Rapid generation and selection of Cas9-engineering TRP53 R172P mice that do not have off-target effects
Genetic mutations cause severe human diseases, and suitable animal models to study the regulatory mechanisms involved are required. The CRISPR/Cas9 system is a powerful, highly efficient and easily manipulated tool for genetic modifications. However, utilization of CRISPR/Cas9 to introduce point mutations and the exclusion of off-target effects in mice remain challenging. TP53-R175 is one of the most frequently mutated sites in human cancers, and it plays crucial roles in human diseases, including cancers and diabetes.
Here, we generated TRP53-R172P mutant mice (C57BL/6 J, corresponding to TP53-R175P in humans) using a single microinjection of the CRISPR/Cas9 system. The optimal parameters comprised gRNA selection, donor designation (silent mutations within gRNA region), the concentration of CRISPR components and the cellular sites of injection. TRP53-R172P conversion was genetically and functionally confirmed. Combination of TA cloning and Sanger sequencing helped identify the correctly targeted mice as well as the off-target effects in the engineered mice, which provide us a strategy to select the on-target mice without off-target effects quickly and efficiently.
A single injection of the this optimized CRISPR/Cas9 system can be applied to introduce particular mutations in the genome of mice without off-target effects to model various human diseases.
KeywordsHuman genetic diseases Mouse model TRP53-R172P mutation CRISPR/Cas9 system Off-target effects
CRISPR-associated protein 9;
Clustered Regularly Interspaced Short Palindromic Repeats
double strand break
in vitro transcription
mouse double minute 2 homolog;
Online Mendelian Inheritance in Man
protospacer adjacent motif
Humans suffer from thousands of genetic disorders, which arises from various mutations in the genome. Among them, single-gene mutations account for over 6000 human monogenic disorders according to Online Mendelian Inheritance in Man (OMIM, https://www.omim.org/). Therefore, suitable animal models are urgently needed to elucidate the regulatory mechanisms of genetic mutations in the development and progression of human diseases.
p53 mutations in cancers
Cancers that involve abnormal cell growth and have the potential to spread throughout the body, affected 90.5 million people and caused 8.8 million deaths [1, 2] in 2015. Oncogenes and tumour suppressor genes are the two major groups of genes contributing to the transformation of normal cells into malignant cells. The Tp53 tumour suppressor gene is the most frequently mutated gene in cancers [3, 4], highlighting its importance in cancer generation. Normally, TP53 is sequestered by the negative regulator, mouse double minute 2 homolog (MDM2). Oncogenic events, such as DNA damage, or other stresses release the TP53 protein from the MDM2 complex. Subsequently, TP53 induces cell cycle arrest, initiates DNA damage repair processes to fix the damage or eliminates irreparable cells through senescence or apoptosis . Activated TP53 transactivates the expression of the downstream gene p21, which directly binds to cyclin/cyclin-dependent kinase (CDK) complexes and inhibits their kinase activity, thereby leading to cell cycle arrest at the G1/S transition checkpoint . While a variety of mutations of Tp53 have been found to contribute to malignant progression, the most common ones are all single nucleotide missense mutations which are corresponding to the DNA-binding region of TP53 . Among the 6 hotspot amino acids, Arg175Pro (R175P) substitution leads to completely defective initiation of apoptosis but partially retains the function of cell cycle arrest [8, 9]. The mouse model of this TP53 mutant escapes the early onset of spontaneous tumorigenesis  but develops diabetes  as well as colon adenocarcinomas  upon the deficiency of nonhomologous end-joining (NHEJ). These findings suggest that the mouse model of the human TP53 R175P mutant is valuable to explore the influences of TP53’s ability of cell cycle arrest in human diseases, including cancer and diabetes.
Genetic editing via CRISPR/Cas9
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) has been demonstrated to be a powerful tool in genomic editing since its first application in human cells [13, 14]. CRISPR system is the prokaryotic immune system and has been identified in 40% of sequenced bacteria and 90% of sequenced archaea . The CRISPR associated protein 9 (Cas9) endonuclease is a simple CRISPR system from Streptococcus pyogenes that contains four components, and it was re-engineered into an even simpler two-component system . The efforts of geneticists have shown that the Cas9 system is highly efficient, easily manipulated, and flexible. This system has been widely used in genomic editing of various organisms, including mice . The genome-editing system includes Cas9, gRNA, and a donor (an optional section of DNA repair template). The gRNA of 20 nucleotides can be designed to target the genomic sites of interest with a 5′-NGG-3’protospacer adjacent motif (PAM) sequence . The gRNA directs Cas9 to target sites where it cleaves the DNA and produces double strand breaks (DSBs). The DNA damage is repaired by NHEJ which causes insertions or deletions randomly, or by homology-directed repair (HDR) with a donor to generate the desired mutations in the genome . While the engineering of the mice via CRISPR/Cas9 system has been widely adopted and reported, the functional confirmation of genetic mutations as well as the identification and excluding of the off-target effects in descendant generations remain unexplored. In this study, a new insight of the application of CRISPR/Cas9 system was provided for murine genetic modification which minimizes the off-target effect and can be recommended to future application of modelling human diseases.
This study was to explore the practicability of constructing genetically modified mice of TRP53-R172P by a single injection of the CRISPR/Cas9 system. The single amino acid substitution would be validated genetically and functionally, and the excluding of the off-target effect in the descendant generation would be confirmed.
Generation of TRP53R172P mice with a single microinjection of CRISPR/Cas9 system
Single targeting of TRP53-R172P mice with CRISPR/Cas9 system
Survival/ Injected zygotes
Transferred embryos/ Recipients
KO (double peaks)
Identification of the designated TRP53 R172P mice
Confirmation of the TRP53 R172P substitution in various tissues and descendants
Examination of the off-target effects of targeting mice
Analysis of the off-target effects of descendants arising from different parents
Validation of UV radiation hypersensitivity of TRP53 R172P mutant
The present study generated tumour suppressor gene TRP53 R172P mutant mice by a single injection of CRISPR-cas9 system. The present results suggested that microinjection of 200 zygotes is sufficient to produce knock-in mice with genetic point mutations. The efforts to obtain this mouse line included optimization of the designation of gRNA and donor as well as confirmation of the results step by step. It has demonstrated that the procedure worked well and can be applied to generate mouse models for other human genetic diseases.
gRNA and donor designation
The selected gRNA has great influence on the targeting efficiency of CRISPR-Cas9 in mice. To generate a point mutation in the genome, mutation sites must be close enough to gRNA. Mutation sites closer to the Cas9 cleavage site (nucleotide4 before PAM) will have higher genomic knock-in efficiency. Donor designation was also optimized through introduction of synonymous mutations in the gRNA region, which abolished the effect of secondary targeting of Cas9 on successfully knock-in genomic sites. To ensure escape from Cas9 targeting, the donor comprised one mutation at PAM and mutations of at least 3 nucleotides in the gRNA in this study.
Injection concentration and cellular sites
For genomic knock-in, the mixture of RNA and DNA needs to be injected into both the nuclei, where the homologous directed recombination (HDR) of donor occurs, and cytoplasm, where the Cas9 mRNA is translated into enzyme, of fertilized eggs. A balance of the concentrations of injection components also exists. Higher concentrations produce higher targeting efficiency but lead to higher percentage of zygote death. The concentration used in the present study was optimized to result in good targeting efficiency but also to generate a sufficient number of surviving mice.
Elimination of the off-target effects
Several publications have reported that most mice generated from direct introduction of Cas9 mRNA and sgRNA into zygotes are genetic mosaics, that is, one mutant mouse is composed of cells carrying different sets of mutations [19, 20, 21]. Similarly, the genotypic mosaicism was found in founder mice derived from injected zygotes in the present study. To identify 1st generation KI mice, TA cloning technology, which can effectively and efficiently dissect the detailed genomic information of mosaic mice, was applied [22, 23]. DNA sequencing of 6–8 clones offered 3–4 different genotypes in the mosaic mice. One common criticism of CRISPR/Cas9 system is the off-target effects. Same as the genetic modification, the off-target effects of CRISPR/Cas9 system can be traced, identified (Additional file 3: Figure S3) and passed on to offspring mice. To exclude the off-target effects of the CRISPR/Cas9 system, we develop a new rapid approach (Fig. 6): screen the potential off-target loci in generation 1 mice and select those that do not carry off-target effects for further applications. This approach resolves the off-target problems in a short time and at an efficient manner, suggesting its wide utilizations in future for engineering mice based on CRISPR/Cas9 system.
The advantages of Cas9 over conventional methods
Before the discovery of Cas9 editing system, homologous recombination is commonly carried out to introduce the inherit mutations into genome. The procedure comprises several complicated steps, which makes it time consuming and high cost. Comparably, it is straight forward and easy forCas9 mediated genetic editing that does not need the cloning of large genomic fragments. And the identification of correct targeting in Cas9 editing with PCR and Sanger sequencing is much easier than that in homologous recombination with drug selection and Southern blotting. More importantly, the targeting efficiency is significantly higher with Cas9 system and no exogenous elements will be introduced into the genome. Also, the potential off-target effects of Cas9 should be taken into account but can be excluded.
The Cas9 system is a powerful tool to generate mice carrying genetic mutations for studying the pathology of cancers and other human genetic diseases. To overcome the difficulty of generating KI mice, the optimizations of the donor designation, cellular injection sites and the injection concentration are required to increase the efficiency of successfully targeting. More importantly, we developed an approach to determine and avoid the off-target effects in Cas9 engineered mice in a short term and an efficient manner. The approach can be applied in any engineered mouse derived from Cas9 targeting, no matter for gene knockout or donor substitution. In future, the benefit of Cas9-mediated production of KI alleles needs to be carefully and systematically evaluated. And It is likely that the Cas9 mouse will have more applications beyond cancer field.
In vitro transcription of Cas9 and gRNA
The Cas9-coding region was PCR amplified with Phusion DNA polymerase from the pX260 plasmid (Addgene) using the Cas9-F primer containing the T7 promoter and Cas9-R primer (Additional file 6: Data 1). Cas9 PCR products were purified with phenol-chloroform. Following the manufacture’s manual, in vitro transcription (IVT) of Cas9 was performed using the mMESSAGE mMACHINE T7 Ultra Kit (Ambion, AM1345). Agar gel electrophoresis and nanodrop analysis were used to verify the quality and concentration of the obtained mRNA purified with MEGAclear kit (Ambion, AM1908). Purified PCR products of T7-gRNA for Trp53 were used as a template for IVT using the MEGAshortscript T7 kit (Ambion, AM1354). gRNA was purified with the MEGAclear kit (Ambion, AM1908) and resuspended in endonuclease-free water. Agarose gel electrophoresis and nanodrop analysis were used to verify the quality and concentration.
Source of animals
The female and male C57BL/6 mice, ICR mice used in this study were housed and bred in the Animal Center of Tsinghua University.
Zygote injection of Cas9 the system
C57BL/6 J female mice and ICR mouse strains were used as embryo donors and foster mothers, respectively. Superovulated 8-week-old female C57BL/6 J mice were mated to C57BL/6 J males, and the fertilized eggs from oviducts were collected. Cas9 mRNA (40 ng/μl), Trp53 gRNA (17.5 ng/μl), and donor oligos (60 ng/μl) were mixed in 20 μl of nuclease-free H2O and centrifuged at 12000 rpm for 2 min. The supernatant was transferred into a new Eppendorf tube and microinjected into both the nuclei and cytoplasm of zygotes at the pronuclei stage in M2 medium (Sigma).210 Injected zygotes were cultured in KSOM medium at 37 °C and 5% CO2 for 1 day. The surviving embryos at the 2-cell stage were transferred into uterus of 6 pseudopregnant female mice. Totally, 26 mice were born.
Euthanasia of the animals
The euthanasia of adult mice was performed via a gradual fill of CO2 at a rate of about 20% chamber volume per minute, and maintained for more than 5 min. The death of mice was verified before removing the mice from the CO2 chamber.
PCR products of mouse tail amplification were confirmed on an ethidium bromide-stained agarose gel and subjected to Sanger sequencing. The products with continuous overlapping peaks (so called double peaks) in Sanger sequencing were subjected to TA cloning with pEASY®-Blunt Cloning Kit (Transgene). The ligated products were transformed into Trans1-T1 competent cells which were then plated on LB agar dishes containing ampicillin, and 6–8 bacterial clones from every dish were DNA sequenced for the identification of mouse genomic information.
Mouse embryo fibroblast (MEF) preparation and UV treatment
Trp53 R172P Heterozygous mouse was mated with the same genotype mouse, the mother was sacrificed at embryo stage E13.5. The embryonic heads were harvested for genome extraction to genotype the MEFs. After remove of limbs and visceral tissues, the embryos were sectioned into small pieces and digested with 0.25%trypsin at 37 °C for 10 min. The trypsinization was stopped by 10% FBS. The cells were isolated by vigorously pipetting and plated at 10 cm dishes before incubation at 37 °C, 5% CO2until 100% confluence. Simultaneously, wild type (WT), Heterozygous (HET) and Homozygous (HOM) MEF cells were exposed to UV light (GE, G36T5L (39 W) UV-C Ultraviolet Germicidal 254 nm LIGHT Bulb Lamp) for 15 s (The time course of UV treatment was optimized before the experiments.). The cells were collected 24 h after UV treatments with nm UV light equipped in the biosafe incubator.
The MEFs were lysed in RIPA buffer containing 50 mmol/L Tris-HCl pH 8.0, 150 mmol/L NaCl, 1%Nonidet P-40, 1% Na-deoxycholale, 0.1% SDS, 1 mmol/L Na3VO4, 1 mmol/L NaF,1 mmol/L PMSF and a protease inhibitor mixture (Roche Diagnostics, Mannheim, Germany). The cell lysates were subjected to separation with SDS PAGE and immunoblotted with specific antibodies of p53 (Cell signalling technology, CST#2524) and β-Actin (Servicebio).
The raw data collection
All the raw data can be found in the Additional file 7: Data 2.
We would like to thank the members of the Animal Facility of Tsinghua University for their support.
CZ, XL, and GZ produced the study concept and designed the studies. GZ and QZ analysed and interpreted the data. GZ, JD and CZ prepared the manuscript. All authors read and approved the final manuscript.
This work was supported by Chengming Zhu’s start-up funding from Sun Yat-Sen University. The funding body did not play any role in the design of the study, the collection, analysis, and interpretation of data and in writing the manuscript.
Ethics approval and consent to participate
All experiments involving mice in the present study were performed in accordance with protocols #15-LX1 approved by the Animal Committee of Tsinghua University. Date of INSTITUTIONAL ANIMAL CARE AND USE COMMITTEE (IACUC) review and approval is 20150119, and 3-year Renewal date is 20180326.
Consent for publication
The authors declare that they have no competing interests.
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