Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice

Aida et al. Genome Biology Cloning-free CRISPR/Cas system facilitates functional cassette knock-in in mice Tomomi Aida 1 Keiho Chiyo 1 Takako Usami Harumi Ishikubo 1 Risa Imahashi 1 Yusaku Wada Kenji F Tanaka Tetsushi Sakuma Takashi Yamamoto Kohichi Tanaka 0 1 0 The Center for Brain Integration Research (CBIR), TMDU , Tokyo 113-8510 , Japan 1 Laboratory of Molecular Neuroscience, Medical Research Institute (MRI), Tokyo Medical and Dental University (TMDU) , Tokyo 113-8510 , Japan Although the CRISPR/Cas system has enabled one-step generation of knockout mice, low success rates of cassette knock-in limit its application range. Here we show that cloning-free, direct nuclear delivery of Cas9 protein complex with chemically synthesized dual RNAs enables highly efficient target digestion, leading to generation of knock-in mice carrying a functional cassette with up to 50% efficiency, compared with just 10% by a commonly used method consisting of Cas9 mRNA and single guide RNA. Our cloning-free CRISPR/Cas system facilitates rapid one-step generation of cassette knock-in mice, accelerating functional genomic research by providing various in vivo genetic tools. - Background Although gene-targeted knockout and knock-in mice are invaluable tools for understanding the functions of genes in vivo, the production of such genetically modified mice has relied on gene targeting in embryonic stem cells, which is a complicated and time-consuming process [1]. The recent development of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system, a genome editing technology, has allowed for the direct manipulation of the genome in mouse zygotes in vivo (in vivo genome editing) with extremely high efficiency, enabling the highly convenient and ultra-rapid one-step generation of genetically modified mice without embryonic stem cells [2,3]. A flood of studies using CRISPR/Cas-mediated in vivo genome editing have reported the production of knockout mice [4-6] and knock-in mice carrying single nucleotide substitutions combined with oligo DNA donors [5,7,8]. In contrast, there has been only one report on the successful production of knock-in mice carrying reporter gene cassettes [9], essential tools for analyzing complex tissues such as brain in vivo [10], and the efficacy of the targeted insertion of the reporter gene was only about 10% [2,3,9,11]. The low success rates of gene cassette knock-in limit the applicability of CRISPR/Casmediated in vivo genome editing. The CRISPR/Cas system was initially reported as an adaptive immune system in bacteria, consisting of three components including Cas9 nuclease and two small RNAs, CRISPR RNA (crRNA), which guides the Cas9 complex to the target sequence, and trans-activating crRNA (tracrRNA), which binds to crRNA and forms a ribonucleoprotein complex with Cas9 nuclease [12]. When it was harnessed as a genome editing tool [13,14], the dual-crRNA:tracrRNA was engineered as a chimeric single guide RNA (sgRNA) [13]. The CRISPR/Cas system consisting of two components - Cas9 nuclease and sgRNA - is the most common approach in the field of genome editing due to its enhanced convenience and robust targeting [15]. However, it is still unknown whether the commonly used sgRNA works more efficiently than the dual-crRNA:tracrRNA, especially for the production of knock-in mice carrying reporter gene cassettes. Here, we show the highly efficient generation of knockin mice carrying a functional gene cassette by a cloningfree CRISPR/Cas system using Cas9 protein combined with chemically synthesized dual-crRNA:tracrRNA. Results Generation of highly active guide sequence In a previous study, we demonstrated that the insertion of a transgene downstream of the Actb polyadenylation signal allowed for sufficiently high levels of gene induction [16,17]. Thus, we chose the Actb locus as a model to be targeted for the generation of knock-in mice carrying a functional gene cassette. We first designed the guide sequence targeted to the locus 800 bp downstream of the mouse Actb polyA signal (Figure 1a) and inserted it into a bi-cistronic expression vector pX330 plasmid [18,19] containing sequences encoding Cas9 and sgRNA backbone sequences. Then, we determined its high activity of DNA digestion in vitro using a single-strand annealing (SSA) assay with episomal plasmid vectors containing a split luciferase gene and Actb target sequences in human HEK293T cell lines (Figure S1 in Additional file 1), and a Cel-I assay in mouse Neuro2A cell lines to target the endogenous mouse chromosome (Figure S2 in Additional file 1). To further test the activity of Actb sgRNA in vivo, we injected Cas9 mRNA with Actb sgRNA - both in vitro transcribed with T7 RNA polymerase using PCR templates amplified from an Actb pX330 plasmid [11] into one-cell stage mouse zygotes. We obtained 12 newborn mice and found that almost all the newborns Figure 1 Generation of knock-in mice carrying gene cassette by sgRNA combined with Cas9 mRNA injection. (a) Targeting strategy for the generation of Actb-TetO-FLEX-EGFP-polyA knock-in mice. (b) Schematic diagram of pronuclear injection of Cas9 mRNA, Actb sgRNA and TetO-FLEX-EGFP targeting vector. (c) PCR screening of knock-in newborns derived from pronuclear RNA injection. (d) Sequences of boundaries between Actb and TetO-FLEXEGFP-polyA cassette. IF, internal forward primer; IR, internal reverse primer; KI, knock-in; LF, left forward primer; LR, left reverse primer; RF, right forward primer; RR, right reverse primer; L-HA, left homology arm; R-HA, right homology arm; M, molecular marker; WT, wild type. were bi-allelically targeted mutants (Figure S3 and Table S1 in Additional file 1). Thus, the Actb sgRNA we designed is highly active both in vitro and in vivo for target digestion and subsequent induction of nonhomologous end-joining (NHEJ). Generation of reporter knock-in mice by sgRNA combined with Cas9 mRNA As a model for knock-in mice carrying a functional gene cassette, we designed reporter mice highly expressing enhanced green fluorescent protein (EGFP) from the endogenous Actb locus only in a specific cell population intersectionally defined [20] by the expression of Cre recombinase and tetracycline transactivator (tTA). We constructed a 10.5 kb targeting vector for the mouse Actb locus containing a 2.5 kb TetO-FLEX-EGFP-polyA cassette (Tet operator (tetO) sequence concatemers fused to a minimal cytomegalovirus (CMV) promoter, beta-globin intron, inverted EGFP flanked by two pairs of loxP and lox2722 (FLEX switch) and polyA), and 2.0 kb left and right homology arms of the Actb locus (Actb-TetO-FLEX-EGFP-polyA; Figure 1a). Next, we injected the circular Actb-TetO-FLEX-EGFPpolyA targeting vector together with Actb sgRNA and Cas9 mRNA, both in vitro transcribed as above, into one-cell-stage mouse zygotes (Figure 1b). We injected the mixture (5 ng/l Cas9 mRNA, 2.5 ng/l Actb sgRNA, and 10 ng/l of the targeting (...truncated)