies have been done in culturedcells transiently58 or stably59 expressing targeted Cas9 withlittle changes in gene expression beyond those expected. Itmust be stressed, however, that no studies have yet to interrogate the transcriptome or proteome of tissues in mice withchronic expression of Cas9. It is possible that Cas9 bindsother proteins in the cytosol or nucleus affecting subtle perturbations in cell physiology. Furthermore, as a bacterialprotein, Cas9 may elicit an immune response. Thus, untilmore thorough studies are carried out, it is premature to conclude that constitutive expression of Cas9 is not without sideeffects. Efforts should therefore be made to minimize thewindow of time in which Cas9 is expressed in mice. This isbest achieved by microinjecting Cas9 mRNA or protein intothe mouse zygote in combination with the next component ofCRISPR technology.Component 2: sgRNAThe Cas9 endonuclease is inactive until it is bound and ushered to a specific genomic address by the sgRNA. The sgRNAserves as a dual function of both chaperone and activator ofCas9 endonuclease activity through the union of 2 separablesequences transcribed as a chimeric RNA.21 The chaperone (orguiding) function of the sgRNA is achieved by a user-defined20-nucleotide CRISPR RNA (crRNA or protospacer sequencein recognition of similarly functioning sequences in bacteriaand archaea), whereas the activator function is served by theinvariant 80-nucleotide tracrRNA (Figure 2A). The tracrRNA,derived from the S pyogenes genome, comprises many structural modules that serve critical roles for Cas9 binding andactivation.60 The crRNA sequence is complementary to the target sequence to be edited in the mouse genome and althoughsequences longer than 20 nucleotides might intuitively confergreater specificity, they are cleaved to 20 nucleotides duringprocessing.48The 20-nucleotide crRNA sequence must precede aPAM recognition site (NGG or NAG for SpyCas9) locatedimmediately 3′ of the last nucleotide of the crRNA sequence.Binding and cleavage of DNA by Cas9 requires the PAMDSBAsgRNA15’ tolerant8 9 17 18 19 20seed ( 12nt)3’intolerantcrRNA/protospacer (20nt)tracrRNA (80nt)sgRNAB 15ntHDRtemplate5’5’- Homology armCGenomicDNAPAMNXX3’3’- Homology armmutationexonGene RNASpyCas93’Figure 2. Characteristics of single-guide RNA (sgRNA) and homology-directed repair (HDR) repair template. A, The sgRNA is a chimericmolecule comprising a user-defined 20 nucleotide crRNA containing mismatch-tolerant (gray) and intolerant (red) nucleotides and aninvariant 80-nucleotide transactivating crRNA (tracrRNA, green). The red triangle here and below denotes the double-strand break (DSB)that occurs after Cas9 binding (C). B, The HDR template is generally a single-strand oligonucleotide with homology arms of 50 to 70nucleotides on each side. Note position of sequence edit (labeled mutation) at center of HDR template within 15 nucleotides of the DSB.The XX below protospacer adjacent motif (PAM) denotes
nents comprising CRISPR genome editing, each of which is considered next. Components of CRISPR Genome Editing Component 1: Cas9 Endonuclease The most common endonuclease used in CRISPR genome editing is the class II effector protein, Cas9, from S pyogenes (
Adding CRISPR to your Bio ARROW Protocol Page 2 of 6. Work Covered by this Guidance Document: This guidance document covers how to add the use of CRISPR systems (e.g., CRISPR/Cas9, CRISPR/Cpf1) – whether for genome editing or other purposes (e.g., CRISPR-mediated
CRISPR ethics and vice versa. Third, we assess several key ethical considerations. Notably, while some of these concerns are specific to CRISPR technology, many, such as research on human embryos, have been debated long before the CRISPR revolution [15]. Moreover, since CRISPR is still a maturing technology, novel applications in
CRISPR-EZ: CRISPR- RNP Electroporation of Zygotes Chen et al., JBC, 2016 CRISPR-EZ Advantages 100% Cas9 RNP delivery Highly efficient NHEJ and HDR editing indel, point mutation, deletion, insertion 3x increase in embryo viability Easy, economic and high-throughput CRISPR-EZ Challenges Larg
industrial applications. Finally, the efforts of researchers paid off with the development of CRISPR, a robust molecular tool that can edit DNA at virtually any locus. CRISPR technology is igniting a revolution across the life sciences and is quickly becoming a standard tool in many labs. Given its ease-of-use and versatility, CRISPR is already .
CRISPR-Cas mediated knockout of pdgfrB ß and . heterozygous carriers for the mutations are currently being raised. We anticipate that the mutant . CRISPR-Cas9 system is a rapid and efficient
the production of eective compounds [11]. e applica-tion of the CRISPR-Cas system to gene functional studies and metabolic networks regulation of medicinal plants is essential and meaningful, presenting a promising method for improving quality and breeding ideal germplasms in medicinal plants. Here, we review CRISPR-based tools and briey intro-
Cas9 Enzymology The Cas9 protein contains two independent endonuclease domains: one is homologous to the HNH endonuclease . CRISPR/Cas9 Delivery Methods tool, CRISPR was widely used in many experimental settings . RNPs induce editing at. 3 .
Animal Nutrition is a core text for undergraduates in Animal Science, Veterinary Science, Agriculture, Biology and Biochemistry studying this subject. It also provides a standard reference text for agricultural advisers, animal nutritionists and manufacturers of animal feeds. The latest edition of this classic text continues to provide a clear and comprehensive introduction to the science and .
