1. CRISPR/Cas9 Overview
CRISPR/Cas9 use RNA guided engineered nucleases (RGEN) to produce double strand breaks in a chromosome. In order to divide and grow cells must repair the break. Two cellular mechanisms used for this purpose are non-homologous endjoining (NHEJ) and homology directed repair (HDR). When NHEJ repair occurs small deletions and insertions may occur on the chromosome. If these events occur in a critical exon then protein expression will be disrupted and the gene will be “knocked out”. If the cell copies new DNA sequence from a experimental DNA template during HDR then the cell will have a new DNA sequence and can express an altered protein or an exogenous protein such as a fluorescent reporter. If the cell fails to repair the damage then it will fail divide and its daughter cells will be absent from the cell culture.
The microinjection of CRISPR/Cas9 plasmid DNA, mixtures of guide RNA (sgRNA) and Cas9 nuclease mRNA, or mixtures of Cas9 protein and sgRNA into fertilized mouse or rat eggs is a specialized application that differs from cell culture experiments in several ways. The transfection efficiency is 100%, reagents are physically introduced in each egg, one by one. The number of cells that are transfected in a single experiment much lower than can be achieved in cell culture. The absence of daughter cells that do not repair chromosome breaks is manifested as the absence of live mice and rats when pups are born. The pups that are born are euploid and do not carry multiple copies of chromosomes, as is common in long-established cell lines used in routine cell culture work.
The use of the wild type Cas9 enzyme with two functional nucleolytic domains in combination with sgRNA targets that are only 20 bp long or less has drawn interest to the question of off-target cleavage events (Gabriel et al., 2015). In animal models, the backcrossing of founders carrying Cas9-induced mutations to wild type animals will result in the segregation of off-target mutations from the genetic change of interest. In cell culture models this approach is not possible. In order to address concerns raised by off-target hits the use of Cas9 nickase (Cas9n) that carries a mutation from aspartic acid to alanine at residue 10 has been employed. In our hands, there is evidence that the Cas9n has lower activity than Cas9, however the specificity of cleavage is higher and the presence of off-targets hits is near the limit of detection.
The Transgenic Core has microinjects Cas9 plasmid DNA to produce gene knockouts and knockins based on the pX330 plasmid (pX330-U6-Chimeric_BB-CBh-hSpCas9, Ran et al., 2013). This plasmid is available from Addgene.org (Plasmid #42230). The Transgenic Core has used plasmid used to produce gene disruptions, oligonucleotide edited genes, reporter knockins and conditional (floxed) genes in mice. In rats, gene disruptions and oligonucleotide knockins have been produced with several reporter knockins in progress. When concerns regarding off targets are a concern, as in cell lines, the Transgenic Core uses a modified plasmid (pX330A_D10A-1x2) that simultaneously expresses two sgRNA and the Cas9 nickase to introduce specific chromosome breaks (Sakuma et al, 2014). This system can be used to co-express as many as seven sgRNA along with either the Cas9n or Cas9 protein (plasmids are available from Addgene.org).
2. sgRNA Algorithms
The choice of an sgRNA targeting a critical exon for a knockout or a non-coding region for a gene knockin is key to success. For this purpose many algorithms have been published and are available through the internet. A partial list includes the sites listed below. The Transgenic Core prefers to use the algorithm published by Haeussler et al. (2016) because the algorithm ranks sgRNA sequences according to a prediction of activity when delivered from a U6 promoter in a plasmid or when delivered as an RNA molecule.
After sgRNA targets are selected for mouse or rat genes, we clone the targets into a pX330 based plasmid and test it for activity by electroporation of mouse embryonic stem cells, rat fibroblasts or microinjection into fertilized eggs. For an example of the results from one of these tests see
To produce mouse and rat knockouts and knockins we microinject circular plasmid DNA into fertilized mouse or rat eggs. If desired, oligonucleotides or DNA plasmids are co-injected with the Cas9 or Cas9n plasmid to introduce knockins. When designing the HDR templates it is important to modify the donor DNA sequence so that it will not be a substrate for Cas9 or Cas9n cleavage. The length of the homology regions that match to the chromosome vary according to the type of knockin desired and the sequence in the target locus. It should be noted that the frequency of transgenic founders that carry knockins is lower than the number of pups that carry deletions at the site of the double strand break in the chromosome. Efforts to increase the frequency of homology directed repair have centered on the use of the SCR7 small molecule inhibitor of NHEJ (Maruyama et al., 2015, Singh et al., 2014). The Transgenic Core has used SCR7 in experiments, however all oligo knockins and plasmid knockins produced in the Core have not required the use of SCR7 to reduce non-homologous end-joining repair of double strand breaks.
What to expect in your CRSIPR/Cas9 edited founder animal. The frequency of homologous recombination with a plasmid donor is lower than the frequency of indels produced by non-homologous endjoining (Yang et al., 2014). It is our experience and that of others that the founders animals from Cas9 experiments are mosaic. Cas9 is microinjected into fertilized eggs (one cell eggs). Cas9 protein and sgRNA are produced in the egg (one cell) and then persist in the cell as it divides to two, four, eiight, 16, 32, 64 cells three days later. Thus if the Cas9 induces a double strand break in multiple independent cells at the 16 or 32 cell stage embryo the resulting animal will carry more than one allele for the targeted gene. For example, Shen et al. (2013, Fig. S9) subjected genomic DNA from a single founder animals derived by Cas9 gene targeted of EGP to PCR amplification, cloned the PCR product into plasmids, and obtained DNA sequences from 50 cloned DNA fragments. They observed the wild type and six mutant alleles in the genomic DNA of a single founder for a total of 7 different versions of the EGFP gene in a single mouse. As a result is not unusual for a single founder animal to transmit more than one allele to its N1 progeny after mating with a wild type breeding partner. It is also not unusual for all of the N1 pups from such a mating to inherit one mutant allele (from the founder) and one wild type allele.
It is unlikely that all of the N1 littermates will carry the same mutant allele. At this stage it is essential to verify the mutations in the N1 animals by DNA sequence analysis before proceeding with the N2 generation. If the mutation is in-frame (e.g. the deletion of a single codon) gene function may not be knocked out. Ideally the analysis of homozygous animals carrying the same knockout mutation on both chromosomes will be used to assess gene function.
In summary, the Transgenic Core CRISPR/Cas9 Pipeline includes the following steps:
1. Investigator identifies gene of interest to the Core
2. Investigator identifies upstream exon common to all gene isoforms for a knockout, or a genomic location for a knockin to the Core
3. Transgenic Core uses http://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design to find sgRNAs for use with Cas9.
4. Transgenic Core clones two sgRNAs into Cas9 plasmids.
5. Transgenic Core designs a genotyping assay to detect Cas9 activity.
6. Transgenic Core co-electroporates plasmids with a puro-resistance plasmid into mouse ES cells or other appropriate cell line.
7. Transgenic Core purifies genomic DNA from surviving cells and tests for double strand breaks with a Cel I assay.
8. Core purifies active CRISPR/Cas9 plasmid for microinjection and microinjects it into fertilized rat or mouse eggs at 5 nanograms/microliter. (Mashiko et al., 2014).
9. Transgenic Core or Investigator screens for mutant founders by PCR and the Cel I assay or by DNA sequencing
10. Investigator breeds mutant founder mice or rats with wild type mating partners. The mutant founders usually carry multiple mutations of the gene of interest because the Cas9 or Cas9n protein produces chromosome breaks after the microinjected egg has divided several times. Cloning the mutations from the founders that may not be transmitted to offspring is not essential. Cloning the mutations takes place after germline transmission.
11. Investigator clones out the genes from pups produced by the mating of mutant founders and wild type animals to identify the exact DNA sequence of the mutation and to verify transmission of the desired mutation.
12. If desired, Investigator intercrosses heterozygous animals to obtain homozygous mutants for phenotypic characterization.
4. Obtaining CRISPR/Cas9 Genomic Edited Mice and Rats
The Transgenic Core routinley generates new mouse or rat models with CRISPR/Cas9 for biomedical research and guarantees mutants carrying indels produced by non-homologous end joining. If you need a novel rodent model with genetic modifications to establish a pre-clinical model or to test a biological hypothesis, you need only submit a request at our electronic portal. The Transgenic Core team will provide you with a genetic engineering design for your review. Once approved, we will take all necessary steps to work with you to produce transgenic mouse or rat founders for your laboratory.
Unlike conventional transgenic mice, we find that founders produced with CRISPR/Cas9 technology are often genetic mosaics. That is to say that instead of simply being transgene positive or transgene negative a single G0 founder may include in its tissues cells that carry the wild type gene, the gene with the desired knockin, the gene with an insertion/deletion (indel) caused by nonhomologous end joining of CRISPR/Cas9 induced chromosome breaks, and multiple other indels that are genetically distinct (as many as six). Some indels may cause amino acid deletions; others may introduce premature termination codons. Thus the effect of each indel on protein function must be assessed individually.5. Identifying G0 Founder Mice or Rats
To identify G0 founders with the desired genome editing event it is often necessary to use TOPO TA cloning of the G0 founders genotyping PCR products, or to use next-generation DNA sequencing (NGS) approaches. Positive G0 founders are mated with wild type animals to obtain germline transmission of the desired allele. An alternative strategy is to forgo TOPO TA cloning of G0 founders and to simply mate a number of founders that show promising DNA sequence chromatograms with wild type mice. The G1 pups of a mosaic G0 founder will be obligate heterozygotes. That is to say they can only inherit one of the alleles from the mosaic G0 founder and the corresponding wild type allele from their mating partner. It is inadvisable to intercross mosaic G0 founders because of the unpredictability of the genotypes that will be transmitted. DNA chromatograms of the region of interest can be analyzed by subtracting the base pairs of the expected wild type sequence from the chromatogram and thus reveal the DNA sequence of the mutation that was transmitted by the G0 founder. TOPO TA cloning or NGS should be used to validate predicted sequences deduced from chromatograms with overlapping peaks.
Ideally, genotyping PCR assays are established before G0 mosaic founders are born. PCR primers can be tested with wild type DNA or with wild type DNA that has been mixed with an artificial DNA synthesized template to mimic the desired genome editing event. See the copy standards page for more information on setting up artificial gene edited mouse templates for genotyping assay verification. PCR reactions should produce a single band on an agarose gel. The amplicon from the genotyping PCR should be purified and submitted for DNA Sanger sequencing. Please note that the DNA sequence file is often uninformative because the G0 founder mice are often mosaic. Evidence of genome editing will be present in the DNA chromatogram. The presence of multiple alleles in G0 mosaic founders that result from CRISPR/Cas9 NHEJ repair produces overlapping sequences that appear as peaks on peaks in chromatograms. The interpretation of the chromatograms provides the evidence to indicate that the desired genome editing event has occurred.
The Transgenic Core uses the Qiagen QIAquick PCR Purification Kit to prepare amplicons for Sanger Sequencing. If necessary, PCR products can be isolate as bands from an agarose gel for DNA sequencing analysis. Information on submitting samples to the DNA Sequencing Core and software that can be used to view sequencing chromatograms are available at
Primer Design Suggestions for Specific and Sensitive PCR Assays.
Use Primer-Blast to pick primers
Adjust Primer Parameter default settings
Minimum primer melting temperature: change to 60°C
Optimal primer melting temperature: change to 63°C
Maximum primer melting temperature: change to 66°C
Minimum primer melting temperature difference: change to 1°C
Adjust Specificity Checking Parameters
Click box to turn on “Enable search for primer pairs specific to the intended PCR template”
Set Search Mode to “Automatic”
Set Database to “Genome (reference assembly from selected organisms)”
Set Organism to “Mus musculus (taxid: 10090)”
Click on “Advanced Parameters”
Set Primer Size Min to 27
Set Primer Size Opt to 29
Set Primer Size Max to 31
Stratman et al. reported primers of 27-30 nucleotides made up of 50-60% GC content and that will produce a 100-500 bp PCR product uniformly detect genomic templates with single copy sensitivity.
PCR Enhancer Mix
The 5X CES PCR enhancer mix described by Ralser et al. can improve the results obtained from difficult PCR reactions. The authors further recommend that the PCR reaction buffer contain the following final concentrations of components:
65 mM Tris–HCl, 16.6 mM (NH4)2SO4, 3.1 mM MgCl2, and 0.01% (v/v) Tween 20 at a pH of 8.8.
2.7 M betaine
6.7 mM DTT
55 ug/ml BSA
6. Selected Bibliography
Doench JG, Hartenian E, Graham DB, Tothova Z, Hegde M, Smith I, Sullender M, Ebert BL, Xavier RJ, Root DE.2014. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat Biotechnol.32:1262-1267.
Frock RL, Hu J, Meyers RM, Ho YJ, Kii E, Alt FW. 2015. Genome-wide detection of DNA double-stranded breaks induced by engineered nucleases. Nat Biotechnol. 33: 179-186.
Fujii W, Onuma A, Sugiura K, Naito K. 2014. Efficient generation of genome-modified mice via offset-nicking by CRISPR/Cas system. Biochem Biophys Res Commun. 445:791-794.
Haeussler M, Sch÷nig K, Eckert H, Eschstruth A, MiannÚ J, Renaud JB, Schneider-Maunoury S, Shkumatava A, Teboul L, Kent J, Joly JS, Concordet JP. 2016. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 17:148.
Gabriel R, von Kalle C, Schmidt M. 2015. Mapping the precision of genome editing. Nat Biotechnol. 33: 150-2.
Hsu PD, Scott DA, Weinstein JA, Ran FA, Konermann S, Agarwala V, Li Y, Fine EJ, Wu X,Shalem O, Cradick TJ, Marraffini LA, Bao G, Zhang F. 2013. DNA targeting specificity of RNA-guided Cas9 nucleases. Nat Biotechnol. 31:827-382.
Maruyama T, Dougan SK, Truttmann MC, Bilate AM, Ingram JR, Ploegh HL. 2015. Increasing the efficiency of precise genome editing with CRISPR-Cas9 by inhibition of nonhomologous end joining. Nat Biotech. 2015 Mar 23. [Epub ahead of print]
Mashiko D, Young SA, Muto M, Kato H, Nozawa K, Ogawa M, Noda T, Kim YJ, Satouh Y, Fujihara Y, Ikawa M. 2014. Feasibility for a large scale mouse mutagenesis by injecting CRISPR/Cas plasmid into zygotes. Dev Growth Differ. 56:122-129.
Ralser M, Querfurth R, Warnatz HJ, Lehrach H, Yaspo ML, Krobitsch S. An efficient and economic enhancer mix for PCR. Biochem Biophys Res Commun. 2006 Sep 1;347(3):747-51.
Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. 2013a. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 8:2281-2308.
Ran FA, Hsu PD, Lin CY, Gootenberg JS, Konermann S, Trevino AE, Scott DA, Inoue A, Matoba S, Zhang Y, Zhang F. 2013b. Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity. Cell. 154:1380-1389.
Sakuma T, Nishikawa A, Kume S, Chayama K, Yamamoto T. 2014. Multiplex genome engineering in human cells using all-in-one CRISPR/Cas9 vector system. Sci Rep. 4:5400.
Shen B, Zhang J, Wu H, Wang J, Ma K, Li Z, Zhang X, Zhang P, Huang X. 2013 Generation of gene-modified mice via Cas9/RNA-mediated gene targeting.. Cell Res. 23:720-723.
Shen B, Zhang W, Zhang J, Zhou J, Wang J, Chen L, Wang L, Hodgkins A, Iyer V, Huang X, Skarnes WC. 2014. Efficient genome modification by CRISPR-Cas9 nickase with minimal off-target effects. Nat. Methods. 11: 399-402.
Singh P, Schimenti JC, Bolcun-Filas E. 2014. A Mouse Geneticist's Practical Guide to CRISPR Applications. Genetics. 199: 1-15.
Stratman JL, Barnes WM, Simon TC. 2003. Universal PCR genotyping assay that achieves single copy sensitivity with any primer pair. Transgenic Res. 12:521-522.
Tsai SQ, Zheng Z, Nguyen NT, Liebers M, Topkar VV, Thapar V, Wyvekens N, Khayter C, Iafrate AJ, Le LP, Aryee MJ, Joung JK. 2015. GUIDE-seq enables genome-wide profiling of off-target cleavage by CRISPR-Cas nucleases. Nat Biotechnol. 33:187-197.
Wang X, Wang Y, Wu X, Wang J, Wang Y, Qiu Z, Chang T, Huang H, Lin RJ, Yee JK. 2015. Unbiased detection of off-target cleavage by CRISPR-Cas9 and TALENs using integrase-defective lentiviral vectors. Nat Biotechnol. 33:175-178.
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