Journal Article

Easi-CRISPR: a robust method for one-step generation of mice carrying conditional and insertion alleles using long ssDNA donors and CRISPR ribonucleoproteins

Richardson, Guy P., Batra, Surinder K., Sakai, Daisuke, Harms, Donald W., Izu, Yayoi, Ohtsuka, Masato, Seshacharyulu, Parthasarathy, Inagaki, Yutaka, Buckley, Shannon M., Gurumurthy, Channabasavaiah B., Akatsuka, Hisako, Miura, Hiromi, Quadros, Rolen M., Zeiner, Sarah A., Mansour, Suzanne L., Sato, Takehito, Aida, Tomomi, Urness, Lisa D., Redder, Ronald, Thoreson, Wallace B., Jacobi, Ashley M., Behlke, Mark A.

2017

Overview

Background Conditional knockout mice and transgenic mice expressing recombinases, reporters, and inducible transcriptional activators are key for many genetic studies and comprise over 90% of mouse models created. Conditional knockout mice are generated using labor-intensive methods of homologous recombination in embryonic stem cells and are available for only ~25% of all mouse genes. Transgenic mice generated by random genomic insertion approaches pose problems of unreliable expression, and thus there is a need for targeted-insertion models. Although CRISPR-based strategies were reported to create conditional and targeted-insertion alleles via one-step delivery of targeting components directly to zygotes, these strategies are quite inefficient. Results Here we describe Easi- CRISPR ( E fficient a dditions with s sDNA i nserts-CRISPR), a targeting strategy in which long single-stranded DNA donors are injected with pre-assembled crRNA + tracrRNA + Cas9 ribonucleoprotein (ctRNP) complexes into mouse zygotes. We show for over a dozen loci that Easi -CRISPR generates correctly targeted conditional and insertion alleles in 8.5–100% of the resulting live offspring. Conclusions Easi- CRISPR solves the major problem of animal genome engineering, namely the inefficiency of targeted DNA cassette insertion. The approach is robust, succeeding for all tested loci. It is versatile, generating both conditional and targeted insertion alleles. Finally, it is highly efficient, as treating an average of only 50 zygotes is sufficient to produce a correctly targeted allele in up to 100% of live offspring. Thus, Easi- CRISPR offers a comprehensive means of building large-scale Cre- LoxP animal resources.

• Publisher: BioMed Central,Springer Nature B.V,BMC
• Subject: Alleles / Animal Genetics and Genomics / Animal models / Animals / Bioinformatics / Biomedical and Life Sciences / Cell cycle / Clustered Regularly Interspaced Short Palindromic Repeats / Consortia / Cre-LoxP / CRISPR / CRISPR ribonucleoproteins / CRISPR/Cas9 / Custom design / Deoxyribonucleic acid / DNA / DNA repair / DNA, Single-Stranded - genetics / DNA, Single-Stranded - metabolism / Easi-CRISPR / Efficiency / Embryo cells / embryonic stem cells / Endonucleases - genetics / Endonucleases - metabolism / Evolutionary Biology / Founder Effect / Gene Editing - methods / Genes / Genes, Reporter / genetic engineering / Genetic Loci / Genome editing / Genomes / Homologous recombination / Homology directed repair / Human Genetics / Insertion / Integrases - genetics / Integrases - metabolism / knockout mutants / Life Sciences / loci / long ssDNA donors / Mice / Mice, Transgenic - genetics / Mice, Transgenic - growth & development / Microbial Genetics and Genomics / Microinjections / Mutagenesis, Insertional - methods / Plant Genetics and Genomics / progeny / Recombinational DNA Repair / Ribonucleoproteins / Ribonucleoproteins - genetics / Ribonucleoproteins - metabolism / RNA, Guide, CRISPR-Cas Systems - genetics / RNA, Guide, CRISPR-Cas Systems - metabolism / Rodents / Single-stranded DNA / Stem cell transplantation / Stem cells / transactivators / Transcription factors / transgenic animals / Transgenic mice / Trends / zygote / Zygote - growth & development / Zygote - metabolism / Zygotes