How To Ensure That Hdr Template Doesn't Disrupt Gene Expression

Front Genet. 2018; 9: 691.

Methodologies for Improving HDR Efficiency

Mingjie Liu

¹Higher of Veterinary Medicine, Northwest A&F University, Xianyang, China

Saad Rehman

¹College of Veterinary Medicine, Northwest A&F University, Xianyang, People's republic of china

Xidian Tang

¹College of Veterinarian Medicine, Northwest A&F University, Xianyang, China

Kui Gu

¹College of Veterinary Medicine, Northwest A&F Academy, Xianyang, Communist china

Qinlei Fan

^twoChina Brute Health and Epidemiology Centre, Qingdao, Cathay

Dekun Chen

^oneCollege of Veterinarian Medicine, Northwest A&F University, Xianyang, China

Wentao Ma

¹College of Veterinary Medicine, Northwest A&F University, Xianyang, China

Received 2018 Aug 31; Accepted 2018 December 11.

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated poly peptide ix (Cas9) is a precise genome manipulating applied science that can be programmed to induce double-strand pause (DSB) in the genome wherever needed. After nuclease cleavage, DSBs tin be repaired by non-homologous finish joining (NHEJ) or homology-directed repair (HDR) pathway. For producing targeted gene knock-in or other specific mutations, DSBs should exist repaired past the HDR pathway. While NHEJ tin can cause various length insertions/deletion mutations (indels), which tin lead the targeted gene to lose its function past shifting the open reading frame (ORF). Furthermore, HDR has low efficiency compared with the NHEJ pathway. In order to modify the gene precisely, numerous methods arose by inhibiting NHEJ or enhancing HDR, such every bit chemical modulation, synchronized expression, and overlapping homology arm. Hither we focus on the efficiency and other considerations of these methodologies.

Keywords: CRISPR-Cas9, HDR, NHEJ, HDR enhancement, DSB, cell arrest, NHEJ inhibitors

Overview of CRISPR-Cas9 System

Clustered regularly interspaced short palindromic repeats (CRISPR) represents a family of DNA sequences in bacteria and archaea (Barrangou, 2015). This family is characterized by direct palindromic repeats, where sequences are the same in both directions, varying in size from 21 to 37 bp (Barrangou and Marraffini, 2014), interspaced past spacers, which have fragments gathered from viruses or phages that previously tried to infect the prison cell (Horvath and Barrangou, 2010; Morange, 2015). To appreciate this characteristic array, it is termed CRISPR (Jansen et al., 2002; Mojica and Rodriguez-Valera, 2016). CRISPR-associated (cas) genes are invariably located side by side to a CRISPR locus (Jansen et al., 2002). The CRISPR-Cas organisation tin can exist grouped into 3 types: type I, type II, and type III. In add-on, there are 12 subtypes of the CRISPR-Cas system, which are based on their exclusive genetic content and structural differences (Makarova et al., 2015). Cas1 and cas2 are universal across types and subtypes, whereas cas3, cas9, cas10 are signature genes for type I, type 2, and type III, respectively (Makarova et al., 2011). Hither in this review we merely focus on type II. The CRISPR-Cas system functions equally a defense system in bacteria and archaea against bacteriophage infection, conjugation, and natural transformation past degrading strange nucleic acid that enters the cell (Marraffini, 2015). The CRISPR-Cas organization involves three distinct mechanistic stages: adaptation, biogenesis, and interference (Marraffini and Sontheimer, 2010). The adaptation stage involves the integration of fragments of foreign DNA (termed "protospacers," captured, excised, and inserted by Cas proteins) into the CRISPR assortment as new spacers. New spacers are usually added at the kickoff of the CRISPR locus next to the leader sequence, creating a chronological record of viral infections (Sorek et al., 2013) and protecting the cell from farther infection. During the biogenesis phase, the CRISPR assortment is transcribed as a single long transcript (termed "pre-crRNA") containing much of the CRISPR array (Marraffini and Sontheimer, 2010) and is so processed and matured to produce CRISPR RNAs (crRNAs) with only i spacer sequence. As for the interference phase, the spacers in these crRNAs guide cas proteins to foreign DNAs and cleave them (van der Oost et al., 2009; Wiedenheft et al., 2012; Barrangou, 2013). The type II CRISPR-Cas system needs simply cas9 to execute immunity in the presence of an existing targeting spacer sequence (Sapranauskas et al., 2011). It requires two small RNAs: the crRNA and the trans-encoded crRNA(tracrRNA) (Deltcheva et al., 2011). TracrRNA forms a secondary structure that interacts with cas9 protein and it has a complementary region that enables itself to bind to pre-crRNA (Anders et al., 2014; Jinek et al., 2014; Nishimasu et al., 2014). The dsRNA formed between pre-crRNA and tracrRNA is then handled by RNase III to form mature crRNA guides that are used in genome editing. When crRNA and tracrRNA are combined together, they are collectively termed as guide RNA (gRNA) (Jinek et al., 2012). Another important brusque (3–five bp) DNA termed protospacer next motif (PAM) is required for targeting. PAM is a component of the invading virus or plasmid, just it is non a component of the bacterial CRISPR locus. Cas9 volition non successfully bind to or cleave the target Deoxyribonucleic acid sequence if information technology is not followed past the PAM sequence (Mojica et al., 2009). The commencement step in target recognition is the transient bounden of Cas9 to PAM sequences within the target Deoxyribonucleic acid, which promotes the unwinding of the two Deoxyribonucleic acid strands immediately upstream of the PAM (Sternberg et al., 2014), the spacer sequence of the crRNA binds with the unwinded Dna (six–8 bp in length), then forms an RNA-Deoxyribonucleic acid heteroduplex and triggers cleavage at the targeted site (Sternberg et al., 2014; Szczelkun et al., 2014). Subsequently recognition, the CRISPR-Cas9 organization introduces a crRNA-specific DSB in the target sequence, which is further resolved either past homology-directed repair (HDR) or non-homologous terminate joining (NHEJ).

NHEJ Pathway

Typically, cells use 2 master mechanisms to repair DSBs: classical NHEJ and HDR (Symington and Gautier, 2011). In that location are as well many alternative error-decumbent DSB repair pathways: single-strand annealing (SSA) and breakage-induced replication (BIR) (Pardo et al., 2009; Jasin and Rothstein, 2013). SSA does not require a homologous template, and rejoining DNA ends with straight sequence repeats (Symington, 2014). BIR repairs one-ended DSBs, a procedure that is acquired by the collapse of a replication fork (Mayle et al., 2015). When DSBs occur in cells, the get-go reaction is usually carried out in an NHEJ manner. Compared to other DNA repair and DNA recombination pathways, the NHEJ pathway is a robust, error-prone but predominant and fast pathway with high flexibility. It can recognize various end structures at DSBs and reach diverse repair results (Aravind and Koonin, 2001; Gu and Lieber, 2008; Salsman and Dellaire, 2017). NHEJ can be classified into two types: canonical NHEJ (c-NHEJ) and alternative NHEJ (alt-NHEJ), likewise called microhomology-mediated end-joining (MMEJ) (Bae et al., 2014). c-NHEJ is agile throughout the cell cycle and stabilizes the DSB from translocations (Roth et al., 1995; Soutoglou et al., 2007). Based on dissimilar Deoxyribonucleic acid ends, NHEJ is capable of employing different strategies. The whole procedure deals with assembling the core complex, which recognizes broken ends and keeps them together so that the following processing factors can deed (Waters et al., 2014). The core complex is considered to include the Ku heterodimer (Ku80/70), the DNA-dependent protein kinase catalytic subunit (Deoxyribonucleic acid-PKcs), DNA ligase Four, the X-ray repair cross-complementing protein iv (XRCC4), the XRCC4-similar cistron (XLF, or Cernunnos). Ku is a heterodimer, composed of ii subunits (70 and 83 kD), that recognizes and binds to blunt DSBs first (Walker et al., 2001). In c-NHEJ, Ku recruits DNA-PKcs to the DSB site and forms a very stable complex that remains spring to the end (Weterings et al., 2003). Their associates activates the kinase activity of DNA-PK and orchestrates c-NHEJ (Davis et al., 2014; Radhakrishnan et al., 2014). Dna-PK phosphorylates a host of Deoxyribonucleic acid damage response proteins and thus regulates c-NHEJ and DSB processing and recruits Artemis nuclease (Moshous et al., 2001). Nonetheless, DNA-PK mostly phosphorylates itself, which is crucial in DSB processing (Neal et al., 2014). Artemis has v′ to three′ single-stranded DNA exonuclease activity and Dna-PKcs-dependent 5′ and three′ endonuclease activity on hairpins and unmarried-stranded overhangs (Moshous et al., 2001). Ku and DNA-PKcs alone can also promote multiple Deoxyribonucleic acid end-processing activities at the interruption site. The X family of DNA polymerases (politico mu and pol lambda) adds missing nucleotides at the DSB ends (Daley et al., 2005; Paull, 2005). Next, the DSBs volition be ligated by Ligase Four/XRCC4/XLF, which is regulated by DNA-PK. Ligase Four/XRCC4/XLF forms an extended filament that wraps and stabilizes DNA and stimulates ligation (Tsai et al., 2007; Andres et al., 2012). Recent research also showed that a newly identified PAXX (a paralog of XRCC4 and XLF), a fellow member of the XRCC4 superfamily, is another important mediator of c-NHEJ, which interacts directly with Ku. In well-nigh cases DNA is repaired via the c-NHEJ pathway and its efficiency tin can approach near 90% (Yang et al., 2013; Dow et al., 2015), which constitutes the basis of CRISPR/Cas9 technology (Vartak and Raghavan, 2015). The NHEJ process is illustrated in Figure 1.

An external file that holds a picture, illustration, etc. Object name is fgene-09-00691-g0001.jpg

Canonical not-homologous cease joining (c-NHEJ). After CRISPR-Cas9 introduced a DSB, NHEJ is initiated by the binding of the Ku heterodimeric complex. This and then forms the core complex, which is considered to recognize cleaved ends and keeps them together. The ends will then be ligated past various ligases.

HDR Pathway

HDR is a faithful repair pathway. It comes into action mainly in the Southward- or G2-phase of the jail cell cycle and requires homologous DNA sequences. Homologous recombination is the desired mechanism for precise genome editing, which just happens in the presence of a homologous duplex template to repair the broken site. When DSB occurs, pathway choice depends on end resection (Symington and Gautier, 2011). The MRE11-RAD50-NBS1 (MRN, MRX in yeast) complex recognizes dsDNA and first creates a nick 15–xx bp from the 5′-ends of the DSB (Symington, 2014). Exonucleases such equally SGS1-DNA2 and EXO1 complete the resection step (Kim and Mirkin, 2018). It then moves into flanking dsDNA regions and recruits ataxia telangiectasia mutated (ATM) kinase, the key upstream kinase of DSB signaling (Falck et al., 2005), and interacts with CtIP (Makharashvili and Paull, 2015). MRN also tethers Dna ends, which increases its local concentration and thus facilitates ATM activation (Dupre et al., 2006).

The MRN circuitous consists of 3 subunits. MRE11 is a Mn²⁺ dependent nuclease involved in homologous recombination, telomere maintenance, and Deoxyribonucleic acid DSB repair (Paull, 2015). SAE2 activates MRE11 for its dsDNA-specific endonuclease action (Cannavo and Cejka, 2014) and regulates the resection step during appropriate stages of the jail cell cycle (Mathiasen and Lisby, 2014). RAD50 belongs to the structural maintenance of chromosomes (SMC) family, and contains ATPase activity (de Jager et al., 2001). RAD50 becomes dimerized and its Dna-binding activity is activated after ATP binding. Two MRE11 genes will then bind to the ATPase heads of the RAD50 homodimer, enabling itself to collaborate with RAD50 (Williams et al., 2008). RAD50 forms the core of MRN and uses its extended coiled-coil domain to tether DSB ends during HR (Williams et al., 2010; Hohl et al., 2011). NBS1 contains a Fork-Head associated (FHA) domain and BRCA1 C-concluding (BRCT) domain, binds MRE11 and recruits ATM, linking the core MRN activities to DNA damage response (DDR) proteins domains at its North terminus (Glover et al., 2004; Williams et al., 2009).

Histone variant H2AX is phosphorylated by ATM, which becomes γH2AX throughout the area surrounding the breakage within seconds after damage occurs (Rogakou et al., 1998). This sets off elaborate ubiquitylation and SUMOylation cascades to promote recruitment of BRCA1 (Morris et al., 2009) and 53BP1 (Stewart, 2009) but it is non crucial for the activation of ATM substrates such as CHK2 and p53 (Kang et al., 2005). Then MDC1, a large nuclear factor, directly binds to γH2AX and functions as a molecular scaffold that interacts with ATM and NBS1, promoting farther MRN accumulation. In add-on, MDC1 also helps ATM spread on DSB-flanking chromatin and furthers H2AX activation (Spycher et al., 2008). Information technology also mediates the accumulation of many DDR factors, including 53BP1 and BRCA1 (Wang et al., 2002; Stucki and Jackson, 2006; Kim et al., 2007). ATM leads to phosphorylation of DDR cascades such as BRCA1, Chk2, p53, etc., (Shiloh, 2003; Lavin, 2008). DSBs also actuate clutter telangiectasia and RAD3-related protein (ATR). Full ATR activation requires not only itself and DNA damage sensors just besides proteins that function as signal transducers and effectors, such as RPA, RAD17, TopBP1, Claspin, and Chk1. ssDNA overhangs will be coated by replication protein A (RPA) rapidly, and the ssDNA-RPA circuitous acts as a scaffold to concenter ATR/ATR-interacting protein (ATRIP) (Zou and Elledge, 2003) and other DNA damage checkpoint kinases to trigger DDR (Chen and Wold, 2014). Information technology volition then be replaced with adenosine triphosphate (ATP)-dependent recombinase RAD51 (San Filippo et al., 2008) with the assist of BRCA1 and BRCA2 as described in a higher place (Prakash et al., 2015). ssDNA can be generated past nuclease resection, such every bit the MRN-C-terminal binding poly peptide interacting poly peptide (CtIP) for brusque resection, and EXO1/BLM for long resection (Mladenov et al., 2016). In mammals, resection depends on CtIP and needs to be phosphorylated past CDK first (Huertas and Jackson, 2009). BRCA1 contributes to Hr past colocalizing with MRN after DNA damage occurs and interacts direct with the resection factor CtIP (Sartori et al., 2007). BRCA1 assists RAD51 binding to ssDNA by evicting RPA (Zelensky et al., 2014) and promotes the recruitment of BRCA2 to DSBs through the bridging protein PALB2 (Sy et al., 2009). BRCA1 also appears to inhibit the resection suppressor 53BP1 (Bunting et al., 2010). RAD51 is a DNA strand-exchange protein that exists in mammalian cells and forms a filament referred to as the presynaptic complex (van der Heijden et al., 2007; Hilario et al., 2009). The assembly of a RAD51 nucleoprotein filament promotes homologous search by locating and pairing the three′-overhang with a homologous duplex Dna and catalyzing strand invasion (termed single-terminate invasion, SEI) (Morrical, 2015; Ma et al., 2017). The ii ends of the DSB are identical, but one end serves as the "first end," which searches for the homologous sequence and forms a displacement loops (D-loops) structure while the other end waits for the latter process (Kim and Mirkin, 2018). Too RAD51, DNA strand exchange also requires RAD54 and RDH54/TID1, which performs this pace past stabilizing RAD51-ssDNA presynaptic filaments (Mazin et al., 2003).

Resolution of the exchanged Deoxyribonucleic acid strands includes the Holliday Junction (HJ) pathway and the synthesis-dependent strand annealing (SDSA) pathway. Dissolution is the primary pathway for HJ resolution, which involves the BLM helicase-Topoisomerase IIIα-RMI1-RMI2 (BTR) complex. The BTR circuitous promotes co-operative migration of Holliday junctions (Karow et al., 2000) and also acts to suppress crossing over during homologous recombination (Wu and Hickson, 2003). Thus, this dissolution pathway gives rise exclusively to non-crossovers. The other pathways utilise construction-selective resolvases (SLX-MUS complex and GEN1) to process the exchange intermediates and tin can produce both crossover and non-crossover products (Due west et al., 2015). The SLX1 and MUS81-EME1 nucleases bind in close proximity on the SLX4 scaffold and procedure HJs (Castor et al., 2013). SLX1 catalyzes the initial incision and MUS81 introduces the 2nd cut on the opposing strand (Wyatt and Due west, 2014). GEN1 is a member of the RAD2/XPG family and can only access and cleave recombination intermediates when the nuclear membrane breaks down (Rass et al., 2010). GEN1 first forms a dimeric circuitous that contains the two agile sites and then performs a dual symmetric incision at HJs, generating nicked duplex products that can be ligated.

The SDSA pathway besides begins with the generation of a D-loop construction similar the HJ pathway but also includes DNA synthesis in the 3′-direction, which extends the heteroduplex (Daley et al., 2014). The translocating D-loop then collapses, and the other resected DSB finish will anneal to this extended DSB end. Both ends volition get through replicative extension and ligation, which generates non-crossover products.

When it comes to single-stranded template repair (SSTR), the repair mechanism is quite different from the dsDNA repair template scenario. Richardson et al. establish that homo Cas9-induced SSTR requires the Fanconi anemia (FA) pathway, which was previously implicated in responses to interstrand crosslinks rather than nuclease-induced breaks (Richardson et al., 2018). They confirmed that SSTR is RAD51-independent while dsDNA donor HDR is RAD51-dependent. After FA pathway knockdown, the efficiency of SSTR decreased while simultaneously the levels of NHEJ increased, and the total editing stayed relatively constant. This means that the FA pathway can drive the repair events from NHEJ to SSTR. Additionally, FA pathway knockdown specifically inhibits SSTR and has no effect on NHEJ. RAD51C and XRCC3 are required for SSTR, only RAD51B and XRCC2 are not. They too found that FANCD2, a central thespian in the FA pathway, enriched even in the absence of an exogenous homology donor. In short, SSTR is much more efficient than HDR from a dsDNA donor but still needs future investigations. The HDR pathway is demonstrated in Figure ii.

An external file that holds a picture, illustration, etc. Object name is fgene-09-00691-g0002.jpg

Homology-directed repair HDR. When DSB happens in the S- or G2-stage of the cell cycle and homologous sequence exists near the DSB, DSB can be handled through the HDR pathway if the ends of the DSB are resected. Ends will exist coated with various proteins and then invade homologous duplex Deoxyribonucleic acid to course an commutation intermediate: the D-loop construction. Virtually D-loop structures will exist extended by Dna synthesis (dashed arrow). The second end pairs to the D-loop and starts extension. This pathway is called the double Holliday junction pathway. Ligation generates the characteristic double Holliday junction, which may be cleaved by HJ resolvases into either crossover or non-crossover products. The synthesis-dependent strand annealing pathway is illustrated on the right. After D-loop formation, replication and branch migration have place which can lead to D-loop translocation. The translocating D-loop is unstable and collapses easily. After collapse, the extended first end may anneal to complementary ssDNA in the resected second end. Replicative extension of both ends and ligation generates non-crossover products.

Favoring the HDR Pathway Using Chemical and Genetic Modulation

DSBs caused by Cas9 can become through both the NHEJ and HDR pathways, but in most cases they are handled by NHEJ (Frit et al., 2014), and then it seems reasonable to inhibit key enzymes (e.g., Dna ligase Four) of the NHEJ pathway. Maruyama et al. (2015) investigated the effect of SCR7, a putative inhibitor of ligase IV, which targets the DNA bounden domain of ligase IV, impeding its ability to demark to DSBs (Srivastava et al., 2012) in human epithelial (A549) and melanoma (MelJuSo) cell lines. Results showed that SCR7 promotes a 21 bp precise insertion (ssDNA donor with two 100 bp homology arms) in A549 cells 3-fold at 0.01 μM and 19-fold for MelJuSo at 1 μM. They also assessed the result of SCR7 on insertion of a ~800 bp fragment (ssDNA donor with ~80 bp homology flanking sequence on both sides of the DSB) into a murine os marrow-derived dendritic prison cell line (DC2.four cells). After treating the DC2.4 cells with ane μM SCR7, the efficiency of insertion increased past ~13-fold. It is worth mentioning that SCR7 affects lymphocyte evolution and can induce apoptosis. Every bit boosted approaches to DNA ligase IV inhibition, Chu et al. used the adenovirus 4 (Ad4) E1B55K and E4orf6 proteins to suppress NHEJ. These two proteins tin can mediate the ubiquitination and proteasomal degradation of DNA ligase IV (Forrester et al., 2011). Results showed that HDR efficiency was enhanced up to 7-fold (v to 36%) by the Ad4 protein in human HEK293 cells. And in a mouse Burkitt lymphoma jail cell line, the add-on of Ad4 proteins reduced transfection efficiency from xl to 27%, but promoted HDR by five-fold (Chu et al., 2015). Yu et al. identified two small molecules (L755507 and Brefeldin A) that could meliorate HDR efficiency. L755507, a β3-adrenergic receptor agonist, can increment HDR past 3-fold at 5 μM in mouse ESCs. Brefeldin A, an inhibitor of intracellular protein transport from the ER to the Golgi apparatus, promotes HDR by 2-fold at 0.1 μM in mouse ESCs (Yu et al., 2015).

Pinder et al. identified that RS-1 can enhance HDR betwixt 3- and 6-fold, varying with the locus and transfection factor in HEK-293A homo embryonic kidney and U2OS osteosarcoma cell lines (Pinder et al., 2015). RS-ane is a compound that stabilizes the association betwixt Rad51 and DNA. They also constitute that an optimized ratio for Cas9/gRNA to homology donor plasmid doubled the HDR efficiency. Upon BRCA1 over-expression, HDR is increased by 2- to 3-fold.

Too these inhibiting molecules, HDR tin can likewise be promoted using modest interfering RNA (siRNA) to inhibit the expression of Ku protein, which is the pioneer protein in the NHEJ pathway. Li et al. assessed this method on grunter fetal fibroblasts (Li et al., 2018). The result showed that by inhibiting Ku70 or Ku80 expression, Hr tin be promoted by i.six- to 3-fold, likewise as SSA and ssODN-mediated repairs. Yu et al. constructed a Rad51 and a Rad50 co-expression vector to evaluate its performance (Yu et al., 2011). It was determined that Hour efficiency increased 110–245%. Chu et al. used brusk hairpin RNA (shRNA) in HEK293 cells to suppress primal NHEJ pathway proteins such equally KU70, KU80, and Deoxyribonucleic acid ligase Iv. Results showed that HDR efficiency was enhanced from 5 to eight–fourteen% when transfected with unmarried shRNAs confronting KU70, KU80, or Dna ligase IV. Furthermore, they plant that the expression of the target gene diminished in the cells undergoing NHEJ blockade, which may exist caused by local chromatin remodeling through extended Deoxyribonucleic acid damage (Chu et al., 2015).

Timed Commitment of the CRISPR-Cas9 Organisation

While HDR is typically restricted to the S and G2 phases of the prison cell cycle, its efficiency tin be increased by synchronizing and capturing cells at the Due south and G2 phases or using timed delivery. Lin et al. combined cell cycle synchronization techniques, using chemic inhibitors to arrest the cells at specific phases of the jail cell cycle with direct nucleofection of pre-assembled Cas9 ribonucleoprotein (RNP) in HEK293T cells (Lin et al., 2014). Results showed that using lower Cas9/RNP concentrations and cell cycle arrest can improve HDR efficiency to 31% (three.4-fold) at maximum. Consistent with this strategy, Yang et al. successfully enhanced HDR by three- to six-fold using the microtubule polymerization inhibitor nocodazole or ABT-751 (Yang et al., 2016). As for not-proliferating cells, BRCA1 is inhibited by the 53BP1 (Escribano-Diaz et al., 2013) and KEAP1-CUL3 complexes (Orthwein et al., 2015), and RIF1 thus will non demark to DSB. In addition, CtIP tin function normally after beingness phosphorylated by CDK, but CDK is absent when cells stay in the G0/G1 phase (Escribano-Diaz et al., 2013). In social club to overcome this inhibition, Orthwein et al. overexpressed mutated activated CtIP. This depleted the 53BP1 and KEAP1-CUL3 complexes simultaneously, which successfully activated the HDR pathway in G1 cells (Orthwein et al., 2015).

Enhancing HDR by Using Overlapping Sequences

Several types of donor DNA have been used, such as plasmid Dna and constructed oligonucleotides (Carroll and Beumer, 2014). Rational design of homology repair templates strongly enhances HDR efficiency (Renaud et al., 2016). By using a linear repair template with homologous flanks in zebrafish, HDR can increase by almost ten-fold (Irion et al., 2014). Chu et al. assessed the influence of the lengths of homology regions of the repair template on HDR efficiency (Chu et al., 2015). A homologous template is the well-nigh important component of HDR-mediated genome editing. It usually contains intended mutations or insertions flanked by homologous regions. Templates can exist plasmids (up to kilobases modification) or single-stranded oligodeoxynucleotides (ssODN) (50–100 nt modification). It is suggested that sequencing effectually the interested region should be carried out because prison cell-specific mutations and single nucleotide polymorphisms (SNPs) tin influence gRNA targeting up to 6-fold, merely ane mismatch in 100 bases (Tham et al., 2016). This trouble can be overcome past amplifying the homology arm from the genomic Dna extracted from target cells or by synthesize consulting sequence analysis (Salsman and Dellaire, 2017). Information technology is important to retrieve that DSB should ever be as close every bit possible to the region of homology, inside 10 nt upwardly and to a maximum of 100 nt (Elliott et al., 1998). Furthermore, having each homology arm almost 50–100% the size of the payload that can promote HDR (Li et al., 2014).

Enhancing HDR by Using Modified Cas9

As mentioned above, DSB ends must be resected so that they can enter the HDR pathway. CtIP, a central protein in early steps of DSB resection, is essential for HDR initiation. In gild to ensure the presence of functional activated CtIP, Charpentier et al. fused a minimal N-terminal fragment of CtIP to the Cas9 poly peptide (Charpentier et al., 2018). Forcing CtIP to the Deoxyribonucleic acid cleavage site, through fusion to either catalytically dead Cas9 (dCas9) or Cas9 together with 800 bp homology arms, obtained a 2-fold increase in the efficiency of HDR in human fibroblast RG37DR cells, iPS cells, and rat zygotes. However, the expression of dCas9-CtIP is not sufficient by itself to stimulate integration. In addition, the patterns of indels induced by modified Cas9 were different from Cas9. This may be considering the modified Cas9 induced a different residue of the end-joining pathway.

Equally for point mutation, current approaches to target base correction are inefficient and typically induce an affluence of random insertions and deletions at the target locus. Alexis et al. reported a powerful arroyo called a "base-editing (Be) system" to innovate specific signal mutations without introducing DSB or a donor template by linking deaminases and dCas9 together (Komor et al., 2016). dCas9 helps deaminase to locate and deaminases change cytidine to uridine. This conversion will then be repaired through various pathways (Hess et al., 2017). BE2 (optimized BE system) results from the addition of the uracil DNA glycosylase inhibitor, which increases the base-editing efficiency of the C>T substitution 3-fold (Hess et al., 2016). Additionally, the BE3 arrangement was realized by changing the dCas9 to Cas9 D10A. This improvement accomplished a 6-fold increase in efficiency over BE2 merely exhibited a slightly increased indel frequency as nicks tin can atomic number 82 to NHEJ at a depression rate (Certo et al., 2011). Target-activation-induced cytidine deaminase (AID), a similar system, uses nickase Cas9 D10A to recruit the cytidine deaminase PmCDA1 to the target, achieving a mutation frequency of 35%. Adding UGI can obtain ii- to 3-fold increase in efficiency and reduction in deletions (Nishida et al., 2016). In addition, targeted AID-mediated mutagenesis (TAM) (Ma et al., 2016) and CRISPR-10 (Hess et al., 2016) can generate transitions and transversions. These systems can both accomplish the precision editing of single C>T bases with a low charge per unit of indels also every bit sequence diversification. By further Cas9 engineering, the inhibition of HR during the G1 phase volition exist overcome.

The CRISPR-Cas system has been fully studied and adapted for various applications over the decades, which gives us the power to manipulate the genome as we desire. It has certain limitations, such equally off-target effects (which tin can be overcame by rational sgRNA design; Doench et al., 2016) and low efficiency, which can be improved past utilizing the methodologies as described above. These promising strategies have proven their enhancement in the HDR pathway more than one time with results varying from 2- to 30-fold. Combining various approaches can be a potential method of maximizing the rates of HDR. Here we only reviewed NHEJ inhibition by using inhibitors or hindering sure gene expression with siRNA or shRNA, CRISPR-Cas delivery in the G2/Southward phase, calculation homologous arms in donor templets and using modified Cas9. These tactics surely make the CRISPR-Cas system more efficient. In that location is no doubt that more and more ways to boost CRISPR-Cas are imminent.

Author Contributions

ML, WM, and DC designed the construction of this review. ML wrote the kickoff version of the manuscript. SR, XT, KG, and QF helped to revise the manuscript. All authors have reviewed the concluding version of the manuscript.

Conflict of Interest Argument

The authors declare that the research was conducted in the absence of any commercial or fiscal relationships that could be construed as a potential disharmonize of interest.

Footnotes

Funding. This enquiry was supported past the Qinghai Province Major R&D and Transformation Projection (2018-NK-125), Xianyang Science and Technology Major Project (2017K01-34), Key Industrial Innovation Chains of Shaanxi Province (2018ZDCXL-NY-01-06), and the PhD enquiry startup fund of the Northwest Agriculture and Forestry University (00500/Z109021716).

References

Anders C., Niewoehner O., Duerst A., Jinek M. (2014). Structural ground of PAM-dependent target Dna recognition by the Cas9 endonuclease. Nature 513, 569–573. x.1038/nature13579 [PMC free commodity] [PubMed] [CrossRef] [Google Scholar]
Andres S. N., Vergnes A., Ristic D., Wyman C., Modesti One thousand., Junop Chiliad. (2012). A homo XRCC4-XLF complex bridges Dna. Nucleic Acids Res. 40, 1868–1878. 10.1093/nar/gks022 [PMC gratis commodity] [PubMed] [CrossRef] [Google Scholar]
Aravind L., Koonin E. V. (2001). Prokaryotic homologs of the eukaryotic DNA-finish-bounden protein Ku, novel domains in the Ku protein and prediction of a prokaryotic double-strand break repair system. Genome Res. 11, 1365–1374. 10.1101/gr.181001 [PMC gratis commodity] [PubMed] [CrossRef] [Google Scholar]
Bae S., Kweon J., Kim H. S., Kim J. S. (2014). Microhomology-based choice of Cas9 nuclease target sites. Nat. Methods 11, 705–706. 10.1038/nmeth.3015 [PubMed] [CrossRef] [Google Scholar]
Barrangou R. (2013). CRISPR-Cas systems and RNA-guided interference. Wiley Interdiscip. Rev. RNA 4, 267–278. 10.1002/wrna.1159 [PubMed] [CrossRef] [Google Scholar]
Barrangou R. (2015). The roles of CRISPR-Cas systems in adaptive immunity and across. Curr. Opin. Immunol. 32, 36–41. ten.1016/j.coi.2014.12.008 [PubMed] [CrossRef] [Google Scholar]
Barrangou R., Marraffini Fifty. A. (2014). CRISPR-Cas systems: prokaryotes upgrade to adaptive immunity. Mol. Jail cell 54, 234–244. x.1016/j.molcel.2014.03.011 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Bunting Due south. F., Callen E., Wong N., Chen H. T., Polato F., Gunn A., et al. . (2010). 53BP1 inhibits homologous recombination in Brca1-deficient cells by blocking resection of Dna breaks. Cell 141, 243–254. 10.1016/j.cell.2010.03.012 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Cannavo E., Cejka P. (2014). Sae2 promotes dsDNA endonuclease activity within Mre11-Rad50-Xrs2 to resect DNA breaks. Nature 514, 122–125. 10.1038/nature13771 [PubMed] [CrossRef] [Google Scholar]
Carroll D., Beumer K. J. (2014). Genome technology with TALENs and ZFNs: repair pathways and donor blueprint. Methods 69, 137–141. x.1016/j.ymeth.2014.03.026 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Brush D., Nair N., Declais A. C., Lachaud C., Toth R., Macartney T. J., et al. . (2013). Cooperative control of holliday junction resolution and DNA repair past the SLX1 and MUS81-EME1 nucleases. Mol. Cell 52, 221–233. 10.1016/j.molcel.2013.08.036 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Certo M. T., Ryu B. Y., Annis J. E., Garibov M., Jarjour J., Rawlings D. J., et al. . (2011). Tracking genome technology upshot at individual DNA breakpoints. Nat. Methods viii, 671–676. 10.1038/nmeth.1648 [PMC free commodity] [PubMed] [CrossRef] [Google Scholar]
Charpentier M., Khedher A. H. Y., Menoret S., Brion A., Lamribet K., Dardillac E., et al. . (2018). CtIP fusion to Cas9 enhances transgene integration past homology-dependent repair. Nat.Commun. 9:1133. 10.1038/s41467-018-03475-7 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Chen R., Wold M. S. (2014). Replication protein a: unmarried-stranded DNA's first responder: dynamic DNA-interactions allow replication poly peptide A to direct single-strand Deoxyribonucleic acid intermediates into different pathways for synthesis or repair. Bioessays 36, 1156–1161. ten.1002/bies.201400107 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Chu V. T., Weber T., Wefers B., Wurst W., Sander South., Rajewsky G., et al. . (2015). Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise factor editing in mammalian cells. Nat. Biotechnol. 33, 543–548. ten.1038/nbt.3198 [PubMed] [CrossRef] [Google Scholar]
Daley J. M., Gaines Due west. A., Kwon Y., Sung P. (2014). Regulation of Dna pairing in homologous recombination. Cold Spring Harb. Perspect. Biol. 6:a017954. 10.1101/cshperspect.a017954 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Daley J. 1000., Laan R. L., Suresh A., Wilson T. Due east. (2005). Deoxyribonucleic acid articulation dependence of pol X family polymerase action in nonhomologous stop joining. J. Biol. Chem. 280, 29030–29037. x.1074/jbc.M505277200 [PubMed] [CrossRef] [Google Scholar]
Davis A. J., Chen B. P., Chen D. J. (2014). Deoxyribonucleic acid-PK: a dynamic enzyme in a versatile DSB repair pathway. DNA Repair 7, 21–29. ten.1016/j.dnarep.2014.02.020 [PMC free commodity] [PubMed] [CrossRef] [Google Scholar]
de Jager M., van Noort J., van Gent D. C., Dekker C., Kanaar R., Wyman C. (2001). Man Rad50/Mre11 is a flexible circuitous that can tether DNA ends. Mol. Cell 8, 1129–1135. x.1016/S1097-2765(01)00381-one [PubMed] [CrossRef] [Google Scholar]
Deltcheva Eastward., Chylinski K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A., et al. . (2011). CRISPR RNA maturation by trans-encoded small RNA and host factor RNase Three. Nature 471, 602–607. 10.1038/nature09886 [PMC free commodity] [PubMed] [CrossRef] [Google Scholar]
Doench J. G., Fusi N., Sullender Yard., Hegde Thou., Vaimberg E. West., Donovan Chiliad. F., et al. . (2016). Optimized sgRNA design to maximize action and minimize off-target effects of CRISPR-Cas9. Nat. Biotechnol. 34, 184–191. ten.1038/nbt.3437 [PMC complimentary commodity] [PubMed] [CrossRef] [Google Scholar]
Dow L. Due east., Fisher J., O'Rourke Chiliad. P., Muley A., Kastenhuber E. R., Livshits G., et al. . (2015). Inducible in vivo genome editing with CRISPR-Cas9. Nat. Biotechnol. 33, 390–394. x.1038/nbt.3155 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Dupre A., Boyer-Chatenet L., Gautier J. (2006). 2-stride activation of ATM by DNA and the Mre11-Rad50-Nbs1 complex. Nat. Struct. Mol. Biol. 13, 451–457. 10.1038/nsmb1090 [PubMed] [CrossRef] [Google Scholar]
Elliott B., Richardson C., Winderbaum J., Nickoloff J. A., Jasin One thousand. (1998). Gene conversion tracts from double-strand intermission repair in mammalian cells. Mol. Jail cell Biol. 18, 93–101. [PMC free article] [PubMed] [Google Scholar]
Escribano-Diaz C., Orthwein A., Fradet-Turcotte A., Xing Thousand., Young J. T., Tkac J., et al. . (2013). A cell cycle-dependent regulatory excursion composed of 53BP1-RIF1 and BRCA1-CtIP controls Dna repair pathway pick. Mol. Cell 49, 872–883. x.1016/j.molcel.2013.01.001 [PubMed] [CrossRef] [Google Scholar]
Falck J., Coates J., Jackson S. P. (2005). Conserved modes of recruitment of ATM, ATR and Dna-PKcs to sites of DNA damage. Nature 434, 605–611. 10.1038/nature03442 [PubMed] [CrossRef] [Google Scholar]
Forrester N. A., Sedgwick G. G., Thomas A., Blackford A. N., Speiseder T., Dobner T., et al. . (2011). Serotype-specific inactivation of the cellular DNA damage response during adenovirus infection. J. Virol. 85, 2201–2211. 10.1128/JVI.01748-10 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Frit P., Barboule N., Yuan Y., Gomez D., Calsou P. (2014). Culling end-joining pathway(s): bricolage at Dna breaks. DNA Repair 17, 81–97. 10.1016/j.dnarep.2014.02.007 [PubMed] [CrossRef] [Google Scholar]
Glover J. Due north., Williams R. S., Lee M. S. (2004). Interactions between BRCT repeats and phosphoproteins: tangled up in 2. Trends Biochem. Sci. 29, 579–585. 10.1016/j.tibs.2004.09.010 [PubMed] [CrossRef] [Google Scholar]
Gu J., Lieber M. R. (2008). Mechanistic flexibility as a conserved theme beyond three billion years of nonhomologous Dna stop-joining. Genes Dev. 22, 411–415. ten.1101/gad.1646608 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Hess G. T., Fresard L., Han K., Lee C. H., Li A., Cimprich K. A., et al. . (2016). Directed development using dCas9-targeted somatic hypermutation in mammalian cells. Nat. Methods 13, 1036–1042. ten.1038/nmeth.4038 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Hess Thousand. T., Tycko J., Yao D., Bassik M. C. (2017). Methods and applications of CRISPR-mediated base of operations editing in eukaryotic genomes. Mol. Cell 68, 26–43. 10.1016/j.molcel.2017.09.029 [PMC complimentary article] [PubMed] [CrossRef] [Google Scholar]
Hilario J., Amitani I., Baskin R. J., Kowalczykowski Due south. C. (2009). Direct imaging of human Rad51 nucleoprotein dynamics on individual DNA molecules. Proc. Natl. Acad. Sci. United statesA. 106, 361–368. ten.1073/pnas.0811965106 [PMC free commodity] [PubMed] [CrossRef] [Google Scholar]
Hohl M., Kwon Y., Galvan S. M., Xue Ten., Tous C., Aguilera A., et al. . (2011). The Rad50 coiled-coil domain is indispensable for Mre11 complex functions. Nat. Struct. Mol. Biol. 18, 1124–1131. 10.1038/nsmb.2116 [PMC gratuitous article] [PubMed] [CrossRef] [Google Scholar]
Horvath P., Barrangou R. (2010). CRISPR/Cas, the immune organisation of leaner and archaea. Science 327, 167–170. ten.1126/scientific discipline.1179555 [PubMed] [CrossRef] [Google Scholar]
Huertas P., Jackson Southward. P. (2009). Human CtIP mediates prison cell cycle command of DNA end resection and double strand pause repair. J. Biol. Chem. 284, 9558–9565. 10.1074/jbc.M808906200 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Irion U., Krauss J., Nusslein-Volhard C. (2014). Precise and efficient genome editing in zebrafish using the CRISPR/Cas9 arrangement. Development 141, 4827–4830. 10.1242/dev.115584 [PMC costless article] [PubMed] [CrossRef] [Google Scholar]
Jansen R., Embden J. D., Gaastra W., Schouls L. Yard. (2002). Identification of genes that are associated with Dna repeats in prokaryotes. Mol. Microbiol. 43, 1565–1575. 10.1046/j.1365-2958.2002.02839.x [PubMed] [CrossRef] [Google Scholar]
Jasin M., Rothstein R. (2013). Repair of strand breaks past homologous recombination. Cold Jump Harb. Perspect. Biol. 5:a012740. 10.1101/cshperspect.a012740 [PMC gratuitous article] [PubMed] [CrossRef] [Google Scholar]
Jinek Grand., Chylinski Thousand., Fonfara I., Hauer One thousand., Doudna J. A., Charpentier East. (2012). A programmable dual-RNA-guided Deoxyribonucleic acid endonuclease in adaptive bacterial immunity. Science 337, 816–821. 10.1126/science.1225829 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Jinek 1000., Jiang F., Taylor D. Due west., Sternberg Southward. H., Kaya E., Ma E., et al. . (2014). Structures of Cas9 endonucleases reveal RNA-mediated conformational activation. Science 343:1247997. ten.1126/science.1247997 [PMC free commodity] [PubMed] [CrossRef] [Google Scholar]
Kang J., Ferguson D., Song H., Bassing C., Eckersdorff M., Alt F. W., et al. . (2005). Functional interaction of H2AX, NBS1, and p53 in ATM-dependent DNA damage responses and tumor suppression. Mol. Cell Biol. 25, 661–670. 10.1128/MCB.25.2.661-670.2005 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Karow J. K., Constantinou A., Li J. L., West South. C., Hickson I. D. (2000). The Bloom's syndrome gene product promotes branch migration of holliday junctions. Proc. Natl. Acad. Sci. U.S.A. 97, 6504–6508. 10.1073/pnas.100448097 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Kim H., Chen J., Yu Ten. (2007). Ubiquitin-binding poly peptide RAP80 mediates BRCA1-dependent Dna impairment response. Science 316, 1202–1205. 10.1126/science.1139621 [PubMed] [CrossRef] [Google Scholar]
Kim M. P., Mirkin E. Five. (2018). So similar yet and then different: the 2 ends of a double strand intermission. Mutat. Res. 809, 70–eighty. 10.1016/j.mrfmmm.2017.06.007 [PubMed] [CrossRef] [Google Scholar]
Komor A. C., Kim Y. B., Packer K. S., Zuris J. A., Liu D. R. (2016). Programmable editing of a target base in genomic Dna without double-stranded DNA cleavage. Nature 533, 420–424. x.1038/nature17946 [PMC costless article] [PubMed] [CrossRef] [Google Scholar]
Lavin M. F. (2008). Clutter-telangiectasia: from a rare disorder to a paradigm for jail cell signalling and cancer. Nat. Rev. Mol. Cell Biol. 9, 759–769. 10.1038/nrm2514 [PubMed] [CrossRef] [Google Scholar]
Li G., Liu D., Zhang 10., Quan R., Zhong C., Mo J., et al. . (2018). Suppressing Ku70/Ku80 expression elevates homology-directed repair efficiency in primary fibroblasts. Int. J. Biochem. Prison cell Biol. 99, 154–160. ten.1016/j.biocel.2018.04.011 [PubMed] [CrossRef] [Google Scholar]
Li K., Wang G., Andersen T., Zhou P., Pu W. T. (2014). Optimization of genome engineering approaches with the CRISPR/Cas9 system. PLoS Ane 9:e105779. x.1371/periodical.pone.0105779 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Lin Southward., Staahl B. T., Alla R. K., Doudna J. A. (2014). Enhanced homology-directed homo genome technology by controlled timing of CRISPR/Cas9 delivery. Elife 3:e04766. 10.7554/eLife.04766 [PMC gratis article] [PubMed] [CrossRef] [Google Scholar]
Ma C. J., Gibb B., Kwon Y., Sung P., Greene E. C. (2017). Protein dynamics of human RPA and RAD51 on ssDNA during assembly and disassembly of the RAD51 filament. Nucleic Acids Res. 45, 749–761. 10.1093/nar/gkw1125 [PMC costless article] [PubMed] [CrossRef] [Google Scholar]
Ma Y., Zhang J., Yin W., Zhang Z., Song Y., Chang X. (2016). Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells. Nat. Methods 13, 1029–1035. 10.1038/nmeth.4027 [PubMed] [CrossRef] [Google Scholar]
Makarova Grand. Southward., Haft D. H., Barrangou R., Brouns Southward. J., Charpentier E., Horvath P., et al. . (2011). Evolution and classification of the CRISPR-Cas systems. Nat. Rev. Microbiol. 9, 467–477. 10.1038/nrmicro2577 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Makarova K. Due south., Wolf Y. I., Alkhnbashi O. Due south., Costa F., Shah Due south. A., Saunders S. J., et al. . (2015). An updated evolutionary classification of CRISPR-Cas systems. Nat. Rev. Microbiol. 13, 722–736. ten.1038/nrmicro3569 [PMC gratuitous article] [PubMed] [CrossRef] [Google Scholar]
Makharashvili North., Paull T. T. (2015). CtIP: A Dna damage response protein at the intersection of Deoxyribonucleic acid metabolism. Dna Repair 32, 75–81. 10.1016/j.dnarep.2015.04.016 [PubMed] [CrossRef] [Google Scholar]
Marraffini 50. A. (2015). CRISPR-Cas immunity in prokaryotes. Nature 526, 55–61. x.1038/nature15386 [PubMed] [CrossRef] [Google Scholar]
Marraffini L. A., Sontheimer Eastward. J. (2010). CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea. Nat. Rev. Genet. xi, 181–190. 10.1038/nrg2749 [PMC gratuitous article] [PubMed] [CrossRef] [Google Scholar]
Maruyama T., Dougan Southward. K., Truttmann M. C., Bilate A. M., Ingram J. R., Ploegh H. 50. (2015). Increasing the efficiency of precise genome editing with CRISPR-Cas9 past inhibition of nonhomologous finish joining. Nat. Biotechnol. 33, 538–542. 10.1038/nbt.3190 [PMC gratuitous article] [PubMed] [CrossRef] [Google Scholar]
Mathiasen D. P., Lisby Chiliad. (2014). Cell cycle regulation of homologous recombination in Saccharomyces cerevisiae. FEMS Microbiol. Rev. 38, 172–184. x.1111/1574-6976.12066 [PubMed] [CrossRef] [Google Scholar]
Mayle R., Campbell I. Thousand., Beck C. R., Yu Y., Wilson M., Shaw C. A., et al. . (2015). DNA REPAIR. Mus81 and converging forks limit the mutagenicity of replication fork breakage. Scientific discipline 349, 742–747. 10.1126/science.aaa8391 [PMC complimentary article] [PubMed] [CrossRef] [Google Scholar]
Mazin A. V., Alexeev A. A., Kowalczykowski S. C. (2003). A novel part of Rad54 protein. stabilization of the Rad51 nucleoprotein filament. J. Biol. Chem. 278, 14029–14036. 10.1074/jbc.M212779200 [PubMed] [CrossRef] [Google Scholar]
Mladenov E., Magin S., Soni A., Iliakis Thou. (2016). Dna double-strand-intermission repair in college eukaryotes and its part in genomic instability and cancer: cell cycle and proliferation-dependent regulation. Semin. Cancer Biol. 37–38, 51–64. 10.1016/j.semcancer.2016.03.003 [PubMed] [CrossRef] [Google Scholar]
Mojica F. J., Diez-Villasenor C., Garcia-Martinez J., Almendros C. (2009). Brusque motif sequences determine the targets of the prokaryotic CRISPR defence force organisation. Microbiology 155, 733–740. ten.1099/mic.0.023960-0 [PubMed] [CrossRef] [Google Scholar]
Mojica F. J., Rodriguez-Valera F. (2016). The discovery of CRISPR in archaea and bacteria. FEBS J. 283, 3162–3169. 10.1111/febs.13766 [PubMed] [CrossRef] [Google Scholar]
Morange Yard. (2015). What history tells the states XXXVII. CRISPR-Cas: the discovery of an allowed arrangement in prokaryotes. J. Biosci. 40, 221–223. x.1007/s12038-015-9532-6 [PubMed] [CrossRef] [Google Scholar]
Morrical S. W. (2015). Deoxyribonucleic acid-pairing and annealing processes in homologous recombination and homology-directed repair. Cold Leap Harb. Perspect. Biol. seven:a016444. 10.1101/cshperspect.a016444 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Morris J. R., Boutell C., Keppler M., Densham R., Weekes D., Alamshah A., et al. . (2009). The SUMO modification pathway is involved in the BRCA1 response to genotoxic stress. Nature 462, 886–890. 10.1038/nature08593 [PubMed] [CrossRef] [Google Scholar]
Moshous D., Callebaut I., de Chasseval R., Corneo B., Cavazzana-Calvo M., Le Deist F., et al. . (2001). Artemis, a novel DNA double-strand break repair/V(D)J recombination poly peptide, is mutated in homo astringent combined immune deficiency. Cell 105, 177–186. 10.1016/S0092-8674(01)00309-ix [PubMed] [CrossRef] [Google Scholar]
Neal J. A., Sugiman-Marangos S., VanderVere-Carozza P., Wagner M., Turchi J., Lees-Miller S. P., et al. . (2014). Unraveling the complexities of Deoxyribonucleic acid-dependent protein kinase autophosphorylation. Mol. Prison cell Biol. 34, 2162–2175. 10.1128/MCB.01554-13 [PMC free commodity] [PubMed] [CrossRef] [Google Scholar]
Nishida K., Arazoe T., Yachie North., Banno South., Kakimoto M., Tabata M., et al. . (2016). Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive allowed systems. Science 353:aaf8729. x.1126/science.aaf8729 [PubMed] [CrossRef] [Google Scholar]
Nishimasu H., Ran F. A., Hsu P. D., Konermann S., Shehata S. I., Dohmae N., et al. . (2014). Crystal structure of Cas9 in complex with guide RNA and target DNA. Cell 156, 935–949. 10.1016/j.cell.2014.02.001 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Orthwein A., Noordermeer Due south. Chiliad., Wilson Yard. D., Landry Southward., Enchev R. I., Sherker A., et al. . (2015). A mechanism for the suppression of homologous recombination in G1 cells. Nature 528, 422–426. 10.1038/nature16142 [PMC complimentary article] [PubMed] [CrossRef] [Google Scholar]
Pardo B., Gomez-Gonzalez B., Aguilera A. (2009). DNA repair in mammalian cells: DNA double-strand break repair: how to ready a cleaved relationship. Cell Mol. Life Sci. 66, 1039–1056. 10.1007/s00018-009-8740-3 [PubMed] [CrossRef] [Google Scholar]
Paull T. T. (2005). Saving the ends for last: the role of pol mu in Deoxyribonucleic acid end joining. Mol. Cell 19, 294–296. 10.1016/j.molcel.2005.07.008 [PubMed] [CrossRef] [Google Scholar]
Paull T. T. (2015). Mechanisms of ATM Activation. Annu. Rev. Biochem. 84, 711–738. 10.1146/annurev-biochem-060614-034335 [PubMed] [CrossRef] [Google Scholar]
Pinder J., Salsman J., Dellaire Thousand. (2015). Nuclear domain 'knock-in' screen for the evaluation and identification of small molecule enhancers of CRISPR-based genome editing. Nucleic Acids Res. 43, 9379–9392. 10.1093/nar/gkv993 [PMC gratuitous article] [PubMed] [CrossRef] [Google Scholar]
Prakash R., Zhang Y., Feng Due west., Jasin M. (2015). Homologous recombination and man wellness: the roles of BRCA1, BRCA2, and associated proteins. Common cold Bound Harb. Perspect. Biol. 7:a016600. 10.1101/cshperspect.a016600 [PMC gratis commodity] [PubMed] [CrossRef] [Google Scholar]
Radhakrishnan Due south. K., Jette N., Lees-Miller Due south. P. (2014). Non-homologous end joining: emerging themes and unanswered questions. DNA Repair 17, 2–eight. 10.1016/j.dnarep.2014.01.009 [PMC costless article] [PubMed] [CrossRef] [Google Scholar]
Rass U., Compton S. A., Matos J., Singleton Grand. R., Ip S. C., Blanco M. Yard., et al. . (2010). Mechanism of Holliday junction resolution past the human being GEN1 protein. Genes Dev. 24, 1559–1569. ten.1101/gad.585310 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Renaud J. B., Boix C., Charpentier Thou., De Cian A., Cochennec J., Duvernois-Berthet E., et al. . (2016). Improved genome editing efficiency and flexibility using modified oligonucleotides with TALEN and CRISPR-Cas9 Nucleases. Cell Rep. 14, 2263–2272. x.1016/j.celrep.2016.02.018. [PubMed] [CrossRef] [Google Scholar]
Richardson C. D., Kazane K. R., Feng Southward. J., Zelin East., Bray N. L., Schafer A. J., et al. . (2018). CRISPR-Cas9 genome editing in human cells occurs via the Fanconi anemia pathway. Nat. Genet. l, 1132–1139. 10.1038/s41588-018-0174-0 [PubMed] [CrossRef] [Google Scholar]
Rogakou E. P., Pilch D. R., Orr A. H., Ivanova 5. Southward., Bonner W. Thou. (1998). DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem. 273, 5858–5868. [PubMed] [Google Scholar]
Roth D. B., Lindahl T., Gellert M. (1995). Repair and recombination. how to make ends come across. Curr. Biol. five, 496–499. [PubMed] [Google Scholar]
Salsman J., Dellaire Thousand. (2017). Precision genome editing in the CRISPR era. Biochem. Cell Biol. 95, 187–201. ten.1139/bcb-2016-0137 [PubMed] [CrossRef] [Google Scholar]
San Filippo J., Sung P., Klein H. (2008). Mechanism of eukaryotic homologous recombination. Annu. Rev. Biochem. 77, 229–257. 10.1146/annurev.biochem.77.061306.125255 [PubMed] [CrossRef] [Google Scholar]
Sapranauskas R., Gasiunas Grand., Fremaux C., Barrangou R., Horvath P., Siksnys V. (2011). The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli . Nucleic Acids Res. 39, 9275–9282. 10.1093/nar/gkr606 [PMC free commodity] [PubMed] [CrossRef] [Google Scholar]
Sartori A. A., Lukas C., Coates J., Mistrik M., Fu S., Bartek J., et al. . (2007). Human being CtIP promotes Deoxyribonucleic acid end resection. Nature 450, 509–514. 10.1038/nature06337 [PMC gratis article] [PubMed] [CrossRef] [Google Scholar]
Shiloh Y. (2003). ATM and related poly peptide kinases: safeguarding genome integrity. Nat. Rev. Cancer 3, 155–168. 10.1038/nrc1011 [PubMed] [CrossRef] [Google Scholar]
Sorek R., Lawrence C. M., Wiedenheft B. (2013). CRISPR-mediated adaptive immune systems in leaner and archaea. Annu. Rev. Biochem. 82, 237–266. ten.1146/annurev-biochem-072911-172315 [PubMed] [CrossRef] [Google Scholar]
Soutoglou E., Dorn J. F., Sengupta K., Jasin Thousand., Nussenzweig A., Ried T., et al. . (2007). Positional stability of unmarried double-strand breaks in mammalian cells. Nat. Cell Biol. nine, 675–682. 10.1038/ncb1591 [PMC free commodity] [PubMed] [CrossRef] [Google Scholar]
Spycher C., Miller Due east. S., Townsend K., Pavic L., Morrice N. A., Janscak P., et al. . (2008). Constitutive phosphorylation of MDC1 physically links the MRE11-RAD50-NBS1 complex to damaged chromatin. J. Cell Biol. 181, 227–240. 10.1083/jcb.200709008 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Srivastava Thou., Nambiar M., Sharma Southward., Karki South. S., Goldsmith G., Hegde M., et al. . (2012). An inhibitor of nonhomologous end-joining abrogates double-strand break repair and impedes cancer progression. Jail cell 151, 1474–1487. ten.1016/j.cell.2012.11.054 [PubMed] [CrossRef] [Google Scholar]
Sternberg Due south. H., Redding S., Jinek Chiliad., Greene E. C., Doudna J. A. (2014). Deoxyribonucleic acid interrogation past the CRISPR RNA-guided endonuclease Cas9. Nature 507, 62–67. 10.1038/nature13011 [PMC gratuitous article] [PubMed] [CrossRef] [Google Scholar]
Stewart G. S. (2009). Solving the RIDDLE of 53BP1 recruitment to sites of impairment. Prison cell Wheel 8, 1532–1538. 10.4161/cc.8.ten.8351 [PubMed] [CrossRef] [Google Scholar]
Stucki Grand., Jackson South. P. (2006). gammaH2AX and MDC1: anchoring the DNA-impairment-response machinery to broken chromosomes. DNA Repair five, 534–543. 10.1016/j.dnarep.2006.01.012 [PubMed] [CrossRef] [Google Scholar]
Sy Southward. M., Huen Chiliad. S., Chen J. (2009). PALB2 is an integral component of the BRCA complex required for homologous recombination repair. Proc. Natl. Acad. Sci. U.s.a.A. 106, 7155–7160. 10.1073/pnas.0811159106 [PMC costless commodity] [PubMed] [CrossRef] [Google Scholar]
Symington L. S. (2014). End resection at double-strand breaks: mechanism and regulation. Cold Spring Harb. Perspect. Biol. half dozen:a016436. 10.1101/cshperspect.a016436 [PMC costless article] [PubMed] [CrossRef] [Google Scholar]
Symington Fifty. S., Gautier J. (2011). Double-strand break end resection and repair pathway choice. Annu. Rev. Genet. 45, 247–271. 10.1146/annurev-genet-110410-132435 [PubMed] [CrossRef] [Google Scholar]
Szczelkun G. D., Tikhomirova M. S., Sinkunas T., Gasiunas One thousand., Karvelis T., Pschera P., et al. . (2014). Direct observation of R-loop germination by single RNA-guided Cas9 and Pour effector complexes. Proc. Natl. Acad. Sci. U.s.A. 111, 9798–9803. 10.1073/pnas.1402597111 [PMC gratis commodity] [PubMed] [CrossRef] [Google Scholar]
Tham K. C., Kanaar R., Lebbink J. H. (2016). Mismatch repair and homeologous recombination. Dna Repair 38, 75–83. 10.1016/j.dnarep.2015.11.010 [PubMed] [CrossRef] [Google Scholar]
Tsai C. J., Kim S. A., Chu K. (2007). Cernunnos/XLF promotes the ligation of mismatched and noncohesive Dna ends. Proc. Natl. Acad. Sci. U.S.A. 104, 7851–7856. 10.1073/pnas.0702620104 [PMC complimentary commodity] [PubMed] [CrossRef] [Google Scholar]
van der Heijden T., Seidel R., Modesti Thousand., Kanaar R., Wyman C., Dekker C. (2007). Real-time assembly and disassembly of human RAD51 filaments on individual Deoxyribonucleic acid molecules. Nucleic Acids Res. 35, 5646–5657. 10.1093/nar/gkm629 [PMC gratis article] [PubMed] [CrossRef] [Google Scholar]
van der Oost J., Jore M. 1000., Westra E. R., Lundgren M., Brouns Southward. J. (2009). CRISPR-based adaptive and heritable immunity in prokaryotes. Trends Biochem. Sci. 34, 401–407. 10.1016/j.tibs.2009.05.002 [PubMed] [CrossRef] [Google Scholar]
Vartak S. V., Raghavan S. C. (2015). Inhibition of nonhomologous end joining to increase the specificity of CRISPR/Cas9 genome editing. FEBS J. 282, 4289–4294. 10.1111/febs.13416 [PubMed] [CrossRef] [Google Scholar]
Walker J. R., Corpina R. A., Goldberg J. (2001). Construction of the Ku heterodimer bound to Deoxyribonucleic acid and its implications for double-strand break repair. Nature 412, 607–614. x.1038/35088000 [PubMed] [CrossRef] [Google Scholar]
Wang B., Matsuoka S., Carpenter P. B., Elledge S. J. (2002). 53BP1, a mediator of the Deoxyribonucleic acid damage checkpoint. Scientific discipline 298, 1435–1438. 10.1126/scientific discipline.1076182 [PubMed] [CrossRef] [Google Scholar]
Waters C. A., Strande N. T., Wyatt D. W., Pryor J. M., Ramsden D. A. (2014). Nonhomologous cease joining: a good solution for bad ends. DNA Repair 17, 39–51. 10.1016/j.dnarep.2014.02.008 [PMC free commodity] [PubMed] [CrossRef] [Google Scholar]
West S. C., Blanco One thousand. G., Chan Y. Westward., Matos J., Sarbajna S., Wyatt H. D. (2015). Resolution of recombination intermediates: mechanisms and regulation. Cold Leap Harb. Symp. Q. Biol. fourscore, 103–109. 10.1101/sqb.2015.80.027649 [PubMed] [CrossRef] [Google Scholar]
Weterings Due east., Verkaik N. Due south., Bruggenwirth H. T., Hoeijmakers J. H., van Gent D. C. (2003). The role of Dna dependent protein kinase in synapsis of DNA ends. Nucleic Acids Res. 31, 7238–7246. 10.1093/nar/gkg889 [PMC free commodity] [PubMed] [CrossRef] [Google Scholar]
Wiedenheft B., Sternberg Southward. H., Doudna J. A. (2012). RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331–338. ten.1038/nature10886 [PubMed] [CrossRef] [Google Scholar]
Williams M. J., Lees-Miller S. P., Tainer J. A. (2010). Mre11-Rad50-Nbs1 conformations and the control of sensing, signaling, and effector responses at Deoxyribonucleic acid double-strand breaks. Dna Repair nine, 1299–1306. ten.1016/j.dnarep.2010.10.001 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Williams R. South., Dodson One thousand. E., Limbo O., Yamada Y., Williams J. S., Guenther G., et al. . (2009). Nbs1 flexibly tethers Ctp1 and Mre11-Rad50 to coordinate DNA double-strand intermission processing and repair. Cell 139, 87–99. 10.1016/j.cell.2009.07.033 [PMC gratuitous article] [PubMed] [CrossRef] [Google Scholar]
Williams R. Southward., Moncalian G., Williams J. S., Yamada Y., Limbo O., Shin D. South., et al. . (2008). Mre11 dimers coordinate DNA end bridging and nuclease processing in double-strand-pause repair. Cell 135, 97–109. 10.1016/j.prison cell.2008.08.017 [PMC gratuitous commodity] [PubMed] [CrossRef] [Google Scholar]
Wu 50., Hickson I. D. (2003). The Bloom'southward syndrome helicase suppresses crossing over during homologous recombination. Nature 426, 870–874. 10.1038/nature02253 [PubMed] [CrossRef] [Google Scholar]
Wyatt H. D., West Southward. C. (2014). Holliday junction resolvases. Cold Spring Harb. Perspect. Biol. half-dozen:a023192. 10.1101/cshperspect.a023192 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Yang D., Scavuzzo M. A., Chmielowiec J., Sharp R., Bajic A., Borowiak M. (2016). Enrichment of G2/Thou cell cycle phase in human pluripotent stem cells enhances HDR-mediated cistron repair with customizable endonucleases. Sci. Rep. 6:21264. 10.1038/srep21264 [PMC gratuitous article] [PubMed] [CrossRef] [Google Scholar]
Yang H., Wang H., Shivalila C. S., Cheng A. Westward., Shi L., Jaenisch R. (2013). One-step generation of mice carrying reporter and conditional alleles by CRISPR/Cas-mediated genome technology. Cell 154, 1370–1379. x.1016/j.cell.2013.08.022 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Yu C., Liu Y., Ma T., Liu 1000., Xu S., Zhang Y., et al. . (2015). Small molecules enhance CRISPR genome editing in pluripotent stem cells. Cell Stalk Jail cell sixteen, 142–147. ten.1016/j.stem.2015.01.003 [PMC free article] [PubMed] [CrossRef] [Google Scholar]
Yu S., Vocal Z., Luo J., Dai Y., Li N. (2011). Over-expression of RAD51 or RAD54 only not RAD51/iv enhances extra-chromosomal homologous recombination in the homo sarcoma (HT-1080) jail cell line. J. Biotechnol. 154, 21–24. 10.1016/j.jbiotec.2011.03.023 [PubMed] [CrossRef] [Google Scholar]
Zelensky A., Kanaar R., Wyman C. (2014). Mediators of homologous DNA pairing. Cold Spring Harb. Perspect. Biol. 6:a016451. 10.1101/cshperspect.a016451 [PMC gratuitous article] [PubMed] [CrossRef] [Google Scholar]
Zou L., Elledge South. J. (2003). Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science 300, 1542–1548. 10.1126/science.1083430 [PubMed] [CrossRef] [Google Scholar]

Articles from Frontiers in Genetics are provided here courtesy of Frontiers Media SA