ARTICLE IN PRESS The International Journal of Biochemistry & Cell Biology xxx (2013) xxx xxx

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1 The International Journal of Biochemistry & Cell Biology xxx (2013) xxx xxx Contents lists available at ScienceDirect The International Journal of Biochemistry & Cell Biology journal h om epage: Q Short communication One-step generation of different immunodeficient mice with multiple gene modifications by CRISPR/Cas9 mediated genome engineering Jiankui Zhou a,1, Bin Shen a,1, Wensheng Zhang b,1, Jianying Wang a, Jing Yang a, Li Chen a, Na Zhang c, Kai Zhu c, Juan Xu a, Bian Hu a, Qibin Leng c,, Xingxu Huang a, a MOE Key Laboratory of Model Animal for Disease Study, Model Animal Research Center of Nanjing University, National Resource Center for Mutant Mice, Nanjing , China b Wellcome Trust Sanger Institute, Wellcome Trust Genome Campus, Hinxton, Cambridge CB10 1SA, UK c Key Laboratory of Molecular Virology and Immunology, Institute Pasteur of Shanghai, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 225 South Chongqing Road, Shanghai, China a r t i c l e i n f o Article history: Received 14 September 2013 Received in revised form 20 October 2013 Accepted 22 October 2013 Available online xxx Keywords: Cas9 RGEN Multiple Gene targeting Immunodeficient Mouse a b s t r a c t Taking advantage of the multiplexable genome engineering feature of the CRISPR/Cas9 system, we sought to generate different kinds of immunodeficient mouse strains by embryo co-microinjection of Cas9 mrna and multiple sgrnas targeting mouse B2m, Il2rg, Prf1, Prkdc, and Rag1. We successfully achieved multiple gene modifications, fragment deletion, double knockout of genes localizing on the same chromosome, and got different kinds of immunodeficient mouse models with different heritable genetic modifications at once, providing a one-step strategy for generating different immunodeficient mice which represents significant time-, labor-, and money-saving advantages over traditional approaches. Meanwhile, we improved the technology by optimizing the concentration of Cas9 and sgrnas and designing two adjacent sgrnas targeting one exon for each gene, which greatly increased the targeting efficiency and bi-allelic mutations Elsevier Ltd. All rights reserved Introduction The fundamental understanding of many biological processes in humans has resulted from the development of humanized mouse strains that are based on severely immunodeficient mice which carry multiple mutations in different immune responseassociated genes, including B2m, Foxn1, Il2rg, Prf1, Prkdc, Rag1, Rag2, etc. (Shultz et al., 2007, 2012). Advances in the ability to generate humanized mice have depended on a systematic progression of genetic modifications to develop immunodeficient mice with multiple mutations (Dow and Lowe, 2012; Shultz et al., 2007, 2012). Apart from the formidable task to produce and characterize these mutants, the process of intercrossing individual allele or sequential targeting to generate different immunodeficient mouse models with multiallelic mutations is even more tedious, time-consuming, Corresponding author. Tel.: ; fax: Q2 Corresponding author. Tel.: ; fax: addresses: qbleng@sibs.sc.cn (Q. Leng), xingxuhuang@mail.nju.edu.cn, huangxingxu@yahoo.com (X. Huang). 1 1 These authors contributed equally to this work. and expensive. Therefore, efficient strategies for triggering multiple genetic modifications are extremely desirable. By NHEJ (non-homologous end joining), Cas9, a DNA endonuclease (now referred to as RNA-guided nucleases, RGENs) of the clustered, regularly interspaced, short palindromic repeats (CRISPR) system in bacterial and archaeal immunity (Jinek et al., 2012), provides the potential to engineer the eukaryote genome in a multiplexed way because its DNA-cleaving activity can be programmed with small RNA molecules to recognize specific DNA sequences, thus sparing the need to engineer a new protein for each new DNA target sequence. This potential was first achieved in cells (Cong et al., 2013; Jinek et al., 2012; Mali et al., 2013; Qi et al., 2013). Recently, Wang et al. made the potential become reality in mice (Wang et al., 2013). Such strategy would find broad application in the development of multigenic mouse models. Taking advantage of the multiplexable genome engineering feature of the RGEN system, we sought to generate different kinds of immunodeficient mouse strains by co-microinjection of Cas9 mrna and multiple sgrnas targeting mouse B2m, Il2rg, Prf1, Prkdc, and Rag1 into embryos. We successfully achieved multiple gene modifications, fragment deletion, double knockout of genes localizing on the same chromosome, and got different kinds of immunodeficient mouse models with different heritable genetic /$ see front matter 2013 Elsevier Ltd. All rights reserved.

2 G Model 2 J. Zhou et al. / The International Journal of Biochemistry & Cell Biology xxx (2013) xxx xxx modifications at once, providing a one-step strategy for generating different immunodeficient mice with multiple gene modifications by CRISPR/Cas9 mediated genome engineering. Meanwhile, we improved the efficacy of the CRISPR/Cas9 technique in mouse genome engineering beyond the current standard by optimizing the concentration of Cas9 and sgrnas and designing two adjacent sgrnas targeting one exon for each gene. 2. Materials and methods 2.1. Animals Mice were housed in standard cages in an Assessment and Accreditation Of Laboratory Animal Care credited SPF animal facility on a 12-h light/dark cycle. All animal protocols are approved by the Animal Care and Use Committee of the Model Animal Research Center, the host for the National Resource Center for Mutant Mice in China, Nanjing University DNA constructs pst1374-cas9-n-nls-flag-linker (AddgeneID 44758) was described as before (Shen et al., 2013). sgrna oligos were annealed into the puc57-sgrna expression vector with a T7 promoter. The sequences of sgrna oligos and puc57-sgrna expression vector are listed in Supplementary Table 3 and Supplementary Sequence In vitro transcription The pst1374-cas9-n-nls-flag-linker vector was linearized with Age1 and in vitro transcribed using T7 Ultra Kit (Ambion, AM1345). Cas9-N-NLS-flag-linker mrna was purified using the RNeasy Mini Kit (Qiagen, 74104). sgrna expression vectors were linearized with Dra I and transcribed in vitro using the MEGAshortscript Kit (Ambion, AM1354). sgrnas were purified using the MEGAclear Kit (Ambion, AM1908) and concentrated by alcohol precipitation. For 5 (or 10) sgrnas co-injection experiments, 2 g each of the 5 (or 10) sgrna expression vectors were mixed together and digested with Dra I. Digestion products were cleaned up with PCR Purification Kit (Qiagen, 28004), followed by transcription in vitro T7EN1 cleavage assay and sequencing T7EN1 cleavage assay was performed as described (Shen et al., 2013). For the T7EN1 cleavage assay of Il2rg, which is localized on chromosome X, 50 ng wild-type PCR fragment and 150 ng PCR fragment from all male founders were mixed, then applied T7EN1 cleavage assay. Primers for amplifying B2m, Il2rg, Prf1, Prkdc and Rag1 sgrnas targeted fragments are listed in Supplementary Table 4. PCR products with mutations detected by T7EN1 cleavage assay were sub-cloned into T vector (Takara, D103A). For each sample, at least 24 white colonies were picked up randomly and sequenced by M13-47 primer. Colonies with double peaks were taken out during alignment. Indel and mutation information were listed in Supplementary Table 2. For possible long fragment deletion, new primers were designed to amplify larger amplicon flanking the targeted site. The PCR products were cloned and sequenced by M13-47 and M13-48 primers for each colony Cas9/sgRNA injection of one-cell embryos Cas9/sgRNA co-injection of one-cell embryos was performed as described (Shen et al., 2013). Briefly, mouse zygotes obtained by mating of CBA males with superovulated C57BL/6 J females, were injected with a mixture of Cas9 mrna and sgrnas as indicated in Table 1. Microinjections were performed in the cytoplasm and larger (male) pronucleus of fertilized oocytes. Injected zygotes were transferred to pseudopregnant CD1 female mice Flowcytometry analysis of leukocytes in peripheral blood Three drops of whole blood drawn from 6-week-old founder s tail vein was collected in 30 L of 50 mm EDTA, and red blood cells were lysed with ACK buffer (0.15 M NH4Cl, 1 mm KHCO3, 0.1 mm EDTA, ph 7.2). The remaining cells were washed with cold PBS and stained with fluorescent antibodies against B220 (ebioscience, ), H2-kb (ebioscience, ), and CD90.2 (ebioscience, ) for subsequent analysis by flowcytometry (BD LSR II analyzer). Total 10,000 cell events were collected and analyzed in flowjo software. Even-aged mice in the same genetic background were used as control. 3. Results and discussion 3.1. sgrna:cas9-mediated modifications of 5 immuno-genes by single sgrna targeting Different immunodeficient mouse models are derived from multiallelic mutations of different immune-related genes, including B2m, Foxn1, Il2rg, Prf1, Prkdc, Rag1, Rag2, etc. (Shultz et al., 2007, 2012). For example, the most versatile immunodeficient strain, NSG, carries Prkdc and Il2rg double mutation. A recent paper described the multiplexable genome engineering feature of the RGEN system in mouse (Wang et al., 2013), suggesting the possibility to get different immune-related gene mutation mouse models by embryo co-microinjection of different kinds of sgrnas. To test the possibility, 5 different sgrnas each targeting one exon of mouse B2m, Il2rg, Prf1, Prkdc, and Rag1 (Supplementary Figure 1 and Supplementary Table 1), were selected using previously described criteria (Cho et al., 2013; Cong et al., 2013; Hwang et al., 2013; Mali et al., 2013). The sgrnas were in vitro transcribed as description (Shen et al., 2013). The first test was performed by one-cell-stage mouse embryo co-microinjection with a mixture of 20 ng/ l mrna of NLS-flaglinker-Cas9, a modified Cas9 easy for nuclear localization (Shen et al., 2013), along with 5 ng/ l each of 5 sgrnas targeting B2m, Il2rg, Prf1, Prkdc, and Rag1 (Table 1). A total of 27 pups (Founders #33 59) from 4 litters were born from 81 transferred embryos (Table 1). Modifications of the different loci from randomly selected 7 founders were first assayed by PCR (Fig. 1A), T7EN1 cleavage assay (Fig. 1B), and sequencing (Tables 2 and 3, and Supplementary Table 2). The results showed, all founders harbored gene modifications, Table 1 Summary of embryo microinjection of Cas9 mrna and sgrnas. Inject mixture Embryos injected Embryos transferred Recipient Pups amount Pups No. 1st 20 ng/ l Cas9 mrna #33 #59 5 sgrnas, 5 ng/ l each. 2nd 20 ng/ l Cas9 mrna #60 #68 10 sgrnas, 2.5 ng/ l each. 3rd 20 ng/ l Cas9 mrna #90 #93 10 sgrnas, 5 ng/ l each.

3 J. Zhou et al. / The International Journal of Biochemistry & Cell Biology xxx (2013) xxx xxx 3 Table 2 Summary of the genotyping of the founders. 1st 2nd 3rd Pups No. Rate Pups No. Rate Pups No. Rate Single gene modification #49,# % (2/7) Double gene modification #41,#43,# % (3/7) #60, #62, #67, # % (4/9) Triple gene modification #54,# % (2/7) #63, #64, # % (3/9) Quadruple gene modification #61, # % (2/9) #93 25% (1/4) Quintuple gene modification #90,#91,#92 75% (3/4) Table 3 Summary of target site modification. Mice targeted by sgrna/total mice B2m Il2rg Prf1 Prkdc Rag1 sgr1 sgr2 sgr1&2 sgr1 sgr2 sgr1&2 sgr1 sgr2 sgr1&2 sgr1 sgr2 sgr1&2 sgr1 sgr2 sgr1&2 1st N.A. 3/7 N.A. 2/7 N.A. N.A. N.A. 1/7 N.A. 5/7 N.A. N.A. 3/7 N.A. N.A. 2nd 2/9 3/9 0/9 0/9 0/9 0/9 5/9 0/9 0/9 3/7 7/9 7/9 3/9 7/9 2/9 3rd 3/4 3/4 3/4 3/4 0/4 0/4 4/4 2/4 0/4 3/4 2/4 4/4 2/4 4/4 0/ and the cleavages were detected at all the 5 sites targeted by the tested 5 sgrnas with efficiency of 3/7 at B2m (Founders #54, #58, and #59), 2/7 at Il2rg (Founders #49 and #59), 1/7 at Prf1 (Founder #58), 5/7 at Prkdc (Founders #41, #43, #54, #57, and #58), and 3/7 at Rag1 (Founders #41, #43, and #54), respectively, indicating the RGENs function efficiently at these immuno-related gene loci. Subsequently, genotyping by PCR and T7EN1 assay of the remaining 20 founders was performed. The similar efficiency of gene modifications of the different loci was observed (data not shown). It has been estimated that the generation of a single gene knockout mouse by traditional strategy may cost about 30, ,000 dollars, and take 6 12 months or longer (Capecchi, 2005; Dow and Lowe, 2012). Therefore, generation of different immunodeficient mice bearing different gene mutations either by intercrossing of single-mutant mice or sequential targeting in mouse ES cells is much more time-consuming, labor-intensive, and expensive. Our data showed (Fig. 1B, Tables 2 and 3, and Supplementary Table 2), co-injection of 5 sgrna targeting 5 genes yielded single gene modification in 2 out of 7 founders (Founders #49 and #57), 2 gene modifications in 3 out of 7 founders (Founders #41, #43, and #59), 3 gene modifications in 3 out of 7 founders (#54 and #58). These data confirmed the multiplexable genome engineering feature of the RGEN, and also demonstrated one RGEN reaction can yield multiple genotypes in several weeks and at about 10% of the single gene knockout cost, suggesting RGEN is an extremely powerful technique to get different immunodeficient mouse models Fig. 1. sgrna:cas9-mediated 5 gene modifications by 5 sgrnas. (A) PCR identification of sgrna:cas9-mediated site-specific cleavage of the endogenous B2m, Il2rg, Prf1, Prkdc, and Rag1 loci. The genetic modification analysis was performed by PCR amplification of the targeted fragment in the B2m, Il2rg, Prf1, Prkdc, and Rag1 in founder mice treated with co-microinjection of a mixture of mrna of NLS-flag-linker-Cas9 and 5 sgrnas as described in Table 1. (B) Detection of sgrna:cas9-mediated site-specific cleavage of the endogenous B2m, Il2rg, Prf1, Prkdc, and Rag1 by T7EN1 cleavage assay. PCR products from (A) were subjected to T7EN1 cleavage assay as described in Section 2.

4 G Model 4 J. Zhou et al. / The International Journal of Biochemistry & Cell Biology xxx (2013) xxx xxx Fig. 2. sgrna:cas9-mediated 5 gene modifications by low concentrations of sgrnas. (A) PCR identification of sgrna:cas9-mediated site-specific cleavage of the endogenous B2m, Il2rg, Prf1, Prkdc, and Rag1 loci. The genetic modification analysis was performed by PCR amplification of the targeted fragment in the B2m, Il2rg, Prf1, Prkdc, and Rag1 in founder mice treated with co-microinjection of a mixture of mrna of NLS-flag-linker-Cas9 and 10 sgrnas as described in Table 1. (B) Detection of sgrna:cas9-mediated site-specific cleavage of the endogenous B2m, Il2rg, Prf1, Prkdc, and Rag1 by T7EN1 cleavage assay. PCR products from (A) were subjected to T7EN1 cleavage assay as described in Section Dual sgrnas facilitated multiple gene modifications by sgrna:cas9 One of the major concerns is that the mutations by ZFNs, TALENs, or CRISPR/Cas9 are unpredictable owing to the variable DNA repair by NHEJ. In addition, NHEJ induced in-frame mutations frequently. Therefore, we tested whether using dual sgrnas targeting at one gene enable more efficient gene deletion by CRISPR/Cas9 system. Considering that two sgrnas targeting adjacent sites efficiently deleted the intervening region (Cong et al., 2013), we designed another distinct sgrna targeting to an adjacent site bp away from the tested sgrnas of mouse B2m, Il2rg, Prf1, Prkdc, and Rag1 (Supplementary Figure 1). We performed one-cell-stage mouse embryo co-microinjection with equal amounts of sgrna, 2.5 ng/ l each of 10 sgrnas targeting B2m, Il2rg, Prf1, Prkdc, and Rag1. A total of 9 pups (Founders #60 68) from 4 recipients were born from 93 transferred embryos (Table 1). Similarly, the modifications of the different loci were first analyzed by PCR using tail DNA from 5-day-old founders. Different from the single sgrna, dual sgrnas even yielded visible shorter PCR fragments in tail DNA from 5-day-old founders with efficiency of 6/9 at Prkdc (Founders #60 62, #64, #66, and #68) and 3/9 at Rag1 (Founders #61, #63, and #66) (Fig. 2A), confirming gene modification occurrence. Following T7EN1 cleavage assay and sequencing showed (Fig. 2B, Tables 2 and 3, and Supplementary Table 2), 2 out of 9 founders gave rise to 4 gene modifications (Founders #61 and #66). The mutation efficiency was 22.22%; 3 out of 9 founders gave rise to 3 gene modifications (Founders #63 65). The mutation efficiency was 33.44%; 4 out of 9 founders each yielded 2 gene modifications (Founders #60, #62, #67, and #68). The mutation efficiency was up to 44.44%. Interestingly, two genotypes of two kinds immunodeficient mouse models, Prkdc and Rag1 double modification (Lapidot, 2001) or B2m and Prf1 double modification (Shultz et al., 2003), were generated with high efficiency of 7/9 (Founders #60, #61, #63 66, #68) or 2/9 (Founders #61 and #66), respectively. These data further confirmed the high efficiency of RGENs function at immune response-associated gene loci, demonstrating CRISPR/Cas9-mediated genome engineering is an efficient strategy to generate different immunodeficient mice with multiple gene modifications in one step, and representing the first significant improvement over traditional approaches in generating multiple knockout mice, in which labor-intensive and time-consuming interbreeding or sequential targeting in ES cells is required. The precise determination of modification by sequencing showed, different indels or mutations from imperfect NHEJ were detected in all founders (Table 3 and Supplementary Table 2). Compared with single sgrna, besides single, double, and triple gene modifications, dual sgrnas even induced quadruple modification, and induced cleavage with even higher efficiency of 5/9 at Prf1, 8/9 at Prkdc and Rag1, indicating two sgrnas function better at immune response-associated gene loci. In addition, fragment deletions were observed at Prkdc of 7 founders (Founders #60 64, #66, #68), at Rag1 of 2 founders (Founders #63, #66) (Table 3 and Supplementary Table 2). These results demonstrated dual sgrnas facilitated multiple gene modifications by sgrna:cas9. Our data further confirmed that targeting two adjacent sites efficiently deleted the intervening region, suggesting CRISPR/Cas9 can be used to induce larger DNA fragment deletion by manipulating multiple sgrnas easily, instead of co-transfection of two ZFN pairs (Lee et al., 2010), or two TALEN pairs (Carlson et al., 2012; Kim et al., 2013). By co-microinjection of 2 sgrna and Cas9 mrna into zygotes, we have achieved 5 kb deletion with up to 25% efficiency in mice (data not shown). More intriguingly, 3 of 9 founders (Founders #61, #63, and #66) harbored simultaneously mutations of B2m and Rag1, both are localized on the chromosome 2 (Supplementary Table 1), representing the second significant improvement over traditional approaches in generating multiallelic knockout of genes localizing on the same chromosome, for which labor-intensive and

5 J. Zhou et al. / The International Journal of Biochemistry & Cell Biology xxx (2013) xxx xxx 5 Table 4 Summary of bi-allelic mutations. Mice with mutant bi-alleles/total mice B2m Il2rg Prf1 Prkdc Rag1 1st 0/7 0/7 0/7 0/7 0/7 2nd 1/9 0/9 1/9 8/9 4/9 3rd 4/4 1 * /4 4/4 4/4 4/4 * #91 is female harboring two Il2rg alleles. Fig. 3. sgrna:cas9-mediated 5 gene modifications by high concentrations of sgr- NAs. (A) PCR identification of sgrna:cas9-mediated site-specific cleavage of the endogenous B2m, Il2rg, Prf1, Prkdc, and Rag1 loci. The genetic modification analysis was performed by PCR amplification of the targeted fragment in the B2m, Il2rg, Prf1, Prkdc, and Rag1 in founder mice treated with co-microinjection of a mixture of mrna of NLS-flag-linker-Cas9 and 10 sgrnas as described in Table 1. Large fragment deletion occurred in B2m locus of Founder #90 and Prkdc locus of Founder #92. (B) Detection of sgrna:cas9-mediated site-specific cleavage of the endogenous B2m, Il2rg, Prf1, Prkdc, and Rag1 by T7EN1 cleavage assay. PCR products from (A) were subjected to T7EN1 cleavage assay as described in Section , 2012), were produced via a single targeting attempt. These results demonstrated that high concentrations of sgrna efficiently improved multiple gene modifications by sgrna:cas9. Notably, intact alleles were detected in all samples with single sgrna/gene-induced modifications, but were absent in some of the samples with dual sgrna/gene-induced modifications (Table 4 and Supplementary Table 2), suggesting bi-allelic mutations occurred at dual sgrnas/gene but not single sgrna/gene-induced gene modification. Strikingly, a total 20 reactions at 5 target loci of 4 founders from high concentration of sgrnas treatment, intact alleles were only detected in 3 reactions of Il2rg (Table 4 and Supplementary Table 2), indicating bi-allelic modification occurred in remaining 17 of total 20 reactions with efficiency up to 85%. These results demonstrated that mice carrying bi-allelic mutations in different immune related genes can be generated within one month with high efficiency, representing the third significant improvement over traditional approaches in generating homozygous knockout mice, for which labor-intensive and time-consuming interbreeding or sequential targeting in ES cells is required. Furthermore, no intact B2m and Rag1 were detected, confirming dual sgrna/gene induced bi-allelic mutations in two genes localizing on a single chromosome, further highlighting the impressive efficiency and versatility of the CRISPR/Cas9 system for in vivo genome modification time-consuming interbreeding or sequential targeting in ES cells is required Multiple immuno-gene modifications by sgrna:cas9 resulted in immunodeficiency High concentrations of sgrna improved multiple gene modifications by sgrna:cas9 We further optimized this efficient CRISPR/Cas9-based strategy for immunodificient mouse model generation by increasing the sgrna concentration to 5 ng/ l for each of 10 sgrnas targeting B2m, Il2rg, Prf1, Prkdc, and Rag1. A total of 4 pups (Founders #90 93) from 3 recipient mice were born from 89 transferred embryos (Table 1). The NHEJ-mediated gene mutations were detected via the same method mentioned above. As shown in Fig. 3A, besides Prkdc and Rag1, high concentrations of sgrnas induced shorter PCR fragments of B2m and Prf1, indicating that similar to the scenario with Cas9 mrna (Wang et al., 2013), higher concentration of sgrna resulted in more efficient gene modifications. Interestingly, the B2m locus in Founder #90 and Prkdc locus in Founder #92 cannot be amplified (Fig. 3A), indicating large fragment deletion. To confirm these results, larger amplicon flanking the targeted sites were amplified, and indeed, the PCR results showed there harbored large fragment deletion (Supplementary Figure 2). The subsequent T7EN1 assay and sequencing showed that (Fig. 3B, Tables 2 4, and Supplementary Table 2), more strikingly, higher concentrations of sgrnas induced modifications of all 5 genes in Founder #90 92, or 4 genes in Founders #93. Therefore, different immunodeficient mouse models, including NSG model with Prkdc and Il2rg mutation, BRG model with Rag1 and Il2rg mutation, NSG B2m / model with B2m, Prkdc, and Il2rg mutation (Shultz et al., Disruptions in different immuno-related genes results in different immunodeficiency. For example, mutations of Prkdc and Rag1 lead to lack of mature T and B cells (Shultz et al., 2007, 2012); B2m disruption causes MHC class I expression failure (Shultz et al., 2007, 2012); Il2rg knockout results in NK cell absence (Shultz et al., 2007, 2012). We characterized the possible immunodeficiency from sgrna:cas9-medicated multiple immuno-gene modifications by examining of the effect of these mutations on the MHC-I expression of leukocytes and proportions of T and B cells in the peripheral blood of Founders # Consistent with the deletion of both Prkdc and Rag1, we found that the proportions of T and B cells in all the founders dramatically decreased in comparison to control mice (Fig. 4A). Meanwhile, MHC-I expression was almost undetectable in peripheral blood cells of all the founders (Fig. 4B), suggesting the deletion of B2m. These results indicated that multiplexed RGEN targeting of critical genes associated with immunity results in significant immunodeficiency, confirming the efficient bi-allelic immuno-gene modifications. In addition, we determined the transmission of gene modifications by breeding 2 F 0 mutants (Founders #61 & #91) with wild-type C57BL/6 J and genotyping the resulting F 1 offspring. The offspring yielded PCR product of same size as founders (Supplementary Figure 3A and C). The PCR products were further analyzed by sequencing. Sequencing results showed the same mutations appeared in offspring as founders (Supplementary Figure 3B and D and Supplementary Table 2), demonstrating that mutations were successfully transmitted through germline

6 G Model 6 J. Zhou et al. / The International Journal of Biochemistry & Cell Biology xxx (2013) xxx xxx Fig. 4. Flowcytometry analysis of peripheral blood nucleated cells from founders and control mice. (A) The proportions of T cells (CD B220 ) and B cells (B220 + CD90.2 ) of 6-week-old founders and control. (B) Histograms of MHC-I (H2-Kb) expression in total nucleated cells in 6-week-old founders and control Taken together, taking the advantages of CRISPR/Cas9, and improving the technology by optimizing the concentration of Cas9 and sgrnas and designing two adjacent sgrnas targeting one exon for each gene, we successfully achieved multiple gene modification, fragment deletion, double knockout of genes localizing on the same chromosome, and got different kinds of immunodeficient mouse models with multiple heritable genetic modifications in one single targeting attempt. This strategy for immunodeficient mouse models showed significant advantages over traditional approaches, for which labor intensive and time consuming interbreeding or sequential targeting are required to generate multiple knockout mice, multiallelic knockout of genes localizing on the same chromosome, and homozygous knockout mice. Conflict of interest statement The authors declare no competing financial interests

7 J. Zhou et al. / The International Journal of Biochemistry & Cell Biology xxx (2013) xxx xxx Acknowledgements We thank Dr. William C. Skarnes for discussion and critical reading of the manuscript. We thank the entire Huang Lab for their support and advice. We also thank Prof. Jianghuai Liu (Nanjing University) for careful reading and editing of the manuscript. This work was supported by grants from the 973 program of the Ministry of Science and Technology of China to XH (2009CB918703) and the National Natural Science Foundation of China to QL ( ). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at References Capecchi MR. Gene targeting in mice: functional analysis of the mammalian genome for the twenty-first century. Nat Rev Genet 2005;6: Carlson DF, Tan W, Lillico SG, Stverakova D, Proudfoot C, Christian M, et al. Efficient TALEN-mediated gene knockout in livestock. Proc Natl Acad Sci USA 2012;109: Cho SW, Kim S, Kim JM, Kim JS. Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 2013;31: Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013;339: Dow LE, Lowe SW. Life in the fast lane: mammalian disease models in the genomics era. Cell 2012;148: Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, et al. Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 2013;31: Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-rna-guided DNA endonuclease in adaptive bacterial immunity. Science 2012;337: Kim Y, Kweon J, Kim A, Chon JK, Yoo JY, Kim HJ, et al. A library of TAL effector nucleases spanning the human genome. Nat Biotechnol 2013;31: Lapidot T. Mechanism of human stem cell migration and repopulation of NOD/SCID and B2mnull NOD/SCID mice. The role of SDF-1/CXCR4 interactions. Ann NY Acad Sci 2001;938: Lee HJ, Kim E, Kim JS. Targeted chromosomal deletions in human cells using zinc finger nucleases. Genome Res 2010;20:81 9. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, et al. RNA-guided human genome engineering via Cas9. Science 2013;339: Qi LS, Larson MH, Gilbert LA, Doudna JA, Weissman JS, Arkin AP, et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 2013;152: Shen B, Zhang J, Wu H, Wang J, Ma K, Li Z, et al. Generation of gene-modified mice via Cas9/RNA-mediated gene targeting. Cell Res 2013;23: Shultz LD, Banuelos S, Lyons B, Samuels R, Burzenski L, Gott B, et al. NOD/LtSz- Rag1nullPfpnull mice: a new model system with increased levels of human peripheral leukocyte and hematopoietic stem-cell engraftment. Transplantation 2003;76: Shultz LD, Ishikawa F, Greiner DL. Humanized mice in translational biomedical research. Nat Rev Immunol 2007;7(2): Shultz LD, Brehm MA, Garcia-Martinez JV, Greiner DL. Humanized mice for immune system investigation: progress, promise and challenges. Nat Rev Immunol 2012;12(11): Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell 2013;153: