ZOOLOGICAL RESEARCH In vivo genome editing thrives with diversified CRISPR technologies Xun Ma1, Avery Sum-Yu Wong1, Hei-Yin Tam1, Samuel Yung-Kin Tsui1, Dittman Lai-Shun Chung1, Bo Feng1,2,3,* 1 Key Laboratory for Regenerative Medicine in Ministry of Education, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Hong Kong SAR, China 2 Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou Guangdong 510530, China 3 SBS Core Laboratory, CUHK Shenzhen Research Institute, Shenzhen Guangdong 518057, China ABSTRACT replace a selected endogenous genome DNA segment with a foreign DNA donor carrying homology sequences in living cells Prokaryotic type II adaptive immune systems have (Vasquez et al., 2001). Subsequently, by combining this with been developed into the versatile CRISPR mouse embryonic stem cell (ESC) technology established at technology, which has been widely applied in site- the same time, traditional gene targeting technology was specific genome editing and has revolutionized developed to generate genetically modified mice (Koller et al., biomedical research due to its superior efficiency 1989). Since 1989, genetic modification by HR-based gene and flexibility. Recent studies have greatly diversified targeting in living mammals has become a fundamental CRISPR technologies by coupling it with various approach to analyze gene functions and has revolutionized our DNA repair mechanisms and targeting strategies. understanding of mammalian development, metabolism, and These new advances have significantly expanded genetic diseases (Capecchi, 2005; Koller et al., 1989). the generation of genetically modified animal Traditional HR-based gene targeting is associated with low models, either by including species in which targeted efficiency and requires laborious clonal expansions and genetic modification could not be achieved sophisticated selections to identify target cells carrying the previously, or through introducing complex genetic desired modifications (Koller et al., 1989). With pioneering studies modifications that take multiple steps and cost years finding that the introduction of double-strand breaks (DSBs) in to achieve using traditional methods. Herein, we target DNA by rare-cutting endonuclease I-Sce-1 could increase review the recent developments and applications of HR efficiency by several orders of magnitude in the subsequent CRISPR-based technology in generating various DNA repair process (Rouet et al., 1994), extensive effort has animal models, and discuss the everlasting impact of been made to develop programmable endonucleases. 1 this new progress on biomedical research. Zinc finger nuclease (ZFN), which was first reported in 1986 as an artificial nuclease to carry a zinc finger domain and a catalytic Keywords: CRISPR/Cas9; Genome editing; Animal domain from restriction enzyme FokI, was suitable for introducing models DNA cleavage and enhancing HR-dependent gene targeting (Bibikova et al., 2002; Kim et al., 1996). However, the laborious INTRODUCTION work involved in the design and identification of an efficient ZFN to a newly selected target sequence significantly limited its utility. Genome editing by manipulating functional DNA sequences in Transcription activator-like effector protein (TALE), which the host genome is a fundamental strategy for biomedical research. Starting from the discovery of the basic principles of Received: 17 September 2017; Accepted: 17 November 2017 DNA structure and genome organization, scientists have Foundation items: This study was supported by funds provided by the investigated various strategies for many decades to improve Research Grants Council of Hong Kong (CUHK 14104614, genome editing technology for different research and application purposes. TBF16ENG007 and TBF17MED002 to B.F.; and 3132966 to W.Y.C.), In the 1980s, gene targeting methods emerged together with and in part by funds from the Croucher Foundation (CAS16CU01/ a deepening understanding of DNA repair mechanisms. Back CAS16401 to W.Y.C.) and the National Basic Research Program of then, DNA conversion was found to occur between homology China (973 Program, 2015CB964700 to Y.L.) sequences, often termed homologous recombination (HR) (Zinn *Corresponding author, E-mail: [email protected] & Butow, 1985). Early studies took advantage of this finding to DOI:10.24272/j.issn.2095-8137.2017.012 58 Science Press Zoological Research 39(2): 58-71, 2018 originated in plant pathogen Xanthomonas sp., was found to has been demonstrated in large mammals such as pigs and recognize target DNA with highly conserved yet variable repetitive monkeys to establish disease or genetic models for organ elements, each showing a preference to bind to specific transplantation (Niu et al., 2014; Yu et al., 2016). At the same time, nucleotides (Boch et al., 2009; Moscou & Bogdanove, 2009). CRISPR/Cas9 technology has also been applied in various lower Fusion of the programmable TALE domains and FokI catalytic vertebrate and invertebrate models (Irion et al., 2014; Shi et al., domain thus yielded TALE-nuclease (TALEN), which is easier to 2015; Wen et al., 2016). The success of CRISPR technology is construct and can introduce DNA cleavage and targeted genome particularly valuable in lower vertebrate models, such as Xenopus modification equally efficiently as ZFN (Christian et al., 2010). and zebrafish (Irion et al., 2014; Shi et al., 2015), in which More recently, an RNA-guided DNA-targeting approach was targeted genome editing could not be achieved previously. developed from the type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR) adaptive Molecular mechanisms for various genome editing strategies immune system (Bhaya et al., 2011; Wiedenheft et al., 2012). In Sequence-specific DNA cleavage induced by any of the above this system, a programmable small guide RNA (sgRNA) engineered nucleases will elicit endogenous cellular responses complexes with Cas9 nuclease and anneals with a 20-nt target to repair the damaged DNA in target cells. Utilizing various DNA DNA sequence, at the presence of the adjacent NGG PAM repair mechanisms to induce mutations/deletions or to incorporate (proto-spacer adjacent motif) sequence in a base-pairing insertions of foreign DNA lays the foundation for genome manner. This process allows Cas9 to introduce DSB at the editing. Cellular repair of DNA damage is mediated by two main target region and enables genome modification in a site-specific pathways, namely, homology-directed repair (HDR) and non- manner (Jinek et al., 2012). The ease of constructing a homologous end joining (NHEJ). Despite their varied activities sequence-specific sgRNA and the highly specific RNA-DNA in different cell types and species, both pathways are highly recognition has made the CRISPR/Cas9 system superior to conserved, from yeasts to mammals (Taylor & Lehmann, 1998). ZFN and TALEN, becoming the most popular tool for The HDR pathway mediates a strand-exchange process to introducing programmed DNA cleavage as well as site-specific repair DNA damage based on existing homologous DNA genome modifications in cells and animals (Barrangou & sequences (Heyer et al., 2010), allowing precise insertion of Doudna, 2016; Mali et al., 2013; Ran et al., 2013). foreign DNA at target regions by replacing endogenous These recent advances in engineered nucleases, especially genomic segments with donor DNA. CRISPR/Cas9-introduced the CRISPR/Cas system, have opened new prospects for site-specific DNA cleavage triggers DNA repair and greatly accomplishing robust gene targeting in previously non-permissive promotes HR at nearby regions, thus enhancing the efficiency cell contexts. More importantly, it has widely revolutionized of HDR-based genome editing (Yang et al., 2013). In contrast, biomedical research by promoting quick generation of various the conventional NHEJ pathway initiates DNA repair with quick animal models, which either carry complex genome modifications occupation by the Ku70/Ku80 complex at DNA broken ends, or are derived from species that could not be genetically modified followed by recruitment of other components for end processing previously (Dow et al., 2015; Swiech et al., 2015; Yin et al., 2016). and subsequently DNA ligase IV for ligation. NHEJ-based DNA Such progress has provided a wide range of methods as well as repair is a homology-independent and mechanistically flexible advanced animal models to study gene function and biological process, which often results in random insertions or deletions processes, significantly promoting research under in vivo (indels) of a small number of nucleotides (Lieber, 2010). Hence, conditions. Hence, in this review, we focus on summarizing the CRISPR/Cas9-induced NHEJ repair has been employed to recent developments and applications of CRISPR-based generate loss-of-function alleles in protein-coding genes (Wang technology in generating various animal models. et al., 2013). In general, the NHEJ pathway mediates rapid DNA repair and plays an important role in various cellular OVERVIEW OF RECENT DEVELOPMENTS IN CRISPR- contexts. Therefore, CRISPR/Cas9-induced NHEJ repair offers BASED ANIMAL MODELS high efficiency and has been exploited to develop a variety of targeting strategies. Since early 2013, when the first successful CRISPR-based More recently, in addition to the conventional HDR and NHEJ genome editing was demonstrated in mammalian cells (Mali et pathways, studies have discovered the microhomology-mediated al., 2013), the number of studies using the CRISPR system has end joining (MMEJ) pathway, which is also termed as alternative grown dramatically. Among the CRISPR-based in vivo studies, NHEJ (Alt-NHEJ) pathway (Lieber, 2010; McVey & Lee, 2008). the majority (61.2%) have been conducted using mouse models This MMEJ pathway repairs DNA damage by initiating single- (Figure 1, left panel). With the comprehensive knowledge and strand resection similar to the HDR process, followed by technologies established so far, research investigations using microhomology-based alignment and ligation by DNA ligase III. In CRISPR technology in mouse models have covered various general, the MMEJ pathway mediates an error-prone repair areas of biomedical research, including inherited metabolic process and plays a minor role to complement DNA repair by disorders (Xue et al., 2014; Yang et al., 2016), cancer (Maddalo HDR and NHEJ (McVey & Lee, 2008). Collectively, the coupling et al., 2014; Platt et al., 2014), neurology and neuroscience (Li of different DNA repair mechanisms with various strategies to et al., 2015c; Swiech et al., 2015), and virus-related studies design donor templates or select target sites in genomes, has (Jiang et al., 2017; Zhu et al., 2016). resulted in a variety of targeting approaches, each having distinct In addition to mouse models, CRISPR-based genome editing advantages in different species (Table 1). Zoological Research 39(2): 58-71, 2018 59 Table 1 Summary of CRISPR-based in vivo genome editing in zygotes via different DNA repair mechanisms in different species Genome modifications and Species ESCs Efficiency*# References targeting strategies involvement NHEJ-based knockout by Mouse: introducing indels Multiple genes No 50%–80% Dow et al., 2015; Mandasari et al., 2016; Swiech et al., 2015; Wang et al., 2013; Xu et al., 2017 Single gene 8%–90% Challa et al., 2016; Hay et al., 2017; Helsley et al., 2016; Hinze et al., 2017; Ishikawa-Fujiwara et al., 2017; Jiang et al., 2017; Kasparek et al., 2016; Kim et al., 2017b; Li et al., 2015d; Mandasari et al., 2016; Meyer et al., 2016; Mianné et al., 2017; Miyata et al., 2016; Sweeney et al., 2017; Wang et al., 2015a; Zhang et al., 2017b; Zhong et al., 2015; Zhu et al., 2016 Rat: Single gene No 28.6%–45.5% Rannals et al., 2016; Wang et al., 2016c; Yoshimi et al., 2014 Pig: Multiple genes No N/A Wang et al., 2016a Single gene 60%–83%% for embryos Park et al., 2017; Petersen et al., 2016; Wang et al., 2015e; Yu et al., 2016 50%–100% for offspring Monkey: Single gene No 10.2% for offspring Niu et al., 2014 Sheep: Single gene No 37.4%–87.6% for embryos Crispo et al., 2015; Li et al., 2017b; Niu et al., 2017a; Zhang et al., 2017a 59.1%–83.3% for offspring Goat: Single gene No 15%–28.6% Malpotra et al., 2017; Wang et al., 2015c; Zhou et al., 2017 Zebrafish: Single gene No ~32.9% Ablain et al., 2015; Anelli et al., 2017; Fujii et al., 2016; Gallardo et al., 2015; Gui et al., 2017; He et al., 2015; Homma et al., 2017; Hoodless et al., 2016; Lee et al., 2016; Narayanan et al., 2016; Perles et al., 2015; Shah et al., 2015; Varshney et al., 2015; Vejnar et al., 2016; Wang et al., 2014; Yuan et al., 2016a; Zhang et al., 2014 Drosophila: Multiple genes No N/A Port et al., 2014 Single gene Bassett et al., 2014; Gao et al., 2015; Wakabayashi et al., 2016 Rabbit: Single gene No 98.7% for embryos Lv et al., 2016; Yuan et al., 2016b 100% for offspring Mosquito: No N/A Dong et al., 2015 NHEJ-based knockout via Mouse Yes 10%–90% Han et al., 2014; Kraft et al., 2015; Seruggia et al., 2015; deletion Wang et al., 2015a，2017; Zhang et al., 2016 NHEJ-based knock-in Zebrafish No 4%–54% Auer et al., 2014; Hisano et al., 2015; Kimura et al., 2014; Li et al., 2015a Frog No 8%–12% Shi et al., 2015 Sheep No 34.7% Ma et al., 2017 HDR-based knockout Drosophila No 47% Wen et al., 2016 HDR-based knock-in Mouse: dsDNA No 10%–88% Aida et al., 2015; Chu et al., 2016; Guo et al., 2016; Han et al., 2015; Ishizu et al., 2016; Lewis et al., 2016; Li et al., 2016; Mashiko et al., 2014; Wang et al., 2015b; Wu et al., 2013 ssODN 6%–66% Inui et al., 2014; Zhu et al., 2017 60 www.zoores.ac.cn Continued Genome modifications and Species ESCs Efficiency*# References targeting strategies involvement HDR-based knock-in Rat: ssODN No 7.7% Yoshimi et al., 2014 Pig: dsDNA No 5%–18.2% Peng et al., 2015 ssODN 28.6%–80% Zhou et al., 2016 Goat: ssODN No 24% Niu et al., 2017b Zebrafish dsDNA No 1.7%–3.5% Irion et al., 2014 C. elegans: ssODN No 4.9%–62.8% Paix et al., 2016 Drosophila No 4.3%–10.8% Li et al., 2015e; Lin & Potter, 2016; Liu et al., 2016; Ukken et al., 2016; Voutev & Mann, 2017; Yu et al., 2014 Frog No N/A Sakuma et al., 2016 MMEJ-based knock-in Zebrafish No N/A He et al., 2015 Mouse No 12% Aida et al., 2016 *: Data were converted into percentages, without normalization or additional statistical analysis. #: Data presented were based on records at the offspring stage, if stage not indicated. genome targeting in zygotes has also overcome the limitations Enhanced genome editing via CRISPR-induced HDR of ESC unavailability, and made genome editing possible in HDR is a major DNA repair mechanism broadly employed in many previously inaccessible organisms, such as pigs and CRISPR-based genome editing (Heyer et al., 2010). In the monkeys (Peng et al., 2015). Furthermore, the introduction of presence of Cas9 nuclease and specific sgRNA targeting a site-specific DNA breaks allows the use of much shorter selected sequence in the genome, site-specific DNA cleavage homology arms to achieve successful genetic modifications. is introduced at the target genomic locus, which then will trigger Around 1 000 bp homology fragments are usually sufficient, and DNA repair. When the target cells are given a large quantity of around 100 bp single-stranded oligodeoxynucleotides (ssODN) donor templates carrying homology sequences, HDR-based carrying a 50–60 nt homology sequence at each side are repair will utilize the donors as templates to repair the damaged effective in introducing small mutations/insertions to produce genome, thus introducing foreign DNA included in the donor genetically modified animals (Inui et al., 2014; Zhou et al., construct into the recipient genome (Heyer et al., 2010). 2016). The traditional gene targeting approach succeeded before CRISPR-coupled HDR-mediated in vivo genome editing has the establishment of engineered nucleases. To accomplish been broadly used to introduce knock-in or knockout in the sequence replacement in the genome, this approach relies on genome of various animal models for studying gene functions, the HDR repair process triggered by spontaneous DNA damage modeling diseases, or developing novel treatment by correcting that randomly occurs near target regions, (Koller et al., 1989). disease-associated mutations. Direct injection of Cas9 mRNA, The desired targeting events occur at low frequency. Hence, sgRNA targeting only the mutant allele, and donor ssODN successful genome targeting requires long homology arms in carrying a wild-type allele sequence into mouse zygotes donor constructs, and needs sophisticated selection and clonal carrying a heterozygous dominant-negative cataract-causing expansion in mouse ESCs before generating chimeric animals mutation in the Crygc gene resulted in cataract-free progeny and genetically modified offspring (Koller et al., 1989; Thomas & (Wu et al., 2013). Besides rodents, large animals like pigs have Capecchi, 1987). It often takes more than one year to establish also been used for disease modeling (Peng et al., 2015; Wang a knock-in or knockout strain of mouse. et al., 2015d; Zhou et al., 2016). In these studies, together with Site-specific DNA breaks trigger DNA repair around a target the use of the single blastocyst genotyping system and/or region. Hence, coupling this to the CRISPR system can greatly ssODN donors, researchers can assess sgRNA efficiency at enhance the efficiency of HDR-based genome targeting and the embryonic stage and achieve up to 80% targeting efficiency result in a high success rate of desired targeting. This in producing animals carrying the desired genetic modification. improvement has bypassed the usage of ESC cells, allowing Furthermore, successful targeting has also been reported in direct genome targeting in mouse zygotes (Yang et al., 2013). lower vertebrates and invertebrates (Irion et al., 2014; Li et al., The direct genome targeting in zygotes via CRISPR-coupled 2015e; Lin & Potter, 2016; Liu et al., 2016; Paix et al., 2016; HDR can produce a high percentage of chimeric animals and Sakuma et al., 2016; Ukken et al., 2016; Voutev & Mann, 2017; genetically modified mouse strains within 3–6 months, a much Yu et al., 2014). Targeted gene modification and tagging has shortened period of time (Yang et al., 2013). Moreover, direct been achieved in Drosophila based on the CRISPR/Cas9- Zoological Research 39(2): 58-71, 2018 61 coupled HDR approach (Li et al., 2015e; Lin & Potter, 2016; Liu Gene correction in somatic tissues has also been performed et al., 2016; Ukken et al., 2016; Voutev & Mann, 2017; Yu et al., using the CRISPR system and donor DNA (Table 2). Targeting 2014), with a similar method also applied in zebrafish, of deficient ornithine transcarbamylase in the mouse model producing up to 50% targeted mutations in larvae (Irion et al., showed more than 10% correction of the deficient gene in liver 2014). With modified ssODN templates and CRISPR cells and significantly improved the survival rate in target groups components, gene editing efficiency has reached 85% in C. (Yang et al., 2016). Similarly, somatic correction of Duchenne elegans (Paix et al., 2016). Targeted genes or long noncoding muscular dystrophy (DMD) caused by a mutation in the gene RNA (lncRNA) can be precisely replaced with fluorescence encoding dystrophin has been reported, showing a 70% reporters to deplete target genes by inserting visible markers increase in functional dystrophin and apparent improvement in (Platt et al., 2014; Wen et al., 2016). the mouse model (Bengtsson et al., 2017). Table 2 Summary of CRISPR-based in vivo genome editing in somatic tissues Genome modifications and Species Delivery system Efficiency*# References targeting strategies NHEJ-based knockout via Mouse Virus 14.8%–86% Cheng et al., 2014; Chiou et al., 2015; de Solis et al., 2016; indel formation Ding et al., 2014; El Fatimy et al., 2017; Guo et al., 2017; Heckl et al., 2014; Hung et al., 2016; Kaminski et al., 2016; Kim et al., 2017a; Li et al., 2017a; Monteys et al., 2017; Ortinski et al., 2017; Tabebordbar et al., 2016; Wang et al., 2015a, 2016b; Yin et al., 2017 Hydrodynamic injection N/A Liang et al., 2017; Weber et al., 2015; Xue et al., 2014 Electroporation N/A Kalebic et al., 2016; Latella et al., 2016; Maresch et al., 2016; Shinmyo et al., 2016; Straub et al., 2014 Cell injection 90% Courtney et al., 2016; Katigbak et al., 2016; Wu et al., 2017 Chicken Electroporation N/A Véron et al., 2015 NHEJ-based knockout via Mouse Virus N/A Long et al., 2016; Nelson et al., 2016 deletion Hydrodynamic injection 30% Pankowicz et al., 2016 HDR-based knockout Mouse Virus 85% Platt et al., 2014 HDR-based knock-in Mouse Virus 2.3%–6% Bengtsson et al., 2017; Xie et al., 2016; Yang et al., 2016; Yin et al., 2016 Electroporation Chen et al., 2016 Cell injection Ou et al., 2016 MMEJ-based knock-in Mouse Virus 20% Yao et al., 2017 NHEJ-based knock-in Mouse Virus 3.4%–10% Suzuki et al., 2016 Chromosomal rearrangement Mouse Virus N/A Blasco et al., 2014; Maddalo et al., 2014 *: Data were converted into percentages, without normalization or additional statistical analysis. #: Data presented were based on records at somatic tissue level, if stage not indicated. introduce loss-of-function effects (Figure 1). To date, most Diverse targeting strategies through CRISPR-induced animal models established using CRISPR technology have NHEJ-mediated DNA repair employed this strategy to knockout a specific gene, especially Double-strand DNA breaks due to the disruption of model organisms that are incompatible with the traditional HDR- phosphodiester bonds between adjacent nucleotides in double- based strategy, such as zebrafish or Xenopus (broadly noticed helix DNA. While HDR repairs a broad range of DNA damage, via personal communications) (Table 1 and 2) (Auer & Del NHEJ is the primary mechanism for repairing DSBs in Bene, 2014; Irion et al., 2014; Won & Dawid, 2017). mammalian cells. With site-specific DSBs able to be introduced Furthermore, due to its simple principles and procedures, at almost any target site in the genome with high efficiency and CRISPR-NHEJ-based mutagenesis has been applied in high- accuracy using the CRISPR system, the NHEJ repair throughput studies. Xu et al. reported successful loss-of- mechanism has been broadly employed to introduce random function screening to identify genes essential to tumorigenesis mutations at selected target sites. This CRISPR-coupled NHEJ- in mice using pre-constructed sgRNA libraries (Xu et al., 2017). based mutagenesis approach can disrupt protein coding Interestingly, in vivo application of a sgRNA library has also potential of a target gene by causing frame shift or premature been reported in zebrafish (Shah et al., 2015). Combining termination, and therefore deplete functional proteins and CRISPR-based high-throughput screening with excellent 62 www.zoores.ac.cn accessibility to embryonic development, straight-forward frequencies of NHEJ- and HDR-mediated knock-in after phenotyping has allowed large scale analysis of gene function. coupling with the CRISPR system (He et al., 2016). We found Shawn M. Burgess and colleagues have verified more than 50 that knock-in via CRISPR/Cas9-induced NHEJ is superior to genes by this method (Varshney et al., 2015), and Stefania the commonly used HDR-based method in all human cell lines Nicolia’s team has succeeded in a similar screening using the examined (He et al., 2016). This NHEJ-based knock-in sgRNA pool-targeting miRNA family (Narayanan et al., 2016). approach has been applied in precise reporter knock-in in In addition, NHEJ repair has been found to be highly efficient zebrafish (Auer et al., 2014; Hisano et al., 2015; Irion et al., in re-ligating DNA ends from DSBs concurrently produced by 2014; Kimura et al., 2014; Li et al., 2015a) and Xenopus (Shi et the CRISPR system at two different genome loci, despite the al., 2015), with such gene targeting previously impeded by the long distance in genome. In support of these observations, the deficiency of the HDR pathway. More recently, CRISPR/Cas9- CRISPR-coupled NHEJ repair mechanism has also been induced NHEJ has been shown to mediate high efficiency employed to delete selected large DNA fragments by targeting knock-in in mouse somatic tissues (Suzuki et al., 2016), but two regions in the same chromosome (Dow et al., 2015; Han et success in targeting zygotes or blastocysts to generate al., 2014; Wang et al., 2015b) or catalyzing the desired genomic genetically modified mice has not yet been reported. rearrangements by targeting two selected regions from different Through CRISPR-coupled NHEJ repair, various genome chromosomes (Blasco et al., 2014). These strategies have targeting strategies have been established and utilized in succeeded in generating mouse models carrying a 353-kb generating genetically modified animal models. From studies intragenic deletion of Laf4, which recapitulates a human published since early 2013, 75.9% (110/145) of in vivo genome malformation syndrome (Kraft et al., 2015), and engineering editing studies have employed NHEJ-based targeting strategies. mouse models that harbor chromosomal rearrangements Extensive evidence has shown that NHEJ-based genome recurrently found in lung cancer to model carcinogenesis targeting is simpler, more flexible, and more efficient compared (Blasco et al., 2014; Maddalo et al., 2014). The functional study with HDR-based approaches. Without homology sequences of lncRNA genes is another important application of NHEJ- involved, the design and system construction for NHEJ-based mediated large fragment deletion. Knockout of the lncRNA gene strategies are less laborious. On the other hand, however, the Rian through a large deletion of up to 23 kb demonstrated random nature of NHEJ repair incurs disadvantages including efficiency as high as 33% (Han et al., 2014) can be achieved, the unpredictability of indel-based mutagenesis as well as off- with similar results reported for the tyrosinase (Tyr) associated target cleavage and insertion. lncRNA gene (Seruggia et al., 2015). Rather strikingly, CRISPR-coupled NHEJ repair has also Genome editing by CRISPR-induced MMEJ repair enabled high-efficiency knock-in of exogenous DNA at pre- Distinct from NHEJ and HDR, the two common forms of DNA selected locations. This is consistent with common observations repair, MMEJ requires microhomologous sequences of only 5– that NHEJ is the predominant repair mechanism in mammalian 25 bp for the repair of DSBs in DNA. Sakuma et al. devised a cells. Since the early 1980s, transgenic technology has been detailed protocol for CRISPR-based gene knock-in using established and applied broadly to render stable ectopic MMEJ, termed Precise Integration into Target Chromosomes expression by introducing foreign DNA fragments carrying (PITCh) (Sakuma et al., 2016). complete gene cassettes into host genomes (Palmiter et al., In this system, DSBs are needed in both the genomic DNA 1982). Later studies have found that the NHEJ repair and donor vector to insert a DNA fragment from the donor into mechanism is responsible for capturing foreign DNA fragments the genome. As MMEJ repair requires the presence of at spontaneously occurring DSBs in the genome, resulting in microhomology both upstream and downstream of the DSB random integrations (Lin & Waldman, 2001). Consistently, site, two microhomologous sequences need to be added to the traditional gene targeting studies have also shown that the donor vector at both sides of the purpose sequence (Sakuma et frequency of random DNA integration via the NHEJ repair al., 2016). For the CRISPR system, two sgRNAs are required to mechanism is significantly higher (over 1 000-fold) than targeted generate DNA cleavages near the microhomology sequences insertion mediated by the HDR pathway (Vasquez et al., 2001). on both sides, while one sgRNA is used to induce DSBs on the Due to the unavailability of programmable site-specific genome DNA (Figure 2). Longer microhomologies of around 20 nucleases and their erroneous nature, the potential of the NHEJ bp are currently used to improve accuracy. After alignment mechanism in targeted DNA knock-in was largely neglected for between microhomologous sequences, the unmatched non- a long time. homologous sequences at the 3'-parts on both sides of the Until recently, after ZFN was successfully established, short donor appear as single-strand tails and are removed. This oligonucleotides (<100 bp) were able to be inserted efficiently at results in the loss of a small part of the genome sequence at ZFN-induced DSBs via NHEJ repair (Orlando et al., 2010). the target sites. Therefore, MMEJ-based genome editing is Subsequently, inclusion of a ZFN or TALEN target sequence in associated with deletion/insertions that are often larger than donor vectors showed that simultaneous cleavage of donor and NHEJ-introduced indels (Villarreal et al., 2012). genome DNA could enable targeted integration via NHEJ repair Targeted integration mediated by CRISPR-coupled MMEJ (Cristea et al., 2013; Maresca et al., 2013). Using promoterless has been demonstrated in cultured cells and the generation of fluorescence reporters followed by direct quantification using genetically modified zebrafish (He et al., 2015; Hisano et al., fluorescence-activated cell sorting (FACS), we compared the 2015; Nakade et al., 2014). Moreover, one-step knock-in of Zoological Research 39(2): 58-71, 2018 63 gene cassettes and floxed alleles has also been achieved in Although the intrinsic MMEJ pathway often plays a minor role in human cells and mouse zygotes through MMEJ (Aida et al., overall DNA repair, the MMEJ-based targeting strategy has 2016). Recently, precisely targeted gene integration in somatic shown efficiency up to 10-fold higher than that of the HDR- tissues to correct mutation of the Fah gene and rescue liver based approach (Yao et al., 2017). Lastly, the NHEJ repair failure in Fah−/− mice has also been demonstrated (Yao et al., mechanism, which is completely independent of any homology 2017). sequences, offers the easiest path to modify an existing design for a new target site in the genome. In our recent study, a Comparison between different targeting strategies universal donor was established with the use of artificial sgRNA Conventional NHEJ repair does not require the presence of ,which did not target any sequence in mice and humans (He et homology sequences and involves minimal processing of DNA al., 2016). With the minimum work involved in constructing the broken ends. The activity of the NHEJ pathway is high and new sgRNA to the genome, the whole system was easily stable throughout the cell cycle. Distinctly, the HDR repair orientated for targeting a new locus (He et al., 2016). However, mechanism relies on long homology sequences (> 500 bp in the random errors potentially present at the integration/ repair general) to repair DNA lesions, and is only active from the late junctions with NHEJ-based targeting approaches should be S phase to G2 phase during the cell cycle. The MMEJ pathway considered during the design. depends on microhomology sequences (5–25 bps) for DSB repair and is active during the M to early S phase (Taleei & CONCLUSIONS Nikjoo, 2013). These differences explain why the activities of the different DNA repair pathways vary in different cell contexts. The recent advent of CRISPR technology has offered the The intrinsic activities of the two major pathways, HDR and simplest and possibly ultimate solution for introducing site- NHEJ, also vary in different species, despite high conservation specific DSBs in genome DNA, which was once an of these pathways across a broad range of organisms. Lower insurmountable challenge in genome editing. Through coupling vertebrates, such as zebrafish and Xenopus, are deficient in with different DNA repair mechanisms present in the HDR-based DNA repair. Hence, modification of genome endogenous cellular system, various targeting strategies have sequences in these models has mainly succeeded with NHEJ- been developed to introduce a wide range of modifications in based strategies, such as transgenesis, indel-based targeted the genome through sequence-based editing. While further mutagenesis/deletion, or the recent knock-in approach based research is needed to evaluate the off-target issues and on coupling TALEN- or CRISPR-induced DNA cleavage to the overcome the risks by developing improved CRISPR systems, NHEJ repair mechanism (Auer et al., 2014; Hisano et al., 2015; the above technological advances have undoubtedly revolutionized Irion et al., 2014; Kimura et al., 2014; Li et al., 2015a; Shi et al., biomedical research. The CRISPR-based genome editing 2015) (Table 1). In mammalian systems, although HDR was approaches have significantly promoted studies on gene first employed to produce genetically modified mice, evidence function via the rapid generation of animal models that carry shows that the NHEJ repair mechanism is predominant genetic deficiencies of single or multiple genes. In addition, they (Vasquez et al., 2001). Thus, the efficiency of NHEJ-based have also enabled modeling of genetic diseases caused by genome editing is generally superior to HDR-based approaches chromosomal rearrangement or large deletions. Therefore, (He et al., 2016). rapid progress could be foreseen in establishing various animal Scientists have attempted to manipulate the balance between models for disease modeling or therapeutic intervention, which the HDR and NHEJ pathways. Through inhibiting DNA ligase IV, will significantly improve our understanding of human diseases a key component of the NHEJ pathway, studies have shown and promote the development of new therapeutic strategies. that the efficiency of HDR-based gene targeting can be increased substantially (Chu et al., 2015). Similarly, silencing COMPETING INTERESTS KU70, KU80, or DNA ligase IV largely suppressed NHEJ- mediated introduction of indels at the junction and enhanced The authors declare that they have no competing interests. HDR-mediated genome editing (Pierce et al., 2001). To date, this type of approach has not been applied for in vivo gene AUTHORS’ CONTRIBUTIONS targeting. Besides efficiency, accuracy is another major concern. The X.M. A.S.W., H.Y.T., S.Y.T, and D.L.C. wrote different parts of the manuscript; HDR-based targeting strategy requires homology sequences as B.F. compiled and revised the manuscript. All authors read and approved the a template for DNA replication to repair induced DNA cleavage. final manuscript. It involves the cloning of homologous DNA and multi-step construction of donor plasmids. In return, the designed ACKNOWLEDGEMENTS modifications can be introduced into the genome with high We thank Xiang-Jun He and Chen-Zi Zhang for critical comments on the accuracy and off-target integrations can be largely reduced manuscript. compared to other knock-in strategies. MMEJ-based targeting requires microhomologous sequences, which can be easily REFERENCES introduced into donor vectors through synthesized oligos, or during PCR amplification of the desired DNA for insertion. Ablain J, Durand EM, Yang S, Zhou Y, Zon LI. 2015. A CRISPR/Cas9 64 www.zoores.ac.cn vector system for tissue-specific gene disruption in zebrafish. Developmental Chiou SH, Winters IP, Wang J, Naranjo S, Dudgeon C, Tamburini FB, Brady Cell, 32(6): 756–764. 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