Genome editing efficiency using spacers varies depending upon the genomic region from where they were derived. Editing is highest for promoter regions followed by the exonic regions and intronic regions. This is because of the variability in retrieving spacers to the relevant target region, which further depends upon the variable GC content of the spacers (Labuhn et al., 2017), as different regions of the genome adopt various conformations based upon variation in their GC content (Pozzoli et al., 2008; Amit et al., 2012).

The GC content of the spacer region of designed sgRNA indirectly indicates the strength of the interaction between the spacer and protospacer sequences in various life domains that may affect the overall endonuclease activity of various Cas enzymes. In animals, spacers with average GC content tend to be more effective unlike those with unusually low or high GC content (Wang et al., 2014). Similarly, spacers in plants with GC content between 30 and 80% have been practically validated (Liang et al., 2016). However, lesser editing efficiency is observed in plants with 40% GC content as compared to those with higher GC content (Pan et al., 2016b). In mammalian cells, spacers with very low or very high GC content are less effective; however, 40–60% content is favorable for efficient genome editing (Doench et al., 2014; Wang et al., 2014; Liu et al., 2016c). Overall, spacers with more than 50% GC content often show high genome editing efficiency in microbes, animals, and plants (Jiang et al., 2013a; Jiang et al., 2013b; Feng et al., 2014; Wang et al., 2014; Zhang et al., 2014; Pan et al., 2016a). Inefficient sgRNAs can also be excluded by monitoring the GC content in the PAM distal region (4–13 nucleotides) (Labuhn et al., 2017). However, bulges arise between the sgRNA-DNA hybrid when GC content equals 70% (Lin et al., 2014c). DNA bulges formed due to mismatches 7–10 bp from PAM; thus, the 3′end or 5′end can result in mismatches, so these must be avoided (Lin et al., 2014b).

Nucleotide composition (purine or pyrimidine) of spacers may also affect Cas9 binding and nuclease activity. In animal models, it has been reported that the 3′ sequence of the spacer contributes to Cas9 binding preference, and purines are better to recruit the Cas9 enzyme than pyrimidines which eventually affects its nuclease activity (Wang et al., 2014). Doench et al., 2014) reported Cas9 preference for guanine and against cytosine at position 20; for cytosine and against guanine at position 16; for adenine in the middle and against cytosine at position three of the spacer sequence. Similarly, Xu et al. (2015a) revealed Cas9 preference for guanine at positions 19 and 20; against thymine from positions 17 to 20; for cytosine at position 18 (CRISPR/Cas9 complex DNA cleavage site); for adenines from positions nine to sixteen; and for guanines from positions four to seven. Moreover, PAM downstream nucleotides, unlike spacer upstream sequences, contribute to the editing efficiency of spacer (Doench et al., 2014; Xu et al., 2015a). The sequences rich in guanine can form noncanonical stable structures in vivo, called as G-quadruplexes (Huppert, 2008). Moreno-Mateos et al. (2015) injected in vitro transcribed guide sequences into zebrafish model and reported that sequences with more than eight guanines undergo G-quadruplex structures which are more efficient to edit due to their increased stability. However, this modification is not essential in case the guide is being expressed from constitutive promoter (like U6) after lentiviral transduction. On the other hand, these G-rich guides are not efficient in cultured mammalian cells (Malina et al., 2015). In contrast, no such nucleotide preference is observed in plants revealing a major difference in spacer designed to use in plants and animals (Liang et al., 2016).

Transcription from the U6 promoter by RNA polymerase III is inhibited when there is TTT stretch in the DNA, so guide RNAs with a UUU stretch (particularly in the seed region) are inefficient under the U6 promoter, and editing is more favorable when a stable duplex is formed between the target DNA and the guide RNA (Wong et al., 2015). Moreover, particular sequence motifs that affect the synthesis of the tracrRNA structures required for Cas9 interaction may also decrease guide activity. The presence of uridine at four nucleotide proximal to PAM makes it difficult for guide RNA to interact with Cas9 eventually resulting in lower activity (Wang et al., 2014; Hart et al., 2015).

To optimize sgRNA structure, researchers found that 1) substituting one of the T’s in four consecutive T’s with an A (to distort the string) shortly downstream of the spacer and 2) extending the sgRNA duplex region by five-nucleotides changes the sgRNA transcription rate and its structure, respectively, resulting in improved efficiency (Chen et al., 2013; Dang et al., 2015). This strategy also produced promising results with SaCas9 sgRNAs, signifying that this could be applied to other Cas9 orthologues to achieve maximum efficiency for different applications (Chen et al., 2016; Tabebordbar et al., 2016).

Ma et al. (2013) targeted the PVALB gene of humans with various sgRNAs and concluded that the absence of sgRNA secondary structures increases binding with the target gene, leading to more efficient genome editing. However, subsequent studies supported the existence of a secondary structure. Briefly, sgRNA consists of a crRNA sequence {guide/spacer [20 nucleotides (nt)] + repeat region [12 nt]} and a tracrRNA sequence [anti-repeat (14 nt)] and three stem loops (stem loops 1, 2, and 3). The fourth loop (called the RAR loop) is formed by the bases of repeat and anti-repeat regions that trigger RNase III-mediated processing of precursor CRISPR RNA (pre-crRNA). Stem loop 1 is important for formation of the Cas9-sgRNA-DNA functional complex while the other two loops provide stability to the complex and enhance in vivo function (Nishimasu et al., 2014). Liang et al. (2016) reported that all stem loops (except stem loop 1) must have an intact secondary structure for efficient genome editing.

GGX20 modification of sgRNA involves addition of two additional mismatched guanines at the 5′ end of the designed guide RNA. Several studies have shown that this sgRNA modification enables them to differentiate the on-target site from other homologous sites differing by two or more nucleotides eventually increasing on-target specificity of sgRNA 10–100 times, but it also affects editing efficiency at the on-target and off-target locations (Cho et al., 2013; Fu et al., 2013; Cho et al., 2014; Sung et al., 2014; Kim et al., 2015). Moreover, only certain guides are compatible with such a modification, so there is a need to further validate this modification with different lengths and chemically modified sgRNAs.

Another specificity mediator of CRISPR/cas9 technology is the length of the designed sgRNA. Extending Spcas9 guide sequence from 20 nt to 30 nt to increase specificity results in its processing back to natural 20 nt length, so it is useless to increase length (Ran et al., 2013a). Short length (17 or 18 nt) spacer sequences, called truncated spacers or guides, have been reported to potentially increase Cas9 binding sensitivity to mismatches present within a smaller complementary sequence which causes more accurate but less active genome editing (Pattanayak et al., 2013; Fu et al., 2014). Even after eliminating off-target activity at many sites, truncated guides produce new off-target sites due to their shorter length (Fu et al., 2014; Tsai et al., 2015; Wyvekens et al., 2015; Slaymaker et al., 2016). Interestingly, these truncated guides show very low on-target efficiency when used with modified SpCas9 versions, such as eSpCas9 and SpCas9-HF (Kleinstiver et al., 2016; Slaymaker et al., 2016). Improvements in specificity need to be evaluated in other Cas9 orthologues with intact editing efficiency using these guide modifications (Hou et al., 2013; Friedland et al., 2015; Ran et al., 2015). Jinek et al. (2012b) detected efficient in vitro cleavage using truncated crRNA and tracrRNA but failed to detect cleavage at various loci previously modified using crRNA-tracrRNA duplexes with identical guide sequences (Cong et al., 2013b). Another study revealed that this cleavage difference is due to the length of tracrRNA sequence, as they used sgRNAs with tracrRNA tails extended to +67 and +85 nt to mediate cleavage at all previously tested target sites and found a five-fold increase in the level of indels than in the corresponding crRNA-tracrRNA duplex. They also observed increased expression of sgRNAs having longer tracrRNA sequences, predicting that higher sgRNA stability or expression is responsible for improved cleavage of the target sequence (Ran et al., 2013a).

In one study, full length 100 nt sgRNAs (crRNA + tracrRNA) were synthesized with three various chemical modifications of 2′-O-methyl (M), 2′-O-methyl 3′phosphorothioate (MS), or 2′-O-methyl 3′thioPACE (MSP) at the 5 and 3′ termini, which improved editing efficiency in CD34 ⁺ hematopoietic progenitor and stem cells, and human primary T-cells along with off-target activity at a few sites (Hendel et al., 2015). In another study, a 29 nt crRNA (scrRNA) phosphorothioate (PS) backbone was synthesized with chemical substitutions of 2′-fluoro (2′-F), 2′-O-methyl (2′-O-Me) and S-constrained ethyl (cEt) which increased their specificity but not on-target efficiency in human cells (compared to unmodified crRNA) due to its increased binding affinity to tracrRNA and metabolic stability (Rahdar et al., 2015). These contrasting results indicate that more exploration of chemically modified sgRNAs or only crRNAs is required to reach a conclusion.

The efficiency of guide RNA is difficult to predict due to its sequence dependency (Liu et al., 2016a), so a good option is to choose the best one from three tested gRNAs (Ran et al., 2013a) after confirmation by the T7E1 endonuclease assay (Larcher et al., 2014; Vouillot et al., 2015) and direct Sanger sequencing of the target sequence PCR products, particularly for knock-in experiments (Brinkman et al., 2014), DNA capillary electrophoresis of amplified products (Dahlem et al., 2012; Yang et al., 2015), or using fluorescent reporters (Kim et al., 2011). In many studies, guide efficiency correlating scores and sequence criteria have been identified which helps to reduce guide RNA number to test for genome editing. A list of online tools to predict the efficiency of sgRNA is shown in Supplementary Table S2. After predicting sgRNA using any of these tools, the highest score value should be obtained to increase efficiency and specificity.

It depends upon the type of promoter and the host cell line. Most studies have employed the RNA polymerase III promoter (mostly constitutive and consisting of a few cellular promoters) for sgRNA expression which makes conditional or induced expression impossible (Orioli et al., 2012). Researchers have expressed sgRNA from artificial gene RGR which produces sgRNA mRNA with a ribozyme sequence at the flanking ends after transcription following cleavage and generation of a mature sgRNA with high in vitro and yeast genome editing (Gao and Zhao, 2014). Multiplex genome editing in human cells has been achieved using a cell-type specific promoter and the Csy-4 dependent method to form separate mature gRNAs from the same precursor mRNA to improve genome editing efficiency (Nissim et al., 2014). A synthetic hybrid promoter consisting of tRNA and RNA polymerase III has been employed in Yarrowia lipolytica to enhance gRNA expression, resulting in 100% transformants by inhibiting the NHEJ process (Schwartz et al., 2016).

The sgRNA expression level is one of the determinants for increased on-target efficiency of Cas9. Higher expression levels of sgRNA and the repair template increase genome editing in rice (Sun et al., 2016), tobacco (Baltes et al., 2014), S. cerevisiae (Stovicek et al., 2015), Yarrowia lipolytica (Schwartz et al., 2016), and mammals (Ran et al., 2013a). In contrast, higher sgRNA level limited genome editing efficiency in Arabidopsis (Ma et al., 2015b) and tomato (Pan et al., 2016a). Xie et al. (2015) reported that expression levels of Cas9 and sgRNA are lower in transgenic plants or the callus compared to the protoplast, which may affect editing efficiency. Ranganathan et al. (2014) reported less off-target activity than on-target activity in a human cell line when guide RNA was expressed from a weaker H1 promoter. These results indicate species-specific editing-efficiency based on the sgRNA expression level which needs to be further confirmed using more advanced approaches.

Specificity and kinetics of gene editing can be affected by the level of Cas9 protein (Ran et al., 2013a; Pattanayak et al., 2013; Fu et al., 2014). For example, five-time drop in the level of Cas9 protein increased its specificity seven-times by affecting the on-target efficiency for just two-times (Ran et al., 2013a). Despite extensive application of CRISPR/Cas9 technology in plants for genome editing, there is variation in editing efficiencies, as the Cas9 expressing promoter, Cas9 codon optimization, and the positional effect can directly affect the Cas9 expression level and targeting efficiency (Wang et al., 2015a; Ma et al., 2015b; Yan et al., 2015; Mao et al., 2016). Constitutive Cas9 expression results in higher genome editing in mice (Platt et al., 2014), mammalian cells (Koike-Yusa et al., 2014), S. cerevisiae (DiCarlo et al., 2013), and rice (Mikami et al., 2015). In contrast to rice and some other higher plants, a higher Cas9 level reduces editing efficiency in mosaic Arabidopsis (Mao et al., 2013; Feng et al., 2014; Jiang et al., 2014; Ma et al., 2015b; Yan et al., 2015) and toxicity in Chlamydomonas reinhardtii (Shin et al., 2016) while no toxicity was observed in Aspergillus fumigatus with Cas9 constitutive expression (Fuller et al., 2015). Moreover, reduced search efficiency of Cas9 for heterochromatic regions indicates that access to DNA target site also contributes to specificity and efficiency (Knight et al., 2015).

Besides constitutive Cas9 expression, induced (chemical and light) and tissue-specific expression has also been evaluated in various studies to improve genome editing efficiency and specificity. In the case of CRISPR/Cas9 technology, chemically induced expression is achieved by using either an inducible promoter (e.g., doxycycline) or a Cas9 variant. Doxycycline-inducible Cas9 (iCas9) has been reported in vivo by genetic screening studies in humans (Wang et al., 2014); genome editing studies in mice (Dow et al., 2015); and human iPSCs (Zhu et al., 2015). This iCas9 system has also been reported in vitro for cell lineage specific reprogramming studies in a mouse cell line (Chakraborty et al., 2014); genomic loci imaging studies in human cell lines (Chen et al., 2013); reversible gene silencing studies in human iPSCs (Mandegar et al., 2016); multiplexed gene activations studies in cells and zygotes (Cheng et al., 2013); and biallelic gene knockout studies in hiPSCs (González et al., 2014). Cas9 variants have been developed to induce their activity in the presence of small cell-permeable 4-hydroxytamoxifen (4-HT) by combining Cas9 with either 4-HT responsive intein (Davis et al., 2015) or hormone-binding domain of the estrogen receptor (ERT2) (Liu et al., 2016b). Similarly, a light induced Cas9 system has been validated in mammalian cells to study: 1) transcriptional regulation using the p65-CRY2/dCas9-CIB1 construct (Nihongaki et al., 2015b) and the VP64-CRY2/CIB1-dCas9-CIB1 construct (Polstein and Gersbach, 2015) and 2) optogenetic control of genomic editing using the Cas9N713-pMag/nMagHigh1-Cas9C714 construct (named photoactivatable Cas9 (paCas9-1)) (Nihongaki et al., 2015a). The p65-CRY2/dCas9-CIB1 photoactivatable system constitutes sgRNAs and two fusion proteins. The first fusion protein constitutes dCas9 and CIB1; which acts like a genomic anchor probe and binds to target sequence with the help of sgRNAs. The second fusion protein constitutes transcriptional activator domain and photolyase homology region of CRY2 (CRY2PHR) which acts like an activator probe. The CIB1 and CRY2PHR are heterodimerized with the blue light irradiation and transcription is activated with the recruitment of activator domain to the target locus (Nihongaki et al., 2015b). Similarly, VP64-CRY2/CIB-dCas9-CIB1 construct constitutes light-inducible heterodimerizable proteins CRY2 and CIB1 attached to transactivation domain (VP64) and either C- or N-terminal catalytically inactive Cas9 (dCas9), respectively. This system is also directed to the target site with the help of sgRNAs and activates the transcription with blue light (Polstein and Gersbach, 2015). The Cas9N713-pMag/nMagHigh1-Cas9C714 construct constitutes photoinducible dimerization domains (named as Magnets) and split Cas9 fragments. Upon blue light irradiation, paCas9 induces genome editing by involving both homology-directed repair and nonhomologous end joining (Nihongaki et al., 2015a).

To improve spatial genome editing, tissue-specific promoters are being used in zebrafish (Ablain et al., 2015), drosophila (Xue et al., 2014b), mammalian cells (Yoshioka et al., 2015), and plants (Osakabe et al., 2016). Similarly, the mosaic effect in plants is reduced by using specialized promoters such as INCURVATA 2 promoter (Hyun et al., 2015), the meristem-specific YAO promoter (Yan et al., 2015), the germline-specific SPL promoter (Mao et al., 2016), the egg-cell-specific DD45 (Mao et al., 2016), and EC1.2 promoters (Wang et al., 2015b). For effective Cas9 expression in human iPSCs, EF1α promoter is proved stronger than CAG promoter (Matsui et al., 2014), while CMV and SV40 are not recommended due to transcriptional silencing (Hotta and Ellis, 2008). Programmable and multiplexed regulation of various gene networks in human cells is achieved by combining the RNA regulatory strategies with Cas9 based transcription factors (Nissim et al., 2014). For example, immune responses against transgene products can be minimized by using the mir-142-3p which can repress the expression of associated cellular transcripts by binding to their target sequences (Majowicz et al., 2013).

Cas9 is a sequence-specific endonuclease that is delivered to the host cell or organism as DNA, mRNA, or protein for a genome editing experiment. The first human genome editing was performed in 2013 in which all CRISPR components were delivered in the form of DNA plasmids and/or expression cassettes (Mali et al., 2013b). With advancements in the field, various vectors and methods have been attempted to achieve maximum editing efficiency and precision. For example, five-fold more HDR efficiency was achieved with a particle bombardment technique in rice (Sun et al., 2016) and maize (Svitashev et al., 2015) compared to Agrobacterium-mediated transformation. Enhanced genome editing was observed by 1) introducing components using adenovirus vector (AV) or adeno-associated vector (AAV) in mice (Senís et al., 2014; Swiech et al., 2015; Rodriguez et al., 2016) and a lentivirus into melanoma cell lines (Shalem et al., 2014) and 2) using the hydrodynamic injection technique in mice (Xue et al., 2014a). Mostly transfection methods such as electroporation or lipofectamine are used to transfer CRISPR components into host cell (Costa et al., 2007) but nucleofector is commonly used for human iPS cells genome editing (Ran et al., 2013b).

AAV vectors are common delivery tools due to safety and efficiency (Kaufmann et al., 2013; Kotterman and Schaffer, 2014; Kotterman et al., 2015). One problem is the size of the commonly used SpCas9 gene, which cannot be accommodated in wild type AAV, so smaller orthologues (Esvelt et al., 2013) or engineered AAV (Kotterman and Schaffer, 2014) are used as a solution. Moreover, orthologues have been developed to simultaneously perform multiplexed RNA-guided transcriptional repression, activation, and gene-editing (Esvelt et al., 2013). Similarly, AAV vector has shown some efficacy in multiple monogenic disorders including choroideremia (MacLaren et al., 2014), haemophilia B (Nathwani et al., 2011), and Leber’s congenital amaurosis type 2 (Hauswirth et al., 2008; Bennett et al., 2012). Lentiviral vectors have been used to study 1) drug resistant and cell viability genes via loss-of-function mutation in mouse and human cells (Koike-Yusa et al., 2014; Shalem et al., 2014; Wang et al., 2014; Zhou et al., 2014); 2) leukemia causing genes in mouse hematopoietic cells (Heckl et al., 2014); 3) the role of p53 and Pten in the formation of liver tumors using mouse 3T3 cells (Xue et al., 2014a) and 4) Kras gain-of-function mutation (KRAS G12D) in the Neuro-2a neuroblastoma cell line (Platt et al., 2014).

Cas9 and sgRNA show persistent expression when delivered in the form of a cloned vector (Xue et al., 2014a; Yin et al., 2014; Yang et al., 2016); however, it might cause mutation with random integration of CRISPR components at on-target and off-target sites into the host genome (Cradick et al., 2013; Fu et al., 2013). The Cas9mRNA/sgRNA or Cas9protein/sgRNA is injected into the host embryo or zygote for specific genome editing. For example, the injection of sgRNA and Cas9mRNA causes 1) targeted mutagenesis in 88% of embryonically injected flies with a 33% transmittance rate (Bassett et al., 2013), 2) generation of one-step multiple allelic mutated mice via zygotic injection (Wang et al., 2013), 3) correction of mouse crygc allele (Wu et al., 2013) and dystrophin gene (Long et al., 2014) via zygotic injection, and 4) correction of human beta-hemoglobin gene via tripronuclear zygotic injection (Liang et al., 2015). Similarly, genome editing efficiency and specificity can be increased by 1) using the Cas9 protein and sgRNA delivered by electroporation (Kim et al., 2014), protein transduction (Ramakrishna et al., 2014), and lipofection (Zuris et al., 2015), 2) using the MS-modified sgRNA and Cas9 protein complex (Hendel et al., 2015), 3) synchronizing the delivery of ribonucleoprotein (RNP) with the stage of cell cycle in human cells (Lin et al., 2014a) and 4) using a solubilizing salt solution for the fluorescent Cas9/sgRNA complex in zebrafish embryos (Burger et al., 2016). The mixture of Cas9 mRNA and gRNA (Zhang et al., 2016b) or pre-assembled mixture of RNP (Woo et al., 2015; Svitashev et al., 2016; Liang et al., 2017) can be delivered to plant cells for efficient genome editing. These delivery methods are summarized in Supplementary Table S3.

GC content is related to genome size in bacteria, but it is very complex in eukaryotes due to repetitive DNA segments and isochores. There is huge variation in GC content among microbes but less of a range in plants and animals, e.g., monocots and vertebrates have higher GC content than dicots and invertebrates, respectively. There is more GC content in animal chromosomes than that in plants but no correlation between chromosome size and GC content has been observed (Li and Du, 2014). In the case of CRISPR/Cas technology, target sequence GC content affects genome editing efficiency (Zhang et al., 2014; Ma et al., 2015b). For example, higher GC content (>70%) facilitates hybridization between the spacer and the protospacer, which may increase off-targets (Lin et al., 2014b; Tsai et al., 2015; Li et al., 2016). GC content lower than 30% also results in higher off-targets (Fu et al., 2013; Pattanayak et al., 2013). However, no off-targeting was observed with GC content of 57% in soybeans (Jacobs et al., 2015); 50% in Arabidopsis (Sauer et al., 2016); and 50–70% in rice (Ma et al., 2015b).

The genomic sites of the host target sequence that pair six continuous nucleotides with the sgRNA scaffold should not be selected because it results in the formation of a stem loop structure between the sgRNA scaffold and target sequence, which hinders perfect binding with the target sequence ultimately leading to less efficient genome editing (Ma et al., 2015b).

DNA exists in two chromatin states, i.e., euchromatin (less condensed) and heterochromatin (highly condensed); however, accessibility to the latter is a bit difficult. Thus, Cas9 targeting a DNA site in the highly condensed portion leads to decreased binding (Wu et al., 2014; Verkuijl and Rots, 2019), which may be due to less accessibility to the PAM sequence (Hinz et al., 2015). The heterochromatin state of the target sequence can reduce the diffusion of Cas9 (Knight et al., 2015) and CRISPR-Cas9 mediated mutagenesis by 7-fold (Kallimasioti-Pazi et al., 2018). The presence of epigenetic modifications or transcription factors may also affect Cas9 binding following genome editing. It has also been observed that sgRNAs targeting non-transcribed DNA strands are more effective compared to those targeting transcribed DNA strands (Wang et al., 2014). The cleavage efficiency of SpCas9 is independent of methylation of the target region (Ran et al., 2013a).

CRISPRa is achieved by fusing the dCas9 enzyme to transcriptional activators, such as an ω subunit of RNA polymerase in bacterial cells (Bikard et al., 2013) and p65AD or VP64 in mammalian cells (Mali et al., 2013a; Gilbert et al., 2013; Konermann et al., 2013; Perez-Pinera et al., 2013). Various strategies have been adopted to enhance the efficiency of a CRISPRa system by recruiting multiple transcriptional activators: 1) In addition to multiple sgRNAs at a single promotor to recruit multiple activators (Cheng et al., 2013; Maeder et al., 2013; Chavez et al., 2015), strategies to recruit multiple activators to the dCas9 binding site have been developed (Tanenbaum et al., 2014; Chavez et al., 2015; Konermann et al., 2015). For example, the synergistic activation mediator system allows multiple activators to work synergistically by using both sgRNA and dCas9 as scaffolds (Konermann et al., 2015). In this system, sgRNA modified with two MS2 RNA aptamers is combined with dCas9-VP64. Each aptamer recruits a pair of similar RNA binding proteins, MCPs (MS2 bacteriophage coat proteins), which are bound with p65-and HSF1-activating domains (MCP-p65-HSF1) (Konermann et al., 2015; Nishimasu et al., 2015). This system has been applied for large-scale genome screening because of its increased efficiency (Konermann et al., 2015). 2) Multiple VP64 activators can be recruited to a single dCas9 binding site by combining the dCas9 system with multipeptide array, such as SunTag. For example, fusion of dCas9 to the polypeptide array (GCN4s) recruits multiple (10 or 24) copies of its cognate scFv (single-chain variable fragment), an engineered portion of the anti-GCN4 antibody. This scFv was further fused to VP64 which eventually led to strong upregulation of the target gene [chemokine (C-X-C motif) receptor 4 (CXCR4)] because of multiple VP64 copies per dCas9 (Tanenbaum et al., 2014). This system has also been used to reduce cell growth by upregulating the expression of CDKN1B (cyclin-dependent kinase inhibitor 1B) (Tanenbaum et al., 2014) and gain-of-function screening at the genome scale (Gilbert et al., 2014). 3) A tripartite activator system has been developed by fusing three various activators in tandem to dCas9 [VP64-p65-Rta (VPR)] which showed greater activation efficiency than dCas9-VP64 when used with multiple sgRNAs (Chavez et al., 2015). Reprogramming of target cells can be achieved by using multiple approaches (Anwar et al., 2016; Lee et al., 2017) and CRISPR activation system is one of them (Balboa et al., 2015). All of these systems mimic the intrinsic mechanism of gene activation by recruiting multiple activators at the target site (He and Weintraub, 1998; Govind et al., 2005).

Similarly, dCas9 can be used to recruit epigenetic modifiers at a given locus to reshape the epigenome. For example, Neisseria meningitidis dcas9 (Nm dCas9) fused to histone demethylase LSD1 can reduce the expression of genes (which are controlled by the targeted enhancers) by decreasing the epigenetic marks H3K27ac and H3K4me2 near the targeted enhancer region (like Oct4 and Tbx3) (Kearns et al., 2015). Fusing the catalytic core domain of histone acetyltransferase p300 with dCas9 (Nm dCas9-p300 core and Sp dCas9-p300 core) can be used to activate the transcription of various endogenous genes by increasing the H3K27ac level at the targeted enhancer or promoter regions (Hilton et al., 2015).

The dCas9 mediated regulation of gene expression is executed by recruiting various proteins and RNA factors at the target site (Gilbert et al., 2013; Qi et al., 2013). Transcriptional inhibition by the CRISPR machinery is termed CRISPRi and only dcas9 is sufficient in bacterial cells via steric hindrance of the transcriptional machinery (Bikard et al., 2013; Qi et al., 2013). However, it is not very effective in mammalian cells unless dCas9 is fused to a transcriptional repressor domain (such as KRAB of Kox1) (Gilbert et al., 2013; Konermann et al., 2013).

One of the important things in genome biology is to understand the correlation between linear genetic information imprinted on DNA and its three-dimensionally compact organization inside the cell nucleus because many studies have highlighted the impact of organization on the regulation of gene expression and cell differentiation (Lanctôt et al., 2007; Schneider and Grosschedl, 2007; Peric-Hupkes et al., 2010; Dixon et al., 2015). Because of the lack of a proper tool, it was difficult to visualize genomic dynamics in a sequence-specific manner. However, it has become possible with the sequence-specific binding of the dCas9 regardless of the genome architecture and epigenetic state. For example, fusing Streptococcus pyogenes dCas9 (Sp dCas9) to enhanced GFP can be used to visualize the repetitive and nonrepetitive genomic loci in living human cells (Chen et al., 2013). A similar approach was used to label the endogenous telomeres, pericentric regions, and centromeres (Anton et al., 2014). dCas9-based genome imaging has been expanded with various improvements. The SunTag peptide array with dCas9 was exploited to successfully amplify imaging strength (Tanenbaum et al., 2014). Multicolor genome imaging was achieved by individually tagging St1 dcas9, Nm dCas9, and Sp dCas9 with differently colored fluorescent proteins (FPs) and targeting each of them to different loci by the corresponding sgRNAs (Ma et al., 2015a). Fixed cells and tissues can be labeled by in vitro-assembled complexes of fluorescently labeled dCas9-sgRNA, a technique known as CASFISH (Deng et al., 2015). Using a single particle tracking method with fused dCas9 and the HaloTag system, it was found that there is a three-dimensional diffusion based analysis of genome by dCas9, with transient binding at off-targets and reduced efficiency to search at heterochromatic regions (Knight et al., 2015).

The sole or complex of different transcription factors interact with the target DNA by recognizing some consensus sequences (Yesudhas et al., 2017a; Yesudhas et al., 2019). Endogenous proteins interacting with a specific genome region can be found by using the CRISPR-based engineered DNA-binding molecule-mediated ChIP (enChIP) method. The loci specific proteins are pulled down by targeting the affinity-tagged dCas9 at that region using associated sgRNAs. This system has been successfully tested to analyze proteins interacting with an interferon-γ-responsive promoter (Fujita and Fujii, 2013; Fujita and Fujii, 2014).