Generating Mouse Models with CRISPR/Cas9 ABSTRACT Genome editing with CRISPR/Cas9 enables the generation of new mouse models with unprecedented speed and simplicity. The Jackson Laboratory was an early adopter of CRISPR/Cas9 technology for mouse model development and in this brief article we describe the technology and lessons learned using it to generate both knockouts and knockins in more than a dozen different genetic backgrounds. JAX ® MICE, CLINICAL AND RESEARCH SERVICES
Transcript
Generating Mouse Models with CRISPR/Cas9
ABSTRACT
Genome editing with CRISPR/Cas9 enables the generation of new mouse models with unprecedented speed and simplicity. The Jackson Laboratory was an early adopter of CRISPR/Cas9 technology for mouse model development and in this brief article we describe the technology and lessons learned using it to generate both knockouts and knockins in more than a dozen different genetic backgrounds.
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Generating Mouse Models with CRISPR/Cas9
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Generating Mouse Models with CRISPR/Cas9
ContentsCRISPR/Cas9 Model Generation .......................................................................... 4
Advantages over Other Technologies .................................................................... 5
Timelines for Model Generation ........................................................................... 6
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Generating Mouse Models with CRISPR/Cas9
CRISPR/Cas9 Model GenerationCRISPR (clustered regularly interspaced short palindromic repeat) and Cas9 (CRISPR-associated) endonucleases represent a game-changing development in genetic engineering (Cong et al., 2013; Hsu et al., 2014; Jinek et al., 2012; Wang et al., 2013; Yang et al., 2014). The system employs components from bacteria and archaea that protect against invading viruses (Bhaya et al., 2011). Figure 1 illustrates the principle underlying the
methodology, in which double-stranded breaks in DNA are directed by a single guide RNA (sgRNA) attached to a nuclease from the bacterium Straptococcus pyogenes, Cas9. This allows for the creation of random nucleotide insertion and deletion (indel) mutations at the target site involving the error-prone non-homologous end joining repair pathway. More precise gene editing also requires synthesis of an appropriate donor DNA repair sequence or editing template, on which genomic recombination can be directed (Yang et al., 2014).
Cas9
sgRNA
Homology Directed Repair
Editing Template
Knock-in by precise gene editing (point mutation, lox p sites)Knock-out by frame shift mutation
Non Homologous End Joining
Target Gene
DNA Break
DNA Repair
Mutated Allele
Figure 1. Schematic of Cas9 loaded with single guide RNA generating a double strand break that can introduce various mutations through different repair mechanisms. The error-prone non-homologous end-joining pathway introduces indel mutations through misaligned repair or resection that can lead to frameshift mutations and gene knockout. Homology-directed repair involves recruitment of accessory factors that can direct homologous recombination in the presence of an exogenous repair or editing template. Image adapted from Hsu et al., 2014 and Barrangou et al., 2014.
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Generating Mouse Models with CRISPR/Cas9
Advantages over Other TechnologiesThe advent of genetic engineering significantly changed the landscape for basic and translational research, making it possible to engineer the animal model to answer a particular question. However, these technologies have also
had shortcomings. Random insertional mutagenesis made it possible to create transgenic animals to examine the effects of the expression of a foreign gene but relied on a random process that could have unintended consequences as copy number and site of insertion could not be controlled. Homologous recombination in ES cells, though powerful, requires more steps,
Injection
Implantation
Screening
Backcrossing
F0 founder mice screened for transgene
Transgenic mutation(random insertion)
Targeted mutations Endonuclease-mediated mutation
DNA construct
DNA construct
Cas9
Backcrossing to backgroundof interest
Backcrossing to backgroundof interest
Backcrossing not required
F0 chimeras crossed to wild-type mice to test germline transmission
and generate N1
F0 crossed to wild-type mice to test germline transmission
and generate N1
Traditional CRISPR
Figure 2. Traditional vs. CRISPR approaches for the genetic modification of mice. Transgenics are generally produced by injection of a DNA construct into a fertilized egg that is then implanted into a pseudopregnant female. The site of insertion is often unknown and can disrupt other genes. Targeted mutations are produced by introducing a DNA construct into embryonic stem (ES) cells. Positive or negative selection allows the identification of mutant ES cells which have undergone correct homologous recombination that are then injected into a blastocyst that is then implanted in a pseudopregnant female. Chimeric offspring are bred to screen for germline transmission of the targeted mutation. CRISPR/Cas9 mutations can be introduced directly in the fertilized oocyte stage through the use of a guide RNA, endonuclease activity, and repair mechanisms that can be error-prone or homology directed (See Figure 1). The ability to introduce mutations directly on the background of interest obliterates the need for an existing germline-competent ES cell line and thus circumvents the time and cost of backcrossing to the desired inbred background.
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Generating Mouse Models with CRISPR/Cas9
and it takes significantly more time to produce a chimera than it does to produce a founder mouse using CRISPR. This approach is also limited to genetic backgrounds from which germline-competent ES cells can be derived. In contrast, CRISPR/Cas9 is efficient enough that it can be used directly in embryos, eliminating the time, cost and constraints associated with ES cells, but is very precise, providing an advantage over conventional transgenics. Figure 2 shows how the biology of traditional transgenic technologies compared with CRISPR/Cas9 technology.
The rapid adoption of CRISPR-based genome editing technology speaks to its major advantages— increased speed, reduced cost and direct generation of mutants on a large variety of genetic backgrounds. In comparison with more traditional gene targeting, CRISPR/Cas9, bypasses the usage of ES cells, allows the introduction of mutations into virtually any genetic backgrounds, even existing genetically engineered models, creating a powerful platform for generating new mouse models.
Timelines for Model GenerationThe timeline to produce mutants via CRISPR/Cas9 depends on the type of mutation being introduced. Indel mutations can be generated simply by injecting Cas9 mRNA and sgRNA(s) into fertilized oocytes. Founder animals are born 3 weeks after injection, and can be set up for germline breeding around 12 weeks post injection. Heterozygous N1 animals carrying the desired mutation can thus be generated in as little as half a year. The same is true for projects in which smaller single-stranded oligonucleotide repair templates can be used to generate e.g. stop codon insertions, point mutations, and tag insertions. For more complex alleles such as reporter insertions or conditional KO alleles, the cloning of larger double-stranded DNA repair templates is required, adding several weeks to the timeline.
To date JAX has had a greater than 90% success rate with respect to creating knockouts and knock-ins. However, timelines can be extended if no founder animals are produced and injections need to be repeated. Figure 3 outlines major steps from project initiation to completion. Table 1 compares the time to produce mutants using ES cells vs. CRISPR/Cas9.
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Generating Mouse Models with CRISPR/Cas9
Model Desired ES-Cell Based Homologous Recombination CRISPR/Cas9
Estimated Time
Single knockout/point muta-tions
1-1.5 years for germline(+1.5 years for speed congenic breeding)
3-4 months for F0 founders6-8 months for F1 mice
Multiple knockout/point mutations
1-1.5 years(Additional time depends on previous existence of mice and number of alleles to combine)
3-4 months for F0 founders6-8 months for F1 mice
Knock-in (<700 bp) 1-1.5 years for germline(+1.5 years for speed congenic breeding)
3-4 months for F0 founders6-8 months for F1 mice
Knock-in (>700 bp)(limit is 4 kb for insertion or deletion)
1-1.5 years for germline(+1.5 years for speed congenic breeding)
5-6 months for F0 founders8-9.5 months for F1 mice
Advantages • Knock-in/whole gene replacement with large sequences (≥4kb) can be more straightforward
• Well-established methods and procedures
• Any background has the potential to be targeted, eliminating need for congenic breeding
• Mutations introduced directly into embryos (no ES cells required)
• Multiple mutations can be introduced
Limitations • More time-consuming and more expensive, largely due to breeding-dependent selection
• New model generation only feasible on a few inbred backgrounds
• Selection cassettes needed, adding additional steps to construct development
• ES cell challenges (type, timelines, and germline transmission rates)
• Knock-in with large sequences (≥4kb) may be less efficient
• Mosaicism (See section)
• Off-target effects (See section; increases with sub-optimal guide design)
Table1. Comparison of Mouse Model Generation: ES-Cell Based Homologous Recombination vs. CRISPR/Cas
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A Study Director will design your projectand review it with you.
*Timelines are estimates and not guaranteed.
Founder animals are identified, results are
discussed with youthrough a dedicated
Project Manager.
Germline transmissionconfirmed, results are
discussed with youthrough a dedicated
Project Manager.
Additional Services:Speed Expansion
Sperm CryopreservationCohorts
Varies by project14-16 weeks~9-11 weeks
depending on project*Varies by project
Figure 3. Steps involved in the production of mutant mouse models using CRISPR/Cas9 Model Generation Services.
Experience Modifying Multiple StrainsOne of the major advantages of CRISPR/Cas9 is the ability to introduce mutant alleles into strains of any background, including immunodeficient ones, not just strains for which germline-competent ES cells are available.
The depth of knowledge and experience at JAX contributes to our success using CRISPR/Cas9 in multiple strains. The following have been successfully used, including:
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Generating Mouse Models with CRISPR/Cas9
Considerations: Mosaicism, Off-Target Effects and EfficienciesTwo major considerations for investigators using CRISPR/Cas9 are mosaicism and off-target effects. Mosaicism is heterogeneity in the mutation among different cells and tissues, while off-target effects are introduction of a mutation at a site not intended.
Mosaicism arises because the CRISPR/Cas9 complex persists and functions beyond the one-cell embryo stage (Horii et al., 2016; Oliver et al., 2016). As illustrated in Figure 4, the persistent action of Cas9 can lead to multiple and differing mutations arising. Other factors that can influence the degree of mosaicism include the efficiency and specificity of the endonuclease, the specificity of the targeting sgRNA, and whether a provided repair template is used.
Cas9
sgRNA
1 Cell
Cas9
2 Cell
4 Cell
Figure 4. Mosaicism arises when Cas9 remains active across time during development. The timing and action of the Cas9 endonuclease across development can lead to heterogeneous mutations across different tissues, as represented by red and yellow mutant genes within the chromosomes depicted.
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Generating Mouse Models with CRISPR/Cas9
Due to the possibility of heterogeneous mutations, founder animals are screened and sequence-validated. They are also bred to wild-type mice to generate N1 offspring, which are also sequenced. Breeding segregates possible allele variants and establishes germline transmission.
DNA cleavage activity of CRISPR/Cas9 endonucleases can lead to off-target mutations and chromosomal translocations (Cho et al., 2014); however there are several tools to improve project design for on-target efficiency (Graham et al., 2015). Genomic alterations generated by off-target Cas9 activity can be significantly reduced by proper sgRNA design. Other means include using a modified Cas9 with a nickase mutation that reduces non-specific genomic damage caused by the enzyme (Shen et al., 2014), or using a shorter 18bp guide sequence (Fu et al., 2014
While off-target activity does not seem to be a major issue for the use of CRISPR in mice, at The Jackson Laboratory, bioinformatics approaches are used to minimize off-target activity. The genome is searched for homologous off-target sequences to identify sgRNA sequences with minimal predicted off-target activity. In general, if CRISPR projects are well-designed, off-target effects are minimal. Our experience, validated by genome sequencing, has demonstrated that the genome editing method is quite precise in targeting only specific DNA sequences of interest.
Even with tools being improved, not all model types are equally well suited for CRISPR genome editing in vivo.
Efficiency will drop with increasing complexity of the desired genomic modification. While the generation of knockouts by indel formation is very efficient, the introduction of larger deletions by using two sgRNAs already shows lower efficiencies. In addition, indels at the guide target sites compete with the deletion of the segment between the target sites. Efficiencies further decrease if repair templates are to be inserted. Knock-ins of point mutations or small tags encoded by single-stranded oligonucleotides can be inserted with relatively high efficiencies, but larger knock-ins of reporter genes or other cDNAs utilizing double-stranded DNA repair templates can be challenging. The same is true for the generation of conditional knock-out alleles by using CRISPR in vivo. In addition, the larger double-stranded DNA fragments may randomly insert into the genome, and require additional quality controls. Nevertheless, we offer all the above project types at The Jackson Laboratory.
The efficiency of more complex project types, such as the replacement of single exons, or even entire mouse genes by e.g. their human orthologs at this point in time are not yet at a point which allows them to be implemented into a routine model generation pipeline. The correct homologous recombination to incorporate these complex alleles remains a rare event, which would still necessitate the screening of large numbers of individual founder animals. From an animal ethics point of view this
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Generating Mouse Models with CRISPR/Cas9
is not in line with the 3R principle of considerate use of animals. Until we overcome the limitations of increasing the on-target homologous recombination efficiencies, these types of projects may still better be done in ES cells which allow the screening of a larger number of individual clones for the correct event. Improving the recombination efficiencies
e.g. by shifting the repair mechanism from non-homologous end-joining to homology-directed repair currently is an active field of investigation. Figure 5 illustrates the suitability of project types for generation by CRISPR in vivo.
Figure 5. The suitability of project types for generation by CRISPR in vivo.
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Generating Mouse Models with CRISPR/Cas9
Initiating a CRISPR ProjectModel generation with CRISPR/Cas9 at JAX begins with a discussion with your Regional Representative of your goals and determine the feasibility of your project.
Choosing a gene editing approachOur molecular biologists examine each project to understand the genomic structure of a locus and to design the optimal CRISPR strategy for your project. Risks inherent with any given approach will be discussed. For example, the indel approach is the least expensive; however, the repair mechanisms of non-homologous end joining are not predictable and do not always result in production of the desired mutant.
What happens after founder mice are generatedOnce founder animals are obtained, the next step is to breed the mice to produce offspring mice (N1) with defined and heritable targeted mutations. Founder animals can carry a mixture of different mutant alleles, and, therefore, often do not represent the final, desired model. Careful genotyping via sequence analysis of the founder mice is required to select candidate mice for breeding to refine and separate the alleles. It is important to remember that due to mosaicism, alleles present in tissues from the tail or ear may not be present in the gametes, or
the gametes may have mutations that are not present in peripheral tissues. Therefore, breeding more than one founder mouse is done to increase the chances that a desired mutation will be isolated and passed on to progeny.
Selected founder animals are then be bred with wild-type mice, generating N1 mice that must also be sequenced to determine which alleles they carry. Once N1 mice have been genotype-confirmed, they may be bred to generate homozygous mice for phenotypic analysis or cryopreserved for future development.
Breeding strategies become more complicated if a multiplexed gene targeting strategy is employed, but the same principles of carefully genotyping and selecting founder mice for breeding and thoroughly characterizing the phenotype of their N1 (or N2) offspring still apply. Depending upon the success of breeding and genotyping, it typically takes about 6 months to obtain N1 animals carrying the validated desired mutation in heterozygosity
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Generating Mouse Models with CRISPR/Cas9
Complementary ServicesThe Jackson Laboratory offers a comprehensive suite of mouse-related services and products that can integrate with CRISPR model generation, including:
• Cryopreservation to protect the new model(s) from loss and allow easy distribution to colleagues and collaborators.
• Speed Expansion to rapidly produce large cohorts of age-matched animals.
• Custom Breeding Services to:
• Provide a regular supply of the new research model.
• Rapidly cross the new model to other strains, perhaps to generate multi-allelic disease models.
• Model characterization services to analyze the phenotypic effects of the mutation.
• In Vivo Pharmacology Services to perform compound pre-clinical efficacy testing for several disease areas.
For more information about services that compliment your mouse model generation project, please contact your regional representative (jax.org/regional-reps) or visit our website: https://www.jax.org/jax-mice-and-services.
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Bhaya D, Davison W, and Barrangou R. 2011. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu Rev Genet 45, 273-297. PubMed: 22060043
Cho SW, Kim S, Kim Y, Kweon J, Kim HS, Bae S, and Kim JS. 2014. Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases. Genome Res 24(1):132-141. PubMed: 24253446
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F.2013. Multiplex genome engineering using CRISPR/Cas systems.Science. Feb 15;339(6121):819-23. PubMed: 23287718
Fu Y, Sander JD, Reyon D, Cascio VM, and Joung JK. 2014. Improving CRISPR-Cas nucleasespecificity using truncated guide RNAs. Nat Biotechnol 32(3):279-84. PubMed: 24463574
Graham DB, Root DE. 2015. Resources for the design of CRISPR gene editing experiments.Genome Biol. Nov 27;16:260. PubMed: 26612492
Horii T, Hatada I. 2016. Challenges to increasing targeting efficiency in genome engineering. J
Reprod Dev. Feb 20;62(1):7-9. PubMed: 26688299
Hsu PD, Lander ES, and Zhang F. 2014. Development and applications of CRISPR-Cas9 for genomeengineering. Cell 157(6):1262-1278. PubMed: 24906146
Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. 2012. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. Aug 17;337(6096):816-21. PubMed: 22745249
Oliver D, Yuan S, McSwiggin H, Yan W. 2015. Pervasive Genotypic Mosaicism in Founder Mice Derived from Genome Editing through Pronuclear Injection. PLoS One. 2015 Jun 8;10(6):e0129457. PubMed: 26053263
Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins A, Iyer V, Huang X, and Skarnes WC. 2014. Efficient genome modification by CRISPR/Cas9 nickase with minimal off-target effects. Nat Methods 11(4):399-402. PubMed: 24584192
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Generating Mouse Models with CRISPR/Cas9
Wang H, Yang H, Shivalila C, Dawlaty MM, Cheng AW, Zhang f, and Jaenisch R. 2013. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 153, 910-918. PubMed: 23643243
Yang H, Wang H, and Jaenisch R. Generating genetically modified mice using CRISPR/Cas-mediated genome engineering. 2014. Nat Protoc 9(8):1956-68. PubMed: 25058643
Yen ST, Zhang M, Deng JM, Usman SJ, Smith CN, Parker-Thornburg J, Swinton PG, Martin JF, and Behringer RR. 2014. Somatic mosaicism and allele complexity induced by CRISPR/Cas9 RNA injections in mouse zygotes. Dev Biol 393(1):3-9. PubMed: 24984260
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The Jackson Laboratory offers a comprehensive suite of mouse-related services and products that can help you protect, characterize and manage your new mouse model.
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