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Heritable Gene Knockout in Caenorhabditis elegans by Direct Injection of Cas9-sgRNA Ribonucleoproteins [Genetics](Genetics Via Acquire Media NewsEdge) ABSTRACT We present a novel method of targeted gene disruption that involves direct injection of recombinant Cas9 protein complexed with guide RNA into the gonad of the nematode Caenorhabditis elegans. Biallelic mutants were recovered among the F1 progeny, demonstrating the high efficiency of this method. CLUSTERED, regularly interspaced, short palindromic re- peat (CRISPR)-associated, Cas9-derived RNA-guided en- donucleases (RGENs) enable targeted mutagenesis in cells and organisms (Cho et al. 2013a; Cong et al. 2013; DiCarlo et al. 2013; Gratz et al. 2013; Hwang et al. 2013; Jinek et al. 2012; Mali et al. 2013; Wang et al. 2012). In addition to other nuclease-mediated gene targeting methods (Morton et al. 2006; Wood et al. 2011), heritable genome editing was recently achieved in Caenorhabditis elegans using trans- genes, driving the expression of Cas9 and a single guide RNA in C. elegans (sgRNA) (Friedland et al. 2013). Here we report that Cas9 protein, used as an alternative to a Cas9-encoding plasmid or mRNA, which can be silenced in nematodes, can induce efficient genome editing in C. elegans. This article is one of six companion articles (Chiu et al. 2013; Cho et al. 2013b; Katie and Grosshans 2013; Lo et al. 2013; Tzur et al. 2013; Waaijers et al. 2013) that present different approaches to and features of Cas9-CRISPR genome editing in C. elegans. We first chose to target dpy-3, a gene on the X chromo- some, because both homozygous and hemizygous mutations in this gene cause visible phenotypes (Blaxter 1993). We designed two sgRNAs complementary to the coding sequence of dpy-3 (Figure 1, A and B). These sites are unique within the genome, and sequence alignment analysis showed that there were no possible off-target sequences in the entire genome, with fewer than four base mismatches to the target sequences (Supporting Information, File SI, Figure SI, and Table SI). We briefly incubated purified Cas9 protein with the two in vitro transcribed sgRNAs and injected the ribo- nucleoprotein (RNP) complexes into the gonads of adult C. elegans worms (Figure 1A). Among many injected P0 ani- mals, five exhibited bloated gonad after microinjection, which is indicative of successful injection into the gonad. The Fx progeny of these five P0 animals were further exam- ined for mutations. The Fx animals were subjected to the T7 endonuclease I (T7E1) assay (Kim et al. 2009) to detect small insertions or deletions (indels) generated via the error-prone non- homologous end-joining (NHEJ) pathway used to repair double-strand DNA breaks (DSBs) induced at the target site. Mutations were detected in the Fx progeny of two P0 ani- mals at frequencies of 1/24 (labeled as A-l Fi) and 3/33 (labeled as D-l Fi, D-2 Fi, and D-3 Fi). Sequence analysis of PCR products derived from the mutant Fx animals showed small deletions at one site or deletions that spanned both sites, suggesting that the RGENs may have acted on both targeted sequences (Figure IB). We observed more than two mutations in the A-l Fi and D-l Fi worms, suggesting mul- tiple mutational events in these animals, most likely in both the germ cells and somatic cells. Thus it appears that the nuclease maintained its activity in the embryos even after fertilization of the eggs. Notably, we isolated two Ft animals from the same PO animal (labeled D) that exhibited the dumpy (Dpy) pheno- type, which is the expected phenotype of homozygous dpy-3 mutants (Figure 1, B and E). One Dpy Ft animal, D-2 Fq, was examined along with other Ft animals using the T7E1 assay without collecting additional progeny Sequence analysis of the PCR products derived from this animal showed both a small deletion and the wild-type sequence, suggesting that a mosaic mutation had occurred, probably in the hypoder- mis, which conferred the Dpy phenotype. The other animal, labeled D-3 F1; escaped from the plate after laying eggs, making it only possible to examine its F2 progeny Sequence analysis showed that the F2 animals, all of which exhibited the Dpy phenotype, contained two independent mutations (Figure 1C, Table 1). This result suggests that the D-3 Fq animal had two independent mutations that most likely oc- curred in both the oocyte- and the sperm-derived chromo- somes. Based on the phenotype and the sequence analysis, we propose that the D-3 Fq animal contained the biallelic mutations in the dpy-3 gene. In total, we observed 4/121 Fq animals with mutations and at least one case of biallelic heritable mutations in the dpy-3 targeting experiment. The results of the dpy-3 targeting experiment are shown in Table 1. We next chose another X-linked gene, unc-1 (Rajaram et al. 1998; Chen et al. 2007), for targeting with the Cas9-sgRNA RNP complexes. We examined the Fq progeny of four P0 animals injected with the Cas9-sgRNA RNP com- plexes and found that two P0 animals produced mutant Fq progeny at frequencies of 5/32 (labeled as A-l ~5 Fq) and 4/24 (labeled as B-l ~4 Fq). Sequence analysis of PCR prod- ucts derived from four Fq animals out of the nine mutant Fq animals showed that two (B-l Fq and B-2 Fq) contained single mutations, and a third animal (A-l Fq) contained two mutations (Figure ID). The fourth animal, B-3 Fq, con- tained two different mutations as well as the wild-type sequences, suggesting that this animal was a mosaic (Figure ID). Similar to the deletions observed at the dpy-3 locus, deletions that spanned the two RGEN sites were frequently observed, reminiscent of chromosomal deletions induced using zinc finger nucleases (ZFNs) or transcription activa - tor-like effector nucleases (TALENs) (Kim et al. 2009, 2013; Lee et al. 2010, 2012). Although we did not perform direct follow-up analysis of the progeny of Fq animals after unc-1 targeting, we did find progeny that showed the expected uncoordinated (Une) phenotype on the P0 plates from which mutant Fq's were picked after a few generations had passed (Figure IE). This observation suggests that some of the mutations identified in the Fq animals were indeed transmitted through the germline. The results of the unc-1 targeting experiment are shown in Table 1. In summary, we were able to disrupt two endogenous genes in C. elegans using Cas9 protein complexed with in vitro transcribed sgRNAs. It is worth noting that no sub- cloning steps are needed to generate new RGENs. In con- trast, the construction of new ZFNs or TALENs involves recombinant DNA technology. Limited analysis showed that no mutations were observed at the most likely off-target sequences of the dpy-3 gene in the mutant F2 animals (Figure SI), suggesting that this method can induce specif- ically targeted mutations. However, newly formed mutants should still be outcrossed with the wild-type strain to remove any off-target mutations that may have occurred. From our results described above, we propose the following simple and general procedure for generating gene-specific mutations using RGENs: (1) microinject the Cas9-sgRNA complex into ~10 P0 animals, (2) After the P0 animals have laid Fq eggs, clone 10-20 individual Fq animals from each P0 plate and allow them to grow into adults and lay eggs, (3) examine the individual Fq animals using the T7E1 assay, and (4) from the plates with Fq animals with mutations, pick and examine individual F2 animals for germline-transmitted mutations after they have laid eggs. Here, we combine two RGENs to target a single gene and find that RGENs often cleave two sites simultaneously, which gives rise to deletions that span the two sites. Despite the lack of a direct proof of simultaneous hits by the two sgRNAs, this result suggests that it will be worth trying targeting multiple sites simultaneously in different genes in nematodes. The injection of the Cas9 protein into nemat- odes has certain critical advantages over the use of a Cas9- encoding plasmid or mRNA. First, the ribonucleoprotein complexes act on targets immediately after injection. In contrast, mRNA or plasmids must be transcribed and/or translated after injection. Second, unlike exogenous mRNA that can be silenced in C. elegans, protein RGEN complexes remain active in vivo. Third, this procedure can be used in other species, such as parasitic nematodes, because there is no need to change the promoters or to optimize codon usage to express Cas9 in different organisms. Acknowledgments The authors thank H. Lee for technical help and M. S. Choi for critical reading. We also thank the Caenorhabditis Genetics Center for the N2 C. elegans strain. 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Seung Woo Cho,*,1 Jihyun Lee,[dagger],1 Dana Carroll,[double dagger] Jin-Soo Kim,*,2 and Junho Lee[dagger],§,2 Departments of *Chemistry, [dagger]Biological Sciences, and §Biophysics and Chemical Biology, Seoul National University, Seoul 151-742, Korea, and [double dagger]Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84112-5650 Copyright © 2013 by the Genetics Society of America doi: 10.1534/genetics.113.155853 Manuscript received July 28, 2013; accepted for publication August 16, 2013 Available freely online through the author-supported open access option. Supporting information is available online at http://www.genetics.org/lookup/suppl/ doi:10.1534/genetics.113.155853/-/DC1. 1These authors contributed equally to this work. 2Corresponding authors: Department of Chemistry, Seoul National University, Gwanak-ro 1, Gwanak-gu, Seoul 151-742, Korea. E-mail: [email protected]; and Department of Biological Sciences, Institute of Molecular Biology and Department of Biophysics and Chemical Biology, Genetics, Seoul National University, Gwanak-ro 1, Gwanak-gu, Seoul 151-742, Korea. E-mail: [email protected] (c) 2013 Genetics Society of America |