Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins (original) (raw)
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Cell, 2013
The type II bacterial CRISPR/Cas system is a novel genome engineering technology with the ease of multiplexed gene targeting. Here we created reporter and conditional mutant mice by coinjection of zygotes with Cas9 mRNA, different guide RNAs (sgRNAs) as well as DNA vectors of different sizes. Using this one step procedure we generated mice carrying a tag or a fluorescent reporter construct in the Nanog, the Sox2 and the Oct4 gene as well as Mecp2 conditional mutant mice. In addition, using sgRNAs targeting two separate sites in the Mecp2 gene, we produced mice harboring the predicted deletions of about 700 bps. Finally, we analyzed potential off-targets of five sgRNAs in gene-modified mice and ESC lines and identified off-target mutations in only rare instances.
A Mouse Geneticist’s Practical Guide to CRISPR Applications
Genetics, 2014
CRISPR/Cas9 system of RNA-guided genome editing is revolutionizing genetics research in a wide spectrum of organisms. Even for the laboratory mouse, a model that has thrived under the benefits of embryonic stem (ES) cell knockout capabilities for nearly three decades, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 technology enables one to manipulate the genome with unprecedented simplicity and speed. It allows generation of null, conditional, precisely mutated, reporter, or tagged alleles in mice. Moreover, it holds promise for other applications beyond genome editing. The crux of this system is the efficient and targeted introduction of DNA breaks that are repaired by any of several pathways in a predictable but not entirely controllable manner. Thus, further optimizations and improvements are being developed. Here, we summarize current applications and provide a practical guide to use the CRISPR/Cas9 system for mouse mutagenesis, based on published report...
CRISPR–Cas9 gene editing technology has considerably facilitated the generation of mouse knockout alleles, relieving many of the cumbersome and time-consuming steps of traditional mouse embryonic stem cell technology. However, the generation of conditional knockout alleles remains an important challenge. An earlier study reported up to 16% efficiency in generating conditional knockout alleles in mice using 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides (ssODN) (2sgRNA–2ssODN). We re-evaluated this method from a large data set generated from a consortium consisting of 17 transgenic core facilities or laboratories or programs across the world. The dataset constituted 17,887 microinjected or electroporated zygotes and 1,718 live born mice, of which only 15 (0.87%) mice harbored 2 correct LoxP insertions in cis configuration indicating a very low efficiency of the method. To determine the factors required to successfully generate conditional alleles using the 2sgRNA–...
Cell, 2013
Mice carrying mutations in multiple genes are traditionally generated by sequential recombination in embryonic stem cells and/or time-consuming intercrossing of mice with a single mutation. The CRISPR/Cas system has been adapted as an efficient gene-targeting technology with the potential for multiplexed genome editing. We demonstrate that CRISPR/Cas-mediated gene editing allows the simultaneous disruption of five genes (Tet1, 2, 3, Sry, Uty-8 alleles) in mouse embryonic stem (ES) cells with high efficiency. Coinjection of Cas9 mRNA and single-guide RNAs (sgRNAs) targeting Tet1 and Tet2 into zygotes generated mice with biallelic mutations in both genes with an efficiency of 80%. Finally, we show that coinjection of Cas9 mRNA/sgRNAs with mutant oligos generated precise point mutations simultaneously in two target genes. Thus, the CRISPR/Cas system allows the one-step generation of animals carrying mutations in multiple genes, an approach that will greatly accelerate the in vivo study of functionally redundant genes and of epistatic gene interactions.
Scientific reports, 2014
The CRISPR/Cas system, in which the Cas9 endonuclease and a guide RNA complementary to the target are sufficient for RNA-guided cleavage of the target DNA, is a powerful new approach recently developed for targeted gene disruption in various animal models. However, there is little verification of microinjection methods for generating knockout mice using this approach. Here, we report the verification of microinjection methods of the CRISPR/Cas system. We compared three methods for injection: (1) injection of DNA into the pronucleus, (2) injection of RNA into the pronucleus, and (3) injection of RNA into the cytoplasm. We found that injection of RNA into the cytoplasm was the most efficient method in terms of the numbers of viable blastocyst stage embryos and full-term pups generated. This method also showed the best overall knockout efficiency.
Genome Biology
Background CRISPR-Cas9 gene-editing technology has facilitated the generation of knockout mice, providing an alternative to cumbersome and time-consuming traditional embryonic stem cell-based methods. An earlier study reported up to 16% efficiency in generating conditional knockout (cKO or floxed) alleles by microinjection of 2 single guide RNAs (sgRNA) and 2 single-stranded oligonucleotides as donors (referred herein as “two-donor floxing” method). Results We re-evaluate the two-donor method from a consortium of 20 laboratories across the world. The dataset constitutes 56 genetic loci, 17,887 zygotes, and 1718 live-born mice, of which only 15 (0.87%) mice contain cKO alleles. We subject the dataset to statistical analyses and a machine learning algorithm, which reveals that none of the factors analyzed was predictive for the success of this method. We test some of the newer methods that use one-donor DNA on 18 loci for which the two-donor approach failed to produce cKO alleles. We ...
Pipeline for the generation of gene knockout mice using dual sgRNA CRISPR/Cas9-mediated gene editing
Analytical Biochemistry, 2018
Animal models possess undeniable utility for progress on biomedical research projects and developmental and disease studies. Transgenic mouse models recreating specific disease phenotypes associated with β-hemoglobinopathies have been developed previously. However, traditional methods for gene targeting in mouse using embryonic stem (ES) cell are laborious and time consuming. Recently, CRISPR has been developed to facilitate and improve genomic modifications in mouse or isogenic cell lines. Applying CRISPR to gene modification eliminates the time consuming steps of traditional approach including selection of targeted ES cell clones and production of chimeric mouse. This study shows that microinjection of a plasmid DNA encoding Cas9 protein along with dual sgRNAs specific to Hbb-bs gene (hemoglobin, beta adult s chain) enables breaking target sequences at exons 2 and 3 positions. The injections led to a knockout allele with efficiency around 10% for deletion of exons 2 and 3 and 20% for indel mutation.
CRISPR-Cas9 enables conditional mutagenesis of challenging loci
Scientific Reports, 2016
The International Knockout Mouse Consortium (IKMC) has produced a genome-wide collection of 15,000 isogenic targeting vectors for conditional mutagenesis in C57BL/6N mice. Although most of the vectors have been used successfully in murine embryonic stem (ES) cells, there remain a set of nearly two thousand genes that have failed to target even after several attempts. Recent attention has turned to the use of new genome editing technology for the generation of mutant alleles in mice. Here, we demonstrate how Cas9-assisted targeting can be combined with the IKMC targeting vector resource to generate conditional alleles in genes that have previously eluded targeting using conventional methods.
Cellular and Molecular Biology Letters, 2015
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) technology has brought rapid progress in mammalian genome editing (adding, disrupting or changing the sequence of specific sites) by increasing the frequency of targeted events. However, gene knock-in of DNA cassettes by homologous recombination still remains difficult due to the construction of targeting vectors possessing large homology arms (from 2 up to 5 kb). Here, we demonstrate that in mouse embryonic stem cells the combination of CRISPR/Cas9 technology and targeting vectors with short homology arms (~ 0.3 kb) provides sufficient specificity for insertion of fluorescent reporter cassettes into endogenous genes with similar efficiency as those with large conventional vectors. Importantly, we emphasize the necessity of thorough quality control of recombinant clones by combination of the PCR method, Southern hybridization assay and sequencing to exclude undesired mutations. In conclusio...
Cell research, 2017
The CRISPR/Cas9 system is an efficient gene-editing method, but the majority of gene-edited animals showed mosaicism, with editing occurring only in a portion of cells. Here we show that single gene or multiple genes can be completely knocked out in mouse and monkey embryos by zygotic injection of Cas9 mRNA and multiple adjacent single-guide RNAs (spaced 10-200 bp apart) that target only a single key exon of each gene. Phenotypic analysis of F0 mice following targeted deletion of eight genes on the Y chromosome individually demonstrated the robustness of this approach in generating knockout mice. Importantly, this approach delivers complete gene knockout at high efficiencies (100% on Arntl and 91% on Prrt2) in monkey embryos. Finally, we could generate a complete Prrt2 knockout monkey in a single step, demonstrating the usefulness of this approach in rapidly establishing gene-edited monkey models.Cell Research advance online publication 6 June 2017; doi:10.1038/cr.2017.81.