Applications of the CRISPR-Cas9 system in kidney research

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www.kidney-international.org

Applications of the CRISPR-Cas9 system in kidney research Yoshiki Higashijima1,2, Seiichi Hirano3, Masaomi Nangaku1 and Osamu Nureki3 1 Division of Nephrology and Endocrinology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, Japan; 2Isotope Science Center, The University of Tokyo, Bunkyo-ku, Tokyo, Japan; and 3Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan

The recently discovered clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein 9 (Cas9) is an RNA-guided DNA nuclease, and has been harnessed for the development of simple, efficient, and relatively inexpensive technologies to precisely manipulate the genomic information in virtually all cell types and organisms. The CRIPSR-Cas9 systems have already been effectively used to disrupt multiple genes simultaneously, create conditional alleles, and generate reporter proteins, even in vivo. The ability of Cas9 to target a specific genomic region has also been exploited for various applications, such as transcriptional regulation, epigenetic control, and chromosome labeling. Here we first describe the molecular mechanism of the RNA-guided DNA targeting by the CRISPR-Cas9 system and then outline the current applications of this system as a genome-editing tool in mice and other species, to better model and study human diseases. We also discuss the practical and potential uses of the CRISPR-Cas9 system in kidney research and highlight the further applications of this technology beyond genome editing. Undoubtedly, the CRISPR-Cas9 system holds enormous potential for revolutionizing and accelerating kidney research and therapeutic applications in the future. Kidney International (2017) 92, 324–335; http://dx.doi.org/10.1016/ j.kint.2017.01.037 KEYWORDS: CRISPR-Cas9; gene expression; gene therapy; genome editing Copyright ª 2017, International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Correspondence: Osamu Nureki, Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. E-mail: [email protected] Received 14 September 2016; revised 26 December 2016; accepted 9 January 2017; published online 20 April 2017

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The CRISPR-Cas9 system

Clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated (Cas) is a prokaryotic adaptive immune system against invading genetic elements, such as phages and plasmids.1–3 The CRISPR loci consist of Cas genes and a CRISPR array, which encodes CRISPR RNA (crRNAs). The CRISPR-Cas systems can be classified into 6 different types (types I–VI), primarily based on the mechanisms of crRNA biogenesis and target degradation.4 In the type II system, the precursor crRNAs and the trans-activating crRNA (tracrRNA) are transcribed from the CRISPR loci, bound to the Cas9 nuclease, and then processed by RNase III, to form a Cas9-crRNA-tracrRNA effector complex. This complex cleaves DNA targets complementary to the crRNA guide sequence, which is originally derived from previously infected mobile genetic elements.5 In 2012, biochemical studies revealed that Cas9 is a programmable RNA-guided DNA endonuclease, and the Cas9-crRNA-tracrRNA complex cleaves double-stranded DNA targets complementary to the 20-nucleotide guide sequence in the crRNA.6,7 Cas9 contains 2 endonuclease domains, HNH and RuvC, which cleave the DNA strands that are complementary (target DNA strand) and noncomplementary (nontarget DNA strand) to the crRNA guide, respectively (Figure 1a and b). In addition to the crRNA-target DNA complementarity, target recognition by Cas9 requires a protospacer adjacent motif (PAM), a short nucleotide motif immediately downstream of the target sequence.8,9 For example, Streptococcus pyogenes Cas9 (SpCas9) and Staphylococcus aureus (SaCas9) recognize the 50 -NGG-30 and 50 -NNGRRT-30 sequences on the nontarget strand as the PAMs, respectively.6,7,10 In 2013, several groups demonstrated that SpCas9 can be harnessed to edit genomic DNA sequences in eukaryotic cells.11–14 Because a single-guide RNA (sgRNA), a synthetic crRNA:tracrRNA fusion, also directs Cas9 to target cleavage6 (Figure 1a), changing the crRNA guide sequence can allow the simple 2-component Cas9-sgRNA system to edit the DNA target of interest. Thus, SpCas9 is widely used as a costeffective and convenient genome-editing tool that works in a broad range of cell types and organisms. In addition, the smaller SaCas9 can be efficiently delivered to somatic tissues for genome editing.10 Importantly, the orthologous CRISPR-Cas9 systems from different microbes enable simultaneous transcriptional activation, repression, and genome editing,15 because the Cas9 orthologs recognize their cognate guide RNAs in species-specific manners.10,16 Kidney International (2017) 92, 324–335

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a

b

c

Figure 1 | Function and structures of clustered regularly interspaced short palindromic repeat (CRISPR)–associated 9 (Cas9). (a) Schematics of RNA-guided DNA cleavage by Cas9–CRISPR RNA (crRNA)–trans-acting crRNA (tracrRNA) and Cas9–single-guide RNA (sgRNA). (b) Domain organization of Cas9. (c) Crystal structures of Streptococcus pyogenes Cas9 (SpCas9) (Protein Data Bank: 4UN3) and Staphylococcus aureus Cas9 (SaCas9) (Protein Data Bank: 5CZZ). BH, bridge helix; C, C-Terminus; HNH, a Cas9 endonuclease domain; L1, linker 1; L2, linker 2; N, N-Terminus; NGG, 50 -NGG-0 3 PAM sequence of SpCas9; NTS, nontarget DNA strand; NUC, nuclease; PAM, protospacer adjacent motif; PI, PAM-interacting REC, recognition; RuvC, a Cas9 endonuclease domain; TS, target DNA strand; WED, wedge.

Three-dimensional structure of CRISPR-Cas9

The crystal structures of SpCas9 in different functional states advanced our understanding of the mechanism of RNAguided DNA recognition by Cas917–21 (Figure 1c). Cas9 comprises 2 lobes, a recognition lobe and a nuclease lobe. In the absence of the sgRNA, Cas9 adopts an autoinhibited, closed conformation. On sgRNA binding, Cas9 recognizes the sgRNA scaffold and adopts an open conformation, in which the recognition and nuclease lobes form a positively charged, central channel that accommodates the guide RNA-target DNA heteroduplex. The Cas9-sgRNA binary complex initially recognizes the PAM sequence in the target DNA.22,23 In the crystal structure, the PAM-containing duplex (PAM duplex) is bound between the wedge and PAM-interacting domains, where the PAM nucleotides are recognized from the major-groove side by specific amino acid residues in the PAM-interacting domain.21 The PAM recognition induces the unwinding of the double-stranded DNA target, thereby initiating the Watson-Crick base-pairing between the crRNA guide and the target sequence.21,24 The local separation of the Kidney International (2017) 92, 324–335

DNA target likely causes a conformational change in the HNH domain for target cleavage.25 In addition to SpCas9, the crystal structures of SaCas9 and Francisella novicida Cas9 revealed the molecular diversity among the orthologous CRISPR-Cas9 systems26,27 (Figure 1c). The structurally distinct recognition and wedge domains of these Cas9 orthologs recognize their structurally divergent, cognate sgRNA scaffolds in species-specific manners. In the SpCas9 and Francisella novicida Cas9 structures, the 50 -NGG-30 PAMs are recognized by their PAM-interacting domains, in which a distinct set of 2 arginine residues forms direct hydrogen bonds with the GG dinucleotides in the PAM duplex.21,27 In contrast, in the SaCas9 structure, the 50 -NNGRRT-30 PAM is recognized by the PAM-interacting domain, in which 2 arginine and 2 asparagine residues form a hydrogen-bonding network with the GRRT nucleotides.26 Genome editing in a mouse model

Due to the fundamental genetic similarity between mice and humans, phenotypic analyses of mutant mice are widely used 325

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for investigating the in vivo gene functions and the genetics of human diseases. Indeed, for the past several decades, knockout (KO) mice have profoundly contributed toward the identification of the functions of genes underlying human diseases. Gene-targeting technologies using embryonic stem (ES) cells are traditionally used to produce KO mice,28 but the process is extremely long due to several time-consuming and expensive steps, such as the culture and expansion of ES cells and the production of chimeric mice. In particular, homologous recombination in ES cells is a very rare event (<0.01%), and several rounds are required to achieve the germline transmission of ES cells. Thus, alternative approaches that bypass the labor-intensive processes of complicated gene-targeting vector construction and ES cell manipulation have long been awaited. In the last few decades, more rapid and efficient methods to produce KO mice have been developed. The most successful approaches involve engineered nucleases, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs),29–32 which enable genome editing without the use of ES cells. ZFNs and TALENs are composed of sequence-specific DNA-binding modules and a nonspecific restriction endonuclease, Fok I. Once these nucleases are injected into the 1-cell mouse embryo, they can generate double-strand breaks at specific DNA sites in the genome, which are then repaired by error-prone nonhomologous end joining, resulting occasionally in either insertion or deletion mutations. When insertions or deletions are located within protein-coding exons, frameshift mutations are introduced, resulting in KO alleles. Although ZFNs and TALENs have promoted the field of genome editing, they have major limitations. For instance, ZFNs and TALENs require the time-consuming and costly assembly of DNA-binding module proteins, and therefore it is difficult to design a number of these nucleases that target different endogenous sites. Because Cas9-sgRNA can be targeted to a specific genomic locus, through the complementarity between the sgRNA guide and target DNA sequences,5 the CRISPR-Cas9 system is inexpensive and relatively easy to design and construct, as compared with ZFNs and TALENs, making it a powerful tool for creating genetic deletions. In 2013, several groups reported that the CRISPR-Cas9 system can be used for genome editing in mammalian cells,11–14 and subsequent studies demonstrated that the CRISPR-Cas9 system can be used for the efficient generation of KO mice.33,34 Although the microinjection of Cas9-sgRNA into embryos requires a relatively high skill level, recent studies reported the highly efficient introduction of Cas9-sgRNA into embryos by electroporation.35,36 The off-target effects of CRISPR-Cas9 remain a major concern for the use of this technology in genome editing. Several studies reported that modifications of the Cas9 nuclease enhance the DNA-targeting specificity. Cas9 nickase mutants, designed to create a pair of juxtaposed single-strand DNA nicks, achieved 200-fold to >1,500-fold greater 326

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specificity, as compared to the wild-type Cas9.37 A pair of catalytically inactive Cas9 (dCas9) proteins fused to the Fok I nuclease domain also exhibited higher specificity relative to the wild-type Cas9.38,39 Based on the structural information, Slaymaker et al.40 developed “enhanced specificity” SpCas9 mutants, which contain alanine substitutions to neutralize the positively charged residues within the non-target-strand groove and dramatically reduce off-target cleavage. Kleinstiver et al.41 developed a “high-fidelity variant” SpCas9 mutant, by reducing the nonspecific interactions between SpCas9 and the phosphate backbone of the target DNA strand. The marked reduction of the off-target effects along with the high on-target efficiencies was also confirmed in the “high-fidelity variant” SpCas9 system.41 Potential uses of the CRISPR-Cas9 system in mice

In addition to generating frameshift-derived KO mice, the CRISPR-Cas9 system offers many other applications, such as multiplex KO, conditional allele generation (floxed), and reporter gene insertion. In this section, we describe the general considerations of the CRIPSR-Cas9 system for genome editing in mice (Figure 2a–f). Point mutations and small insertions

One of the promising applications of the CRISPR-Cas9 system is the generation of subtle mutations, such as diseasecausing mutations. Traditionally, this requires a great deal of effort, including targeting vector construction, homologous recombination in ES cells, and confirmation of germline transmission in mice. In contrast, the CRISPR-Cas9 system provides a much simpler and faster method to introduce subtle mutations. When the Cas9-sgRNA and the singlestrand oligodeoxynucleotides (ssODNs) carrying the desired mutations are injected into embryos, the ssODNs are used as the homology-directed repair template.42,43 Importantly, ssODN templates with lengths up to 200 base pairs can be commercially synthesized, thus eliminating the need for elaborate targeting vector construction. The ssODNs normally contain 50- to 60-nucleotide homology arms on both sides of the target region. To prevent recleavage of the edited allele by Cas9, the introduced mutations should ideally be located proximally to the PAM sequence in the target site, which facilitates highly efficient genome editing.44–46 Using ssODNs, small epitope tags (V5, HA, FLAG, etc.) can also be introduced to the target sites, thus solving certain problems, such as the lack of specific antibodies to detect a protein of interest in vivo.34 Creating a floxed allele

If a conditional allele is desired, then the Cre recognition site LoxP or flippase recognition target site can be introduced using the CRISPR-Cas9 system with ssODNs.34 Separate sgRNAs and donor oligos are required to flank an exon with LoxP sequences. Importantly, these are independent targeting events and they might not occur on the same copy of the chromosome. Thus, the analysis of more embryos might be Kidney International (2017) 92, 324–335

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a

b

c Cas9

Cas9

+

Cas9

+

+

sgRNA1

sgRNA

sgRNA

NHEJ

LoxP1 LoxP2

sgRNA2

ssODNs

NHEJ

HDR LoxP1 Point mutation or small insertion

Frameshift mutation

d

e Cas9

LoxP2 Floxed allele

f Donor vector (GFP etc)

Cas9

+

Cas9

+

+

sgRNA1

+

sgRNA2

sgRNA1 sgRNA2

sgRNA3

sgRNA

NHEJ

NHEJ

HDR

Multiple mutations Large deletion

Large insertion

Figure 2 | Practical use of the clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated 9 (Cas9) system for genome editing in mice. The CRISPR-Cas9 system offers many targeting applications, including (a) basic knockout, (b) point mutation/small insertion, (c) flox allele, (d) large deletion, (e) large insertion, and (f) multiple mutations. (For details, see main text.) GFP, green fluorescent protein; HDR, homology-directed repair; LoxP, Cre recognition site; NHEJ, nonhomologous end joining; sgRNA, single-guide RNA; ssODNs, single-strand oligodeoxynucleotides.

necessary to achieve a conditional allele. In addition, it is possible that a large deletion between 2 LoxP sites might occur, due to simultaneous genome editing by each sgRNA and Cas9 complex. Although this event can be prevented by dividing the simultaneous LoxP insertion into 2 insertions, studies comparing the efficiency of producing conditional KO mice using the CRISPR-Cas9 system are currently limited, and further research is needed to identify the best way to generate conditional KO mice using the CRISPR-Cas9 system. Large deletions

The ablation of large chromosomal regions is occasionally an appealing experimental strategy. For example, this enables genotyping polymerase chain reaction to be performed, using primers designed to be located outside of the deleted region.34,47 Such deletions are difficult to achieve with conventional targeting techniques, which often require timeconsuming steps including homologous recombination in ES cells. In contrast, the CRISPR-Cas9 system offers a relatively simple way to generate such large deletions. Two sgRNAs are designed to be located outside of a region of Kidney International (2017) 92, 324–335

interest and to generate double-strand breaks flanking large intervening sequences, resulting in the ablation of large chromosome regions through nonhomologous end joining repair. The efficiency and the length of the deletion are influenced by the linear distance between the sgRNAs. Lee et al.48 used digital polymerase chain reaction techniques and reported that the efficiencies of 15 to 33 kilobases (kb), 230 to 835 kb, and 15 megabase deletions were 0.1% to 10%, 0.1% to 1%, and 0.03%, respectively. Indeed, in other published reports, the lengths of the deletions produced using this strategy were <10 kb,34,49,50 and thus further studies still needed to judge whether the CRISPR-Cas9 system can be applied to generate megabase deletions efficiently. Large insertions

Another practical application of the CRISPR-Cas9 system is to introduce large DNA sequences, such as green fluorescent protein and red fluorescent protein, at precise genomic regions. Traditionally, a donor vector plasmid containing >2 kb of homology arms on each side of the desired sequence is required for homologous recombination in ES cells. 327

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Importantly, the double-strand break generated by the CRISPR-Cas9 system increases the frequency of homologous recombination, which enables the homology arms to be shortened to around 500 base pairs to 1 kb, thus simplifying the construction of donor templates. Indeed, a large insertion can be obtained without using ES cells, by injecting the Cas9-sgRNA and a donor vector with shorter homology arms into mouse one-cell embryos.34,51 Despite these successful reports, the generation of a large insertion by the CRISPRCas9 system seems to be remarkably difficult to accomplish in a 1-step procedure in vivo. Therefore, some researchers have reevaluated the CRISPR-Cas9 system as a booster of homologous recombination in ES cells, followed by the traditional blastocyst injection and the generation of chimeric mice. A recent study showed that an anticancer agent, Scr7, increases the frequency of homologous recombination in both cultured cells and mouse embryos, by inhibiting nonhomologous end joining.52,53 Although these effects are influenced by the size of the desired DNA template,51 the inhibition of nonhomologous end joining might improve the production of knock-in mice. Multiplex modification

The genes that are essential for life are often functionally redundant, and the effects of a single gene mutation can be masked by the presence of another gene. For example, the cytochrome P450 proteins, a heterogeneous group of enzymes that catalyze various oxidative reactions in multiple organs, such as liver and kidney, consist of 102 and 57 protein-coding genes (divided into 18 mammalian P450 families) in mouse and human, respectively, and share innumerable cellular functions in our bodies.54 When targeting multiple genomic regions, ZFNs and TALENs require large fusion proteins, consisting of a DNA-binding domain and the Fok I nuclease, against each target genomic region. In contrast, in the CRISPR-Cas9 system, the sgRNAs can share the same Cas9 nucleases, enabling the targeting of multiple genes in a single step by the injection of multiple sgRNAs with Cas9 nucleases

into 1-cell embryos. Although these types of reports are currently limited (Table 1),55–61 the simultaneous targeting of multiple genes by the CRISPR-Cas9 system is likely to become widely used for understanding complex genetic events. Practical use of the CRISPR-Cas9 system in kidney research

Successful uses of the CRISPR-Cas9 system have recently been reported in the field of kidney research. Mandai et al.62 introduced a single nucleotide polymorphism into human kidney cell lines, using Cas9-sgRNAs and ssODNs. Genomewide association studies previously identified serine threonine kinase 39 (STK39), encoding the STE20/SPS-1-related proline/ alanine-rich kinase (SPAK), as 1 of the hypertension susceptibility genes. In addition, a recent meta-analysis confirmed the association of the STK39 intronic polymorphism rs3754777 with essential hypertension, although the biological function of this polymorphism in the development of hypertension has yet to be determined. The rs3754777G>A knock-in cell lines generated by Mandai et al.62 exhibited increases in STK39 mRNA and protein expression and enhanced phosphorylation of SPAK, leading to the increased phosphorylation of the Na-Cl cotransporter 1. They concluded that the common single nucleotide polymorphism rs3754777G>A mutation in STK39 caused hypertension through the SPAK-mediated activation of the Na-Cl cotransporter 1. Recent genomewide association studies have identified hundreds of genetic variants associated with kidney disease,63 and therefore a minor genomic change generated by the CRIPSR-Cas9 system might be a powerful tool to investigate the effects of such variants in the development of kidney disease. Xu et al.64 generated epitope-tagged mice to label the oddskipped related (Osr1) protein. Osr1 is strongly expressed in the nephrogenic mesenchyme and is known to play an important role in early kidney development. Recent studies have revealed that a variant of Osr1 in heterozygotes was associated with reductions in newborn kidney size and function, and the same variant of Osr1 in homozygotes was associated with neonatal lethality with congenital kidney

Table 1 | Multiple-gene KO mice generated by the CRISPR-Cas9 system Number of genes disrupted Double Triple Quintuple Double Triple Triple Double Double

Gene name

Transferred embryos (recipients)

Tet1, Tet2 Tyr, Gdf8, Hprt B2m, Il2rg, Prf1, Prkdc, Rag1 Irx3, Irx5 Slamf5, Slamf6, Slamf1 Slamf5, Slamf6, Slamf1 Tmem176a, Tmem176b Ramp1, Ramp2

144 (7) 60 (3) 89 (3) 105 150–250 Undescribed 375 48 (3)

Newborns (dead)

Pups with biallelic mutation in all targeted genes

Authors

Reference

31 (8) 9 4 27 23 23 81 10c

22 8 3 0a 14 2b 0 0

Wang et al. (2013) Fujii et al. (2014) Zhou et al. (2014) Hara et al. (2016) Hu et al. (2016) Huang et al. (2016) Lemoine et al. (2016) Sakurai et al. (2016)

33 55 56 57 58 59 60 61

B2m, beta-2-microglobulin; Cas9, CRISPR-associated 9; CRISPR, clustered regularly interspaced short palindromic repeat; Gdf8, growth differentiation factor 8 (myostatin); Hprt, hypoxanthine phosphoribosyltransferase 1; Il2rg, interleukin 2 receptor subunit gamma; Irx3, Iroquois homeobox 3; Irx5, Iroquois homeobox 5; KO, knockout; Prf1, perforin 1; Prkdc, protein kinase, DNA-activated, catalytic polypeptide; Rag1, recombination activating 1; Ramp1, receptor activity modifying protein 1; Ramp2, receptor activity modifying protein 2; Slamf1, signaling lymphocytic activation molecule family member 1; Slamf5, CD84; Slamf6, SLAM family member 6; Tet1, tet methylcytosine dioxygenase 1; Tet2, tet methylcytosine dioxygenase 2; tmem176a, transmembrane protein 176A; tmem176b, transmembrane protein 176B; Tyr, tyrosinase. a Due (probably) to embryonic lethal. b Includes mosaic. c 13.5 to 15.5 days post coitum embryo.

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defects.65,66 Xu et al.64 inserted a 2xTY1 epitope tag at the N-terminus of the endogenous Osr-1 in a single step, by injecting the Cas9-sgRNA with ssODNs into 1-cell embryos and successfully generated mice expressing 2xTY1 tagged Ors1. Using these mice, immunologic approaches revealed that Ors1 physically interacts with the nephrogenic transcriptional factor Wt1 in the early metanephric mesenchyme during kidney development. This study suggested the potential use of the CRISPR-Cas9 system to express, purify, and detect recombinant fusion proteins, even in vivo. In vitro organoid models of kidney disease with CRISPRCas9 have also been reported.67 Freedman et al.67 first established the protocols for generating kidney organoids from human pluripotent stem cells (hPSCs), using a 3-dimensional culture method. Next, they simply knocked out podocalyxin using the CRISPR-Cas9 system and investigated the function of podocalyxin during kidney organogenesis. Podocalyxin regulates tight junctions and cell morphology and is highly expressed in podocytes in the adult kidney. The podocalyxin-deficient organoids lacked the linear tracks of other podocyte proteins, synaptoposin and ZO-1, and had smaller gap widths between adjacent podocytelike cells, as compared to wild-type organoids, indicating the essential role of podocalyxin for proper junctional organization in podocytes. In addition, Freedman et al.67 generated the polycystic kidney disease 1 (PKD1) and PKD2 KO hPSCs, using the same system. Biallelic loss-of-function mutations in the PKD1 and PKD2 genes cause human PKD. After organogenesis, large cystlike structures were observed alongside the tubular compartments in the PKD1 and PKD2 KO organoids. Importantly, as the cysts were detected at a low rate (w6% of organoids), some improvements in the hPSC differentiation efficiencies might be required for a more promising approach to model cystogenesis in PKD in vitro. Nevertheless, these studies have highlighted a novel application of the CRISPRCas9 system for exploring gene functions in kidney organoids. Potential use of the CRISPR-Cas9 system in kidney research

One of the advantages of the CRISPR-Cas9 system is the lack of strain dependence in mouse production (Figure 3a). Many published papers have shown that phenotypes and disease susceptibilities can differ between genetic backgrounds, due to the presence of modifier gene loci.68 For example, the phenotypes of diabetic nephropathy in mice markedly depend on the strains,69–71 highlighting the importance of choosing suitable strains for modeling kidney diseases. However, for historical and practical reasons, most KO mice were generated using 129-derived ES cells and then backcrossed with C57BL/ 6 mice,72 which are time-consuming processes and restrict the choice of certain backgrounds for specific applications or disease modeling. The CRISPR-Cas9 system bypasses these laborious processes for ES cell manipulation and provides a direct approach to generate mice with preferred backgrounds. Conceivably, the strain-independent production of KO mice by the CRISPR-Cas9 system will be useful to establish new models suitable for kidney disease studies. Kidney International (2017) 92, 324–335

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Genome editing in rats using the CRISPR-Cas9 system represents another potential application for disease modeling (Figure 3b). The rat was the first mammalian species domesticated for scientific research, and it was the most widely used animal model for physiology, pharmacology, toxicology, nephrology, and drug discovery, before the development of gene-targeting technology in mice.73 The metabolism, physiology, and pathology of rats are more similar to humans than those of mice,74 and the larger size of rats facilitates the collection of specimens, such as urine, as well as more reliable surgical manipulations. There are many superior rat models for cardiovascular disease, diabetes, obesity, rheumatoid arthritis, and neurogenic disorders.75–78 Indeed, for example, spontaneous hypertension rats have contributed greatly to the development of antihypertension drugs, such as angiotensin-converting enzyme inhibitors and angiotensin-receptor blockers.75 In addition, there are many excellent rat models for kidney disease, such as Thy-1 nephritis, Heymann nephritis, puromycin aminonucleoside nephrosis, and 5/6 nephrectomy.79–83 These characteristic features of rats highlight their usefulness in biomedical research. However, over the past several decades, it was much more challenging to produce KO rats as compared to KO mice, because of the difficulty in establishing and manipulating rat ES cells. The emergence of engineered nucleases, such as ZFNs and TALENs, has alleviated this situation by circumventing the use of ES cells.74,84–86 Furthermore, the CRISPR-Cas9 system has accelerated the spread of gene targeting in rats.87–89 The muscular dystrophy model is a successful example of the use of the CRISPR-Cas9 system in rats. The mdx mice are traditionally used as a muscular dystrophy model,90–92 but their degenerative skeletal muscle phenotypes are relatively mild, as compared with human muscular dystrophy. In contrast, the dystrophin-deficient rats generated by the CRISPR-Cas9 system exhibit much more severe degenerative phenotypes and pathological progression in the skeletal muscle, suggesting the advantage of using rat models in muscular dystrophy research.93 The CRISPR-Cas9 system has also been applied in other species. Genetic variants in the APOL1 gene cause a high incidence of focal segmental sclerosis, a podocyte disorder, in African Americans. To clarify the pathogenic role of APOL1, the CRISPR/Cas9 genome editing of apol1 in zebrafish embryos resulted in podocyte loss and glomerular filtration defects.94 Recent studies showed the association between Sec61 translocon alpha 1 subunit (SEC6A1A) variants and autosomal-dominant tubulointerstitial kidney disease by a linkage analysis, and the developmental role of SEC6A1A was confirmed by CRISPR-mediated deletions in zebrafish.95 Currently, the number of animal models that mimic the pathophysiological features of human kidney diseases is limited, and thus genome editing in mice, rats, and other species by the CRISPR-Cas9 system will be helpful to establish new animal models suitable for nephrology research. 329

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a Species-independent b genome editing

Cas9

Strain-independent genome editing

c

d

Genetic disease therapy

Cas9

Cas9

+

Xenogeneic kidney organogenesis

Cas9

+

sgRNA

+

sgRNA

sgRNA

sgRNA

Blastocyst complement approach

Establishing new suitable animal models for kidney research

+

In vivo gene delivery using AAV, lentivirus, nanoparticle, etc.

Potential applications of the CRISPR-Cas9 system as a tool for treating kidney disease

Figure 3 | Applications of the clustered regularly interspaced short palindromic repeat (CRISPR)–associated 9 (Cas9) system in kidney research. The CRISPR-Cas9 system can be applied for strain-independent genome editing (a), species-independent genome editing (b), xenogeneic kidney organogenesis (c), and genetic disease therapy (d). AAV, adenoassociated virus; sgRNA, single-guide RNA.

Potential applications of the CRISPR-Cas9 system to treat kidney disease

Kidney transplantation is the ultimate renal replacement therapy, but the shortage of organs has hampered the wide application of this therapy. The CRISPR-Cas9 system might facilitate the generation of exogenous human organs from hPSCs in large animals (Figure 3c). Despite the successful growth of various types of tissues from hPSCs, organogenesis in vitro has remained a challenge for developmental biology and regenerative medicine.96,97 This is partly due to the difficulty in the in vitro replication of the complicated interactions between cells and tissues during organogenesis. In contrast, xenogeneic approaches in blastocyst complement have succeeded in generating exogenous organs from PSCs. Kobayashi et al.98 injected rat wild-type PSCs into pancreatogenesis-disabled Pdx–/– mouse blastocysts, to developmentally compensate for the absence of the pancreatic “developmental niche” and succeeded in generating a functional rat pancreas in the Pdx–/– mouse. This group also successfully generated an allogeneic kidney in the nephrogenesis-disabled Sal1–/– mouse, using the same methodology.99 Furthermore, these blastocyst complement approaches can generate an exogenous pancreas even in genetically engineered apancreatic cloned pigs,100 suggesting the possibility of producing human organs in large animals. However, genome editing in large animals classically depends 330

on somatic cell nuclear transfer, a complication that has thwarted the development of organogenesis research using large animals. The CRISPR-Cas9 system can be applied to large animals and has already been successfully employed in their genetic engineering with simple and efficient approaches.101,102 In addition, the CRISPR-Cas9 system eliminates the need to employ endogenous porcine retroviruses that may infect human cells, by targeting 62 loci in porcine cells simultaneously.103 Thus, genome editing in large animals using the CRISPR-Cas9 system might promote organogenesis research. However, an important issue of concern is that the exogenous PSCs might migrate into not only the targeted organ but also other organs and tissues, including the brain and gonads. Therefore, the proper control of the differentiation potential of the PSCs is absolutely required to overcome this ethical issue. It is also fascinating to employ the CRISPR-Cas9 technology as a therapeutic tool for treating human genetic diseases (Figure 3d). CRISPR/Cas9 may be used to correct causative gene mutations for therapeutic purposes. Recent studies have succeeded in delivering Cas9 and sgRNAs into mouse liver and muscle tissues by using adenoassociated virus as a vehicle and have demonstrated relatively effective gene targeting in vivo.91,92,104 Importantly, these deliveries of Cas9 to somatic tissues have been achieved by either the i.v. administration or i.p. injection of adenoassociated virus, Kidney International (2017) 92, 324–335

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indicating the potential use of an adenoassociated virus– mediated CRISPR-Cas9 system as a tool for the treatment of genetic disorders. A representative genetic disease in nephrology is autosomal dominant polycystic kidney disease, but there are numerous other critical genetic kidney diseases, such as atypical hemolytic uremic syndrome, Alport syndrome, and so on. Genetic variants of disease-modifying genes represent another potential therapeutic target, as recent genomewide association studies have revealed numerous genes involved in the pathogenesis of kidney disease. The CRISPR-Cas9 systems have many concerns to be addressed, before they can be used for human therapies. The biggest safety concern is precise genome editing. In 2015, a group of Chinese scientists first published the use of CRISPRCas9 system in human nonviable embryos to disrupt the human b-globin gene and found a surprising number of off-target mutations assumed to be introduced by the CRISPR-Cas9 system.105 Following this, another Chinese group also used the CRISPR-Cas9 system in human embryos (which were also not viable) and successfully modified the targeted locus with relatively low efficiency. This group did

a

Transcriptional regulator

not detect any off-site targeting, probably because they only checked 28 of the predicted off-target sites.106 Recently, an advisory committee at the U.S. National Institutes of Health approved a proposal to use CRISPR-Cas9 to engineer immune cells for cancer therapy. This first trial is small and designed to test whether the CRISPR-Cas9 system is safe for use in humans. Undoubtedly, the CRISPR-Cas9 system will be extensively used in human research and clinical situations, and thus further studies are warranted to understand the mechanisms through which CRISPR-Cas9 mediates precise genome editing. A regulatory framework for the use of CRISPR-Cas9 in human studies is obviously important, to meet both technical and ethical requirements. Further applications of the CRISPR-Cas9 system

The ability of Cas9 to target specific genomic regions has been exploited for various applications beyond genome editing. dCas9 has been developed as an RNA-guided DNA targeting platform, to manipulate and probe a specific genomic region (Figure 4a–d). The utility of dCas9 as a tool for sequence-specific gene repression was first demonstrated in bacteria.107 The dCas9-sgRNA complex by itself can repress

b VP16, VP48, etc.

Histone-modifying enzymes DNA-modifying enzymes

Epigenetic regulator

KRAB, etc. ddCas9

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GFP, etc.

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RFP, etc.

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Figure 4 | Catalytically inactive Cas9 (dCas9) as a versatile tool for manipulating and labeling the genome without altering genome sequences. (a) dCas9 fused to transcriptional activation domains, such as VP16 and VP48, or to repressor domains, such as Kruppel-associated box (KRAB) proteins, can function as a transcriptional regulator. (b) dCas9 fused to DNA-modifying enzymes and histone-modifying enzymes can function as an epigenetic regulator. (c) Fluorescent proteins, such as green fluorescent protein (GFP) and red fluorescent protein (RFP), can be fused to dCas9, allowing the imaging of specific genomic loci in living cells. (d) Tag proteins, such as FLAG, V5, and myc, can be attached to dCas9. Antitag antibodies can be used to capture the target genome sequence with its associated DNAs, RNAs, and proteins. These methods are called engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP).

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gene expression by interfering with transcriptional elongation by RNA polymerase and by blocking transcription factor binding.107–109 This approach was highly efficient in suppressing gene expression in bacteria, but only a modest repression was achieved in mammalian cells.107,110 To increase the efficiency of dCas9-mediated gene repression, the transcriptional repressor domain Kruppel-associated box was fused to dCas9, and the fusion protein repressed the reporter gene expression as well as the endogenous gene expression of transferrin receptor CD71 and C-X-C chemokine receptor type 4 (CXCR4) in mammalian cells.110 Similarly, dCas9 has also been applied as a transcription activator. The fusion of the 4 tandem copies of VP16 (VP64) or p65 activation domain can activate both reporter and endogenous gene expression in mammalian cells.110–113 However, these approaches usually require the use of multiple sgRNAs to accomplish significant activation of endogenous genes. Tanenbaum et al.114 have developed a repeating peptide array called SunTag, which can recruit multiple copies of an antibody-fusion protein. They used the SunTag system to increase the efficiency of dCas9-mediated gene activation by recruiting multiple copies of VP64 and confirmed the strong activation of endogenous CXCR4 gene expression with a single sgRNA.114 Chavez et al.115 have also improved the dCas9-mediated gene activation system by fusing 3 different transcriptional activation domains, VP64, p65 activation domain, and the Epstein-Barr virus R transactivator Rta (VPR), to dCas9. As compared to the dCas9-VP68 fusion protein, the dCas9-VPR fusion protein showed 22-fold to 320-fold greater activation of endogenous genes.115 Furthermore, Konermann et al.116 demonstrated that multiple endogenous genes can be activated simultaneously with high efficiency, using (i) dCas9, (ii) an engineered sgRNA, in which the MS2-binding aptamers are fused to the surface-exposed tetraloop and stem loop 2, and (iii) multiple transcription activation domains fused with the MS protein. In addition, dCas9-based epigenome editing tools have recently been developed.117,118 For example, dCas9 fused to the catalytic core of the human histone acetyltransferase p300 can activate target gene expression, by catalyzing the acetylation of histone H3 Lys27 at both the promoter and enhancer regions. Interestingly, when targeted to the promoter regions, the dCas9-p300 fusion protein showed significantly higher levels of transactivation of the targeted genes than did the dCas9-VP64 fusion protein.118 More recently, Liu et al.119 demonstrated that the fusion of methylcytosine dioxygenase TET1 or DNA methyltransferase DNMT3a with dCas9 successfully altered the DNA methylation patterns. The targeted demethylation of the brain-derived neurotrophic factor promoter IV or the myogenic differentiation 1 distal enhancer by dCas9-TET1 increased brain-derived neurotrophic factor or MyoD expression, respectively, and the targeted methylation of transcriptional repressor CTCF motifs by dCas9-DNMT3a altered the CTCF-mediated gene loops. They further confirmed these systems can alter DNA methylation patterns, even in mice. 332

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Finally, dCas9 fused to fluorescent proteins can facilitate the visualization of specific genomic loci in living cells. The dCas9–green fluorescent protein fusion protein with a single sgRNA targeting repetitive elements in telomeres enabled the robust imaging of telomere dynamics in live retinal pigment epithelium cells or Hela cells.120 The visualization of a nonrepetitive sequence requires the use of multiple sgRNAs to target the genomic region of interest.120 The SunTag system might improve the repetitive sequence imaging by amplifying the fluorescent signals.114 Tag proteins, such as FLAG, V5, and myc, can also be attached to dCas9. Pull-down assays using the anti-Flag antibody to capture the dCas9-sgRNA complex with its associated DNAs, RNAs, and proteins (called engineered DNA-binding molecule-mediated chromatin immunoprecipitation) have been recently reported.121,122 In these ways, the potential applications of the CRISPR-Cas9 system are evolving rapidly, and are no longer limited to genome editing. Conclusion

The recently developed CRISPR-Cas9 systems enable the precise modification of the genome in a great variety of organisms and cell types with considerable ease. These systems offer a platform to disrupt 1 or multiple genes, create floxed alleles, and generate endogenously tagged proteins even in vivo. dCas9 also serves as a powerful tool for manipulating (transcriptional control or epigenetic regulation) and labeling a specific genomic region beyond genome editing. The rapid pace of technical improvements and the development of new applications will undoubtedly make the CRIPSR-Cas9 system an integral part of kidney research in the future. DISCLOSURE

All the authors declared no competing interests. ACKNOWLEDGMENTS

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