Cas9-based genome-editing systems

Biochimie 167 (2019) 49e60

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Biochimie journal homepage: www.elsevier.com/locate/biochi

Review

Guide RNA modification as a way to improve CRISPR/Cas9-based genome-editing systems Julia Filippova a, 1, Anastasiya Matveeva a, b, 1, Evgenii Zhuravlev a, Grigory Stepanov a, b, * a b

Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Lavrentiev Avenue, 8, 630090, Novosibirsk, Russia Novosibirsk State University, Pirogova Str, 1, 630090, Novosibirsk, Russia

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 February 2019 Accepted 2 September 2019 Available online 4 September 2019

Genome-editing technologies, in particular, CRISPR systems, are widely used for targeted regulation of gene expression and obtaining modified human and animal cell lines, plants, fungi, and animals with preassigned features. Despite being well described and easy to perform, the most common methods for construction and delivery of CRISPR/Cas9-containing plasmid systems possess significant disadvantages, mostly associated with effects of the presence of exogenous DNA within the cell. Transfection with active ribonucleoprotein complexes of Cas9 with single-guide RNAs (sgRNAs) represents one of the most promising options because of faster production of sgRNAs, the ability of a researcher to control the amount of sgRNA delivered into the cell, and consequently, fewer off-target mutations. Artificial-RNA synthesis strategies allow for the introduction of various modified components, such as backbone alterations, native structural motifs, and labels for visualization. Modifications of RNA can increase its resistance to hydrolysis, alter the thermodynamic stability of RNAeprotein and RNAeDNA complexes, and reduce the immunogenic and cytotoxic effects. This review describes various approaches to improving synthetic guide RNA function through nucleotide modification. © 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

Keywords: Guide RNA Single-guide RNA RNA modification Genome editing CRISPR/Cas9

Contents 1. 2. 3. 4. 5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49 Strategies involving synthetic sgRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 50 Optimization of sgRNA structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Backbone modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52 Multifunctional gRNA modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56 gRNA modifications lead to a decrease in an innate immune response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 Funding . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58

1. Introduction * Corresponding author. Institute of Chemical Biology and Fundamental Medicine, Siberian Branch, Russian Academy of Sciences, Lavrentiev Avenue, 8, 630090, Novosibirsk, Russia. E-mail addresses: fi[email protected] (J. Filippova), anastasiya.maatveeva@ gmail.com (A. Matveeva), [email protected] (E. Zhuravlev), stepanovga@ niboch.nsc.ru (G. Stepanov). 1 These authors contributed to this work equally.

The basic principle of a genome-editing strategy is generation of double-strand breaks (DSBs) in a DNA region of interest. Once the DSBs are generated, they are repaired in the cell either via the mechanism of homologous recombination (HR) or nonhomologous end joining (NHEJ) resulting in local structural changes in the gene.

https://doi.org/10.1016/j.biochi.2019.09.003 0300-9084/© 2019 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.

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J. Filippova et al. / Biochimie 167 (2019) 49e60

There are several classes of genome-editing systems, and the most widely used are transcription activatorelike effector nucleases (TALENs) [1,2], zinc-finger nucleases (ZFNs) [3,4], and the clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system [5,6]. There is a “common” mechanism for all classes, which involves a nuclease responsible for DNA cleavage at a site of interest and a guide element responsible for target recognition. The selection of a genome-editing tool from various classes of sequencespecific nucleases depends on the target DNA region and cell line because some nucleases might yield higher efficiency in each specific case. The CRISPR system, a tool widely used in many experiments on genome editing, represents an engineered analog of the type II CRISPR microbial antiviral immune system first discovered in Streptococcus thermophiles; this system provides prokaryotic acquired immunity against bacteriophage infection [5,7,8]. CRISPR is an array of short repeated sequences, first discovered in 1987 [9]. Later, it has been shown that between the repeats there are short sequences, which match viral genomes [8,10,11]. The main components of the CRISPR/Cas9 system are CRISPR-associated protein 9 (Cas9) and guide RNA (gRNA) composed of two noncoding RNAs: a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). CRISPR/Cas9 requires a G-rich (sequence: NGG) protospacer adjacent motif (PAM) sequence at the 30 end of the target sequence for target recognition and subsequent cleavage. It has been shown that a crRNA and tracrRNA can be fused into a single RNA molecule, namely single guide RNA (sgRNA) (Fig. 1 A) [12]. CRISPR systems can be employed to solve a variety of tasks in the fields of basic and applied science. CRISPR-dependent incorporation of indels (insertions/deletions) into a genome can lead to a frameshift and gene knockout [6]. Transcriptional activation (CRISPRa) or repression via CRISPR interference (CRISPRi) can be achieved too by means of CRISPR [13,14]. Several studies have revealed the possibility of introducing epigenetic modifications into a genome [15e17] and imaging and visualizing genomic elements in living or fixed cells [18e21]. Application of the CRISPR technology holds promise for modifying plants for agriculture [22e25] and fungi, bacteria, and animals for biotechnology [26e28]. Genome editing is used for genome-wide screening [29,30], creation of disease models for further study of molecular pathological pathways, and for their translation into clinical practice [31e33]. In addition, systems for rapid viral diagnostics have been developed on the basis of the Cas13 ribonuclease [34]. The key challenge of CRISPR-based genome editing systems is that desirable efficiency and specificity are not always achievable. The vast majority of studies on genome editing are focused on

vector constructs for intracellular synthesis of sgRNA. Nonetheless, synthetic sgRNAs can be an efficient alternative. This review deals with the approaches to genome editing involving synthetic sgRNAs. We review and summarize various empirical results on the application of RNA modifications for improvement and enhancement of CRISPR genome editing systems. 2. Strategies involving synthetic sgRNAs Synthetic sgRNA enables choosing one of the following genomeediting strategies: co-transfection with a Cas9-encoding plasmid or mRNA, delivery of a ribonucleoprotein (RNP) containing both a recombinant Cas9 protein and sgRNA, and transfection of sgRNA into cells expressing Cas9 from a plasmid vector or a genomic cassette (Fig. 1 B). Genome editing strategies based on synthetic sgRNAs are quite promising and offer a few advantages over the methods including plasmids encoding components of the CRISPR/ Cas9 system. The ability to deliver a CRISPR system into the cell “safe and sound” is important for the implementation of its regulatory effect. One of the approaches can be the improvement of sgRNA stability and activity within the cell through incorporation of chemical modifications into its structure. Among the advantages of synthetic sgRNA over DNA plasmid constructs are the lack of adverse effects of the constitutive presence of exogenous DNA within the cell, as in the case of vector constructs, and more stringent control of the RNA amount delivered into the cell. The use of DNA constructs for a targeted gene knockout leads to constitutive expression of CRISPR/Cas9 components, which eventually results in formation of a large number of off-target DSBs in the genome [35e38]. Moreover, the plasmid DNA introduced into the cell is known to trigger non-specific regulatory cascades, in particular, cyclic GMP-AMP synthase activation [39]. Higher effectiveness of Cas9esgRNA ribonucleoprotein complexes (RNPs) consisting of a synthetic sgRNA and the Cas9 protein (in comparison with plasmid CRISPR/Cas9 constructs) has been demonstrated on a model of slowly dividing cells, namely, fibroblasts and pluripotent stem cells [40]. RNPs are less cytotoxic and yield a greater number of single-cell colonies. In addition, a significant reduction in off-target effects (down to complete absence) is observed: an approximately 10-fold difference in the on-target/ off-target mutation ratio for the RNP method compared to the plasmid constructs. The lifespan of the Cas9esgRNA complex is reported to be quite short but sufficient for implementation of its function within the cell. The Cas9esgRNA complex and its components degrade with time [40]. These features of RNPs apparently allow researchers to obtain modified cell lines and organisms free

Fig. 1. Schematic presentation of (A) CRISPR/Cas9 system and (B) strategies involving synthetic sgRNAs: delivery of a ribonucleoprotein (RNP) complex containing both a recombinant Cas9 protein and a sgRNA (1); co-transfection with a Cas9-encoding mRNA (2) or plasmid (3); transfection of sgRNA into cells expressing Cas9 from a plasmid vector (4).

J. Filippova et al. / Biochimie 167 (2019) 49e60

from the components of the genome-editing system. Thus, strategies involving synthetic sgRNAs have several advantages over the use of plasmid CRISPR/Cas9 constructs. The primary challenge to overcome is poor stability of foreign RNA in a transfected cell; this problem is mainly due to the vulnerability of RNA to nucleases. Therefore, sgRNAs require improvement of their structure, and this alteration can be achieved by various approaches and eventually results not only in higher intracellular stability but also in increased target specificity. 3. Optimization of sgRNA structure There is a set of requirements for successful genome editing with CRISPR/Cas9 systems: high mutation efficiency of the target genome region, a sufficiently small chance of off-target effects (high specificity/selectivity), stability of the complex within the cell, protection of sgRNA from cellular nucleases, minimal activation of the components of innate immunity, and optimized transport of the complex into the cell. To obtain efficient genome-editing complexes, molecular biological and physicochemical aspects of the formation of RNAeprotein and triple-enzymeesubstrate (proteineRNAeDNA) complexes, as well as the mechanisms of their action, should be considered. The mechanism of action of a CRISPR/Cas9 system involves several key stages: recognition of the PAM sequence in a target DNA region, formation of the R-loop between sgRNA and the complementary DNA strand, introduction of DSBs into the DNA molecule at the site of binding by the Cas9 protein. After cleavage of the target DNA region, one of the two pathways of DSB repair can be triggered: 1) HR, which can take place only during one phase of the cell cycle, namely anaphase, and usually does not occur in nondividing cells, or 2) NHEJ, which generates various mutations (deletions and insertions) in the target DNA region. CRISPR/Cas9 systems for a specific gene knockout are based on the latter mechanism of repair. It is known that, for a long period after DSB induction, the sgRNAeCas9 complex remains bound to the target DNA. Hence, DSB ends might be inaccessible to DNA repair factors thus leading to lower genome editing efficiency. Merrill et al. have demonstrated that Cas9 prevents interactions of free DSB ends with other proteins [41]. On the other hand, if the target DNA site is transcribed, the Cas9esgRNA complex is displaced by the RNA polymerase activity from the antisense strand, not from the coding one. Target sites in transcribed DNA, where sgRNA hybridizes with the antisense strand, are edited by CRISPR/Cas9 with higher efficiency: indel frequency at these sites is higher [41]. For this reason, in order to perform efficient cleavage, sgRNA should target the coding strand. After recognition of the PAM region (NGG sequence) by a Cas9esgRNA complex, the sgRNA molecule searches for binding sites in the DNA structure. Note that there are many possible variants of sgRNAeDNA bound structures varying in the number of nucleotides involved in heteroduplex formation (range: 1 to 20 nt). The most optimal sgRNAeDNA structure is the one with the maximal difference in the energy between the unbound state (DNAeDNA duplex and DNA-free sgRNA) and a bound state (a hybrid sgRNAeDNA duplex). The complex with the optimal energetic parameters is the most stable and active [42]. Xu et al. have proposed a method for estimating CRISPR/Cas9 activity for any given sgRNA sequence and predicting the off-target sites [42]. The method is based on the structure of the sgRNAeDNA complex. Keeping in mind that the presence of several mismatches (depending on their number and location) in the structure of sgRNA does not necessarily decrease the activity of a CRISPR/Cas9 complex, those authors endeavored to reveal the sgRNA regions where mutations can or cannot be tolerated. Using experimental data from Refs. [43,44], the authors carried out theoretical calculations

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of energies of the sgRNAeDNA structure via Pearson coefficients for estimation of the correlation between experimentally obtained coefficients of CRISPR cleavage efficiency and theoretical cleavage efficiency values for stable Cas9esgRNA complexes. Analysis of the obtained data indicated that an sgRNAeDNA hybrid helix consisting of 8e17 bp zipped from the PAM region plays the most crucial role in the formation of sgRNAeDNA complexes [42]. It is also worth mentioning that some mutant sgRNAs can have higher cleavage efficiency compared to their unmutated counterparts. Moreover, it has been demonstrated that formation of a fully zipped complex (i.e., the 20 bp structure) does not necessarily ensure effective cleavage of the target DNA [42], as confirmed by the absence of Pearson correlation between the energy states for each sgRNAeDNA heteroduplex and experimentally obtained cleavage yields for each sgRNA. The results of this and similar studies are crucial for designing the algorithms for searching for the off-target effects. In addition to cleavage efficiency, an important criterion of successful genome editing is minimization of the off-target effects. Fu et al. have demonstrated that minimization of off-target effects can be achieved by using a truncated sgRNA, with the protospacer being only 17 nt long [45]. Guide RNAs with a 17- or 18-nt complementary region yield the same cleavage efficiency, whereas truncation to 16 nucleotides sharply decreases or eliminates the activity. The researchers achieved an almost 5,000-fold decrease in off-target cleavage efficiency using truncated sgRNAs, and no additional off-target cleavage sites were noted [45]. Previously described successful strategies for increasing CRISPR efficacy are based on the truncation of the 30 termini of the gRNA (tracrRNA domain) or the addition of two G nucleotides at the 50 termini (the region adjacent to the 20 -nt recognition site) [36,46]. A series of experimental works has shown that mismatches in the protospacer region can be tolerated, and their presence, in some cases, does not affect the sgRNAeDNA interaction. Two main elements can be distinguished in the protospacer structure: the PAM-proximal region (or seed region, 1e10 nt from PAM) and the PAM-distal region (non-seed region, 11e20 nt away from PAM; Fig. 2). The most deleterious mismatches for gRNA function are known to be located in the seed area. Nevertheless, mismatches in the non-seed region can be tolerated because they do not cause such a dramatic decrease in CRISPR/Cas9 efficiency [47]. Fu et al. have reported that, despite the presence of mutations in the sgRNA structure, the Cas9esgRNA system may still hit several targets at the same time [48]. Those authors demonstrated that indels can be formed even in the presence of five mismatches between an sgRNA and the target DNA region. The influence of a 1e5-nt region of non-complementarity between DNA and sgRNA (bulge) on the possibility of generation of off-target effects has been studied [37]. A series of sgRNAs was constructed, each containing a deletion of 1 nt (DNA bulge) at positions 1 to 19 relative to PAM. Having analyzed mutant variants of two sgRNAs targeting two genes, those authors managed to identify a specific relation between the position of a deletion in sgRNA and the frequency of indels (%). Surprisingly, positions 1, 2, 7, 18, and 19 relative to the PAM were shown not only to be the least sensitive to deletions (i.e., these deletions were better tolerated), the absence of nucleotides at these positions (DNA bulge) also had little or no effect on cleavage efficiency and even increased it in some cases (Fig. 2 A). In general, deletions at the 50 end (PAM-distal region) are better tolerated than deletions at the 30 end of the protospacer in sgRNA (PAM-proximal region). This phenomenon may be explained by the lesser significance of the PAM-distal region (as compared to the PAM-proximal region) for sgRNA function. Next, a series of sgRNAs were constructed, each containing an additional nucleotide (insertion) in the guide sequence in the sgRNA (sgRNA bulges).

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Fig. 2. Principles for enhancing editing efficiency and specificity through nucleotide insertion/deletion or modification in sgRNA: A e deletions (either DNA or sgRNA bulges) are tolerated at these positions thus allowing incorporation of modifications at these sites (backbone modifications are not considered); B, C e guidelines for backbone modification (20 O-methyl and phosphate substitutions) of sgRNA (B) or crRNA and tracrRNA (C) [37,49e52]. Such requirements are defined by the direct involvement of the highlighted nucleotides (B) in the formation of Cas9esgRNA complex.

Such nucleotides in the PAM-proximal region inhibited sgRNA activity. Of note, in almost all cases, the addition of U (among all possible nucleotide variants) into the structure increased the frequency of target mutations. In general, an RNA molecule is more amenable to changes in its structure than DNA, as a singlenucleotide bulge in RNA elicits a smaller free energy change as compared to DNA. In this regard, sgRNA bulges have a less negative effect than DNA bulges do. Thermodynamic stability of the sgRNAeDNA complex can be modulated by incorporation of modified nucleotides into the structure of sgRNA. Depending on the type of modification, a chemical modification can decrease or increase the duplex energy, which, in turn, can result in a minimal energy state required for the formation of a catalytically competent RNAeDNAeprotein complex. Thus, it is important to identify precise parameters of the interaction between non-canonical nucleotides and canonical RNA or DNA nucleotides [38,53,54]. Recently, it was demonstrated that incorporation of bridged and locked nucleic acid modifications into the gRNA structure led to the acceleration of the dynamic transitions between the open and zipped conformations on the off-target DNA sequences, resulting in the off-target cleavage decrease [55]. Those authors suggested that not only conformational constraint of the nucleic acid matters for improving Cas9 specificity but other features as well, such as chemical substituents and large steric bulk. It was shown that crRNA modifications could impede Cas9 kinetics suggesting that the reduced rate of the reaction with the off-target DNA substrates resulted in increased specificity. Therefore, kinetic aspects can be taken into account for considering the protospacer sequence and nucleotide modification in gRNA. However, the formulation of theoretical kinetics-based selection criteria requires the accumulation of experimental kinetic data in combination with the analysis of the editing systems in vivo. Another key factor is the secondary structure of the sgRNA itself. According to Chu et al., only sgRNAs with a functional secondary

structure should be selected for gene knockout experiments [56]. A tool for the design of a single guide RNA, namely “CrispRGold,” has been developed to identify a potent sgRNA secondary structure by predicting the minimal sequence elements required for target recognition versus possible off-target sites. This tool also takes into account properties of the sgRNA itself such as the GC content and the folding energy between the target sequence and sgRNA. Nevertheless, Labuhn et al. could not verify the algorithm proposed by Chu et al. in experiments including a dataset of 430 sgRNAs [57]. Thus, the question about the influence of the secondary structure of sgRNA on CRISPR/Cas9 activity requires additional experimental data. Optimization of sgRNA structure is not the only way to improve CRISPR/Cas9 specificity. For instance, a series of studies has revealed that mutant versions of the Cas9 protein can significantly reduce the frequency of off-target mutagenesis/off-target effects [47,58,59]. One of the approaches is based on the fact that Cas9 contains two nuclease domains. If one of them is “turned off” by a mutation, then the protein will act as a nickase and generate only single-strand breaks. When two Cas9esgRNA nickases are used to cleave opposite strands of the neighboring target sites, a staggered break is created in genomic DNA which can be repaired by NHEJ or HR mechanisms [6,36]. Thus, creation of a DSB requires recognition of two target sites rather than one thus resulting in higher specificity as off-target sites tend to be more widely distributed and get nick repaired. Improvement of both sgRNA and nuclease structure and their joined application can lead to a significant breakthrough in the field of genome editing technologies. 4. Backbone modifications Backbone modifications are a standard strategy for enhancing the stability of artificial RNAs. The development of RNA-based technologies for regulation of gene expression by RNAi has led to

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performance improvements by incorporating chemical modifications in the small RNA guides. Although CRISPR development is at its earlier stage, a series of sgRNA modifications that would neither interfere with the formation of a catalytically active complex with the Cas9 protein nor reduce targeting properties of the RNA molecule itself have been studied (Table 1) [49,50,52,60]. To date, various research groups have analyzed lots of common modifications, such as 20 -O-methyl (20 -O-Me) groups, 20 -F-, (S)constrained ethyl (cEt), 20 ,40 -diOMe RNA, 20 -F-40 -OMe RNA, phosphorothioate (PS), 20 -O-methyl-30 -phosphorothioate [MS], 20 -Omethyl-30 -thioPACE [MSP] and some types of groups that can be obtained by solid-phase RNA synthesis (Table 1) [49e52,55,60e62]. Complete ribose modification totally abolished the sgRNA function [49]. Naturally, the largest influence on the guide RNA functioning was caused by the modified monomers in the protospacer region. Similar to mutations, any modifications in the PAM-distal sequence were shown to be better tolerated in comparison to the PAMproximal region (Fig. 2 B). It was demonstrated that up to 10 terminal nucleotides at the 50 - end were available for 20 -O-Me and 20 -F modifications [49,50,52,60,61]. As expected, incorporation of 20 -F modifications into the guide sequence turned out to be less detrimental for gRNA targeting function in comparison to 20 -O-Me modifications at the same positions [49,61]. This phenomenon may be due to the smaller size of the F atom and its potential ability to form a hydrogen bond. PAM-proximal nucleotides were restricted for modification generally in the areas of the structurally predicted 20 -OH contacts with Cas9. Yin H. et al. demonstrated that introduction of 20 -O-Me groups at all nucleotides where such groups are not involved in tertiary interactions with the Cas9 significantly increased the cleavage activity of the sgRNA, and, vice versa, modification of the nucleotides important for the interaction with Cas9 made such sgRNAs inactive (Table 1) [49]. Ribose modifications of the 30 -terminal nucleotides, as well as the 50 -terminal ones lead to the enhanced guide RNA stability. Typically, a few nucleotides (3e5 nt) at the both ends of sgRNA or crRNA are modified at once [49,50,52,60]. Intramolecular hairpins in the tracrRNA can also contain several 20 -O-Me and 20 -F groups in the neighboring positions as well [49]. Moreover, ribose modifications 20 -O-Me, 20 -F and cEt were found to be preferable for the crRNA-tracrRNA interaction regions (Table 1). In addition, Cromwell et al. demonstrated that next-generation bridged nucleic acids (20 ,40 -BNANC[NeMe]) as well as locked nucleic acids (LNA) at specific locations in CRISPR-RNAs (crRNAs) broadly reduced the off-target DNA cleavage [55] (Table 1). Interestingly, not only modification, but also substitution of 20 -OH with H in many positions in crRNAs and tracrRNA, i.e. creation of chimeric RNA-DNA guides, were found to be tolerated for CRISPR/Cas9 activity in vitro. DNA substitutions in the tracrRNApairing region of a crRNA were shown to enhance cleavage activity and improve target specificity [62]. In order to increase the efficiency of RNA-based agents, modifications of the intersite phosphate groups are often used. In general, to achieve the effective target hydrolysis, modifications of phosphodiester bonds should be distributed inside the guide RNA molecule following similar rules (to avoid the area of interaction between the guide RNA and Cas protein), with such modifications having less negative effects at crucial sites of the protospacer region (Table 1). It was found that modification of the entire protospacer area with PS significantly decreased yet did not abolish the activity of a crRNA. However, complete PS modification of the invariable sgRNA part totally abolished its function [49]. It was demonstrated that sgRNA specificity can be also improved by modifying the phosphodiester bonds [51]. In the study Ryan et al., 30 - and 50 -terminal nucleotides were modified with 20 -Omethyl-phosphonoacetate [MP] to protect sgRNA from degradation by exonucleases, and additional modification of other nucleotides

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in the guide sequence of sgRNA structure revealed positions crucial for enhancing the on-target specificity, which was estimated as ontarget/off-target ratio. The most generally useful nucleotides for enhancing specificity were positions 4, 5 or 7 from the 50 terminus of the sgRNA. The set of sgRNAs containing modifications at these positions were characterized by higher specificity with a mostly 10fold reduction in the off-target cleavage. Increased specificity was also noted in some cases for positions 11, 14 or 16 from the 50 -terminus. The findings suggest that there are target agnostic trends for positions where an MP modification may enhance specificity, but the best position(s) in the 20-nt guide sequence for specificity enhancement should be identified empirically. It is worth noting, modifications of phosphodiester bonds can be better incorporatedat the inner region of the DNA sequence, so the results on RNA-DNA chimeric guides are very useful for developing synthetic approaches [62]. Several researches revealed that the most significant increase in the guide RNA efficiency could be achieved by combining different backbone modifications (Table 1). Taking into consideration the general rules described above, various research groups have proposed their own variants for constructing effective modified guide RNAs. Hendel et al. developed a method for editing primary human cells using modified sgRNAs [60]. Three types of chemical modifications (20 -O-Me [M], 20 -O-Me-30 -phosphorothioate [MS], and 20 O-Me-30 -thioPACE [MSP]) were introduced into three terminal nucleotides at the 50 and the 30 ends. All sgRNAs manifested effective target cleavage in the presence of recombinant Cas9 in vitro. All modifications yielded a higher indel frequency in vivo; in addition, MS and MSP modifications led to more effective HR. Studying of delivery conditions showed that MSP achieved the highest level of indels (80%) following a 24 h gap in delivery of Cas9 mRNA after sgRNA, whereas MS modification gave somewhat lower levels (53%) at this interval. By contrast, the M-protected sgRNA lost most of its ability to yield indels when delivered ahead of the mRNA by just 4 h, and unmodified sgRNA yilded no indels by sequential delivery or only a 7% indel frequency when cotransfected with the mRNA. The results are consistent with the predicted relative abilities of the modifications to resist exonucleases, as the expected order of nuclease resistance (MSP > MS > M > unmodified) correlates with the time-course results. In Yin's study, the optimized combination of sgRNA modifications had thiophosphate groups in the 50 - and 30 -terminal regions of the sgRNA, including both 30 terminal stem-loops, as well as the SG-20 -O-Me modifications [49]. Cleavage efficiency exceeded 90% when using such sgRNA. The optimal modification scheme included 20 -O-Me groups at the 50 end of the guide sequence as well as 20 -F- and thiophosphate-modified guide sequence nucleotides not involved in tertiary interactions with the Cas9. Lately, O'Reilly et al. have analyzed the efficiency of crRNAs containing various modifications: 20 -F-RNA, 20 ,50 -RNA, LNA, 20 ,40 -diOMe RNA, 20 -F-40 O-Me RNA and 20 -O-Me groups (Table 1) [61]. The seed area of the crRNA guide region prefers A-form architecture [63], whereas the tracr-binding repeat region is more tolerant to a wider range of modifications, such as DNA, 20 -F-ANA and 20 ,50 -RNA. Incorporation of a minimal number of C20 - or C40 -O-Me groups was also tolerated. The 2-0 F-RNA modification, which is a virtual mimic of natural RNAs, turned out to be the most compatible modification at most positions, including the seed region. Outside of this region, this modification of DNA proved to be useful as well, it enhanced editing efficiency in some cases. Such a result led to a hypothesis that flexibility is an essential factor in Cas9 conformational transitions. The authors suggest using several modification types (e.g., DNA and 20 -F-ANA) since such a strategy allows enhancing editing efficiency through synergistic effects [61]. An attempt to modify crRNA has led to the design of shortened

Modification 20 -O-Methyl [M]

20 -F

Modification type

Preferable or restricted positions in gRNA structure

Reference

Effect and/or application

Backbone

least preferred positions: 1, 12, 15, 16, and 18 in the guide sequence of sgRNA and positions 22e27, 43e45, 47, 49, 51, 58, 59, 62e65, 68, 69, and 82 in the invariable part (numeration starts at the 50 -end) synthetic crRNA (29-nt): 5 modifications at the 30 - and the 50 -ends are preferable crRNA: preferable positions are 1e10, 20, 21, 28-37 tracrRNA: preferable positions are 1e11, 14, 16, 17, 19e22, 25, 29, 33-67 sgRNA: 3 terminal nucleotides at the 50 - and the 30 - ends

Yin H. et al., Nat. Biotechnol. 2017

Moderate increase in editing efficiency, complete modification abolishes activity

Rahdar M. et al., PNAS 2015 Mir A. et al., Nat. Commun. 2018

Higher resistance to nucleases and enhanced efficiency

Backbone

Backbone

Hendel A. et al., Nat. Biotechnol. 2015 sgRNA: same as for the 20 -O-methyl with addition that the Yin H. et al., Nat. Biotechnol. second stem-loop and tetraloop regions as restricted areas 2017 synthetic crRNA (29-nt): 1 modification at 5'-, 30 - or both Rahdar M. et al., PNAS 2015 ends is preferable Mir A. et al., Nat. Commun. crRNA: preferable positions are 11e19, 22-26 tracrRNA: preferable positions are 12, 13, 15, 18, 23, 24, 26 2018 e28, 30-32 crRNA: entire backbone can be modified O'Reilly D. et al. NAR 2019 Yin H. et al., Nat. Biotechnol. sgRNA: restricted positions are between the 1st and the 2nd, the 3rd and the 6th, the 11th and the 20th nucleotides 2017 in the guide sequence; tail region (between the 70th and the 101st) is preferable synthetic crRNA (29-nt): entire backbone can be modified Rahdar M. et al., PNAS 2015 Mir A. et al., Nat. Commun. crRNA: 1e3, 15e16, 19, 22e24, 34e36 phosphodiester 2018 bonds are preferable tracrRNA: 1e3, 64e66 phosphodiester bonds are preferable 0 0 sgRNA: 3 terminal nucleotides at the 5 - and the 3 - ends (in Hendel A. et al. Nat. combination with 20 -O-methyl [MS]) Biotechnol. 2015

(S)-constrained ethyl (cEt)

Backbone

synthetic crRNA (29-nt): seed area is restricted, tracrRNA- Rahdar M. et al., PNAS 2015 binding region is preferable

30 -thioPACE

Backbone

sgRNA: 3 terminal nucleotides at the 50 - and the 30 - ends (in Hendel A. et al., Nat. combination with 20 -O-methyl [MSP]) Biotechnol. 2015

30 -phosphonoacetate

Backbone

sgRNA: preferable positions are 4, 5, 7, 9e12, 14, 16 from the 50 -end (modifications at positions 5 and 11 are especially effective)

Ryan D. et al., NAR 2018

Enhanced stability in combination with 20 F- and PSmodifications, higher efficiency and slight enhancement in specificity Higher efficiency, applicable for editing of human primary cells Increased efficiency, full modification does not abolish the effect Increased efficiency Enhanced stability, higher efficiency and slight specificity improvements High enzyme activity Slight increase in activity for invariable part modifications and significantly higher potency for guide sequence modifications Increased efficiency, enhanced resistance to ribonucleases Enhanced stability, higher efficiency and slight specificity improvements Higher editing efficiency, including homologous recombination, enhanced intracellular stability, improves editing of human primary cells Enhanced efficiency

Higher editing efficiency, including homologous recombination, enhanced intracellular stability, improves editing of human primary cells

Reduced off-target cleavage

J. Filippova et al. / Biochimie 167 (2019) 49e60

Phosphorothioate [PS]

Structure

54

Table 1 A summary of sgRNA modifications, their preferable positions in the structure, and effects.

Backbone

crRNA: guide area is restricted, tracrRNA-binding region is O'Reilly D. et al. NAR 2019 preferable and partial modification is recommended

Enhanced cleavage activity, maintained or enhanced specificity (in combination with each other, 20 F-RNA or 30 ,50 -RNA)

LNA, BNANC

Backbone

crRNA: positions 10e14 from the 50 -end

Cromwell. C. et al. Nature Com. 2018

Enhanced specificity, especially in case of RNA modification, yet reduced activity

Triazole linker

Structural

sgRNA: loop structure of the upper stem

He K. et al., ChemBioChem 2016

Applicable for ligation of both RNAs (tracrRNA and crRNA), allows synthesis of highly modified sgRNAs with complex structure

Cap

Structural

50 -end of in vitro transcribed sgRNA (in combination with polyA tail) 50 -end of sgRNAs processed under RNA polymerase II promoter

Mu W. et al., Protein Cell 2018 Higher permeability, higher editing efficiency, increased intracellular stability; allows editing of primary T-cells Xie C. et al., Sci. Rep. 2017 Loss of nuclease activity (poly(A) tail is not involved), quick transport of such RNA to the cytoplasm

PolyA tail

Structural

30 -end of in vitro transcribed sgRNA (in combination with cap)

Mu W. et al., Protein Cell 2018 Higher permeability, higher editing efficiency, increased intracellular stability; allows editing of primary T-cells

G-quadruplex, G-rich hairpin

Structural

sgRNA: 30 -end

Nahar S. et al., Chem. Commun. 2018

Increased average lifetime, higher editing efficiency, no toxicity and off-target cleavage

Effector-binding aptamers

Structural

sgRNA: tetraloop and loop 2

Shao S. et al., NAR 2016

Method requires dCas9, insertions increase efficiency; labeling is more resistant to photobleaching. Application: imaging of genomic elements

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DNA, 20 F-ANA, 20 ,50 RNA

55

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(up to 29 nt) synthetic CRISPR RNA (scrRNA) containing modifications of the phosphate backbone (phosphorothioate groups, PS), as well as 20 -F- and 20 -O-Me- groups and S-constrained ethyl (cEt) substitutions [52]. Modification of the entire phosphate backbone with PS considerably enhanced the resistance of RNA to ribonucleases. Transfection of such scrRNA into cells resulted in a 4-fold increase in the cleavage efficiency of the target DNA region as compared to the unmodified crRNA. Further modification of scrRNA-PS by addition of five 20 -O-Me- groups at the 30 and 50 ends of the molecule (scrRNA-PS-OMe) led to an even greater nuclease resistance. Cell transfection resulted in a sevenfold increase in the CRISPR/Cas9 efficiency, relative to unmodified scrRNA. Replacement of 20 -OH with 20 -F- groups, as well as replacement of the ribose skeleton with bicyclic nucleotide-cEt, is known to increase the affinity of RNA for DNA. The scrRNA containing one substitution of a canonical nucleotide for 20 -F at the 50 end, 30 end, or both provided an average 20e30% increase in the cleavage efficiency as compared to the sgRNA synthesized in vivo. The introduction of 20 O-Me- groups at the 50 end resulted in a ~40% increase in efficacy as compared to unmodified sgRNA. Additional improvement in the efficiency was observed after the addition of cEt modification, which binds 20 and 40 groups to the methyl bridge, in the structure of 20 -F- or 20 -O-Meemodified scrRNA. Introduction of the same modifications into the seed sequence area had a negative influence by decreasing cleavage efficiency. Substitution of nucleotides for cEt in the tracrRNA-binding region enhanced the cleavage activity [52]. Extensive modification of crRNA and tracrRNA has been performed to maintain efficient SpyCas9 genome editing in human cells [50]. Structure-guided approaches were implemented to introduce 20 -O-Me, 20 -F, and phosphorothioate (PS)-based modifications into the crRNA and tracrRNA strands (Table 1). As a result, active RNPs were obtained separately or in complex with one or both target DNA strands to identify the modification sites where Cas9 did not have any contacts with crRNA or tracrRNA. The authors synthesized a modified crRNA that maintained activity at the level of the unmodified RNA, yet every nucleotide in the modified crRNA had at least one backbone modification. The same optimization procedures were executed to synthesize tracrRNAs with at least 55 of 67 nucleotides carrying modifications as well as active tracrRNAs with at least one chemical modification at each position. Editing efficiency of the fully modified crRNA:tracrRNA pair was twice that of the unmodified pair. The modified crRNAs had insignificant effects on off-target editing, with the fully modified pair providing slightly better results in comparison to the partially modified RNAs. In addition, experiments revealed that most of the terminal modifications analyzed in the study (fluorophores, N-acetylgalactosamine, cholesterol-triethylene glycol) were tolerated well at both ends of the RNAs. Besides CRISPR/Cas9, one of the most common systems that have been adapted for mammalian genome editing is CRISPR/Cpf1. This nuclease is programmed to induce DSBs in the target DNA as well. However, there are several specific distinguishing features, with main of them being 50 -TTTN PAM and a single 43-mer crRNA used for editing. There is a decent amount of articles devoted to the modification of this system. General approaches are similar to those described for Cas9: truncation of crRNA from both 50 - and 30 termini to a 36-mer [64,65], a substitution of crRNA nucleotides with PS, 20 -O-Me, 20 -F, cEt or DNA nucleotides at the 50 - and 30 terminal or internal positions [64,66]. Terminal modifications of synthetic crRNAs (scrRNAs) led to an increased resistance to exonuclease activity as well as allowed crRNAs to be taken up by the cells in culture in the absence of carrier molecules [64]. It should be noted that 20 -F modification at the 30 terminus exhibited higher activity compared to the unmodified crRNA [66]. Secondary

pseudoknot structure of the crRNA was shown to be sensitive to chemical modification. However, terminal and numerous internal positions within the specificity region can be chemically substituted [64]. Interestingly, it was demonstrated that extending the length of crRNA at the 50 -end enhances editing efficiency as well, with chemical modifications on the extended 50 -end leading to the enhanced serum stability [67]. The experiments described above indicate that it is possible to achieve a significant increase in the targeting efficiency of genome editing systems by introducing modifications into the guide RNA nucleotides. In exemplary cases, the number of modified nucleotides (for sgRNA or both crRNA and tracrRNA) can reach ~80%. The basic rule that should be followed during the design of sgRNA is that modification of the phosphate and 20 -hydroxyl groups interacting with the Cas protein should be typically avoided (Fig. 2 (B, C), Table 1). 5. Multifunctional gRNA modifications Backbone and sugar modifications are generally introduced into sgRNA structure through solid-phase synthesis, which is an alternative to sgRNA-expressing plasmids or sgRNA production by in vitro transcription. Long RNAs can be synthesized automatically using 20 -acetoxyethyl orthoesters (ACEs) or 20 -thionocarbamates. The alternative method combines solid-phase synthesis with chemical ligation, for example, click chemistry, which is used to obtain long oligodeoxynucleotides [68,69]. He et al. have reported a novel strategy of solid-phase synthesis for ligating two RNA oligos via alkyne-azide cycloaddition catalyzed by Cu(I) leading to the formation of a long sgRNA containing a triazole linker [70]. The experiment is easy to conduct because conjugation can be performed with various reagents (amine-NHS, thiol-maleimide, or hydrazine-oxime). The method also allows for the synthesis of highly modified sgRNAs with complex structure. The Huisgen 1,3dipolar cycloaddition reaction, which yields a stable triazole linker composed of an alkyne and azide, was employed in one study [70]. The reaction between 20 -ACEeprotected 50 -hexyne tracrRNA and 30 -azide crRNA was carried out in the presence of Cu(I) in an aqueous solution. Unmodified tracrRNA, crRNA, and sgRNA were synthesized in the same manner. Editing efficiency was analyzed: indel frequency for the modified sgRNA was comparable to the level of that for the system composed of unmodified crRNA and tracrRNA and for the unmodified sgRNA (>40%). The most crucial secondarystructure elements in the gRNA were found to be the bulge and nexus. In the modified sgRNA, the triazole linker was placed in the loop structure of the upper stem: a functionally insignificant region (Table 1). Mu et al. have designed a novel modification method for enhancement of the stability of in vitroetranscribed (IVT) sgRNA [71]. First, the backbone was altered via the modifications previously reported to stabilize RNA in cells, e.g., 50 -polyA; stem-loop II, stem-loop IV, and stem-loop at the 30 -UTR of subgenomic flaviviral RNA from Dengue virus and 50 -cap and 30 -polyA structures to imitate mRNA (CTsgRNA) (Table 1). The higher penetration ability in comparison with unmodified sgRNA was manifested only by CTsgRNA, as was the case for editing efficiency: 26.9% (versus 15.29% for unmodified sgRNA). The rate of off-target cleavage remained at the level of unmodified sgRNA. Moreover, no editing was observed using the unmodified sgRNA in primary T cells, whereas CT modification yielded 15.23% efficiency. Intracellular stability also increased, as did the activation of CRISPR/dCas9regulated endogenous genes in K562 cells and primary T cells. Multiplex genome engineering requires expression of multiple sgRNAs at the same time. Mostly, sgRNAs are effectively produced under the control of RNA polymerase III promoters. By means of

J. Filippova et al. / Biochimie 167 (2019) 49e60

specific start and stop sequences, RNA transcripts are produced without any special structures (introns, cap, and polyA tail), and this arrangement is convenient for transcription of short RNAs. The polymerase II promoter enables producing multiple sgRNAs from a single transcript and coordinates and induces regulation, depending on cellular behavioral characteristics (Table 1). Xie et al. have described a novel method for multiple-sgRNA expression in combination with multiple miRNAs (or short hairpin RNAs) from a single transcript under RNA polymerase II control [72]. The polycistron contains miRNAs and sgRNAs and is processed with the help of a microprocessor: a protein complex including RNase III, Drosha, and DGCR8/Pasha. A plasmid was constructed, so that processed sgRNA had a 50 cap. Interestingly, such a structure led to a complete loss of the Cas9esgRNA nuclease activity, with the polyA tail not influencing the activity. Moreover, the described sgRNA had a larger molecular weight than other mature sgRNAs: presumably, Cas9 binds to the sgRNA sequence to prevent its degradation by endogenous RNases. Extended sgRNAs are mostly processed into ~20- nt fragments. The 50 cap and its binding protein are supposed to protect a precursor RNA from degradation. It was also assumed that 50 -capped RNAs are transported to the cytoplasm quickly, while Cas9 remains in the nucleus. The lack of nuclease activity presumably explains why functional sgRNAs cannot be produced directly under the control of RNA polymerase II promoter. By introducing structural changes in the sgRNA via addition of functional elements, various applied tasks can be solved, for instance, visualization of an sgRNA itself or of a genomic locus of interest [73,74]. Shao et al. have described a novel dual-imaging method involving a modified sgRNA and dCas9 protein [21]. Structural modifications have been reported that consist of MS2/ PP7 RNA aptamer inserts that bind tdMCP and tdPCP tagged with fluorescent proteins. The inserts were introduced into the tail, loop 2, or the tetraloop, thereby yielding seven sgRNA variations. Two other sgRNAs had an A-U base flip and extended hairpin as structures necessary for preventing U6 PolII transcription termination and increasing dCas9esgRNA assembly stability [75]. RNA aptamer insertion into the tetraloop or loop 2 has only a weak influence on the ability of the dCas9esgRNA complex to bind to a target site, whereas an insert at the tail region decreases affinity and yields a lower signal/background ratio [21]. An A-U flip and elongated stem-loop increase labeling efficiency. Double insertion into both the tetraloop and loop 2 also increases efficiency in comparison to single insertions; MS2, PP7, lambda, Spinach, and com may serve as inserts. Experiments have revealed that due to a high exchange rate of RNA aptamerebinding effectors, labeling with sgRNA is more resistant to photobleaching than the orthogonal system is [20]. Moreover, the described method is complementary to the orthogonal system. Therefore, it is presumably possible to combine the two strategies. Nahar et al. have supposed that introducing naturally occurring structural modifications might solve the toxicity problem of synthetic sgRNAs, and such alterations should be more suitable for genome editing [76]. The G-quadruplex motif has been chosen because it is known that an intramolecular G-quadruplex at any end of nucleic acids protects phosphodiester bonds from singlestrandespecific endonucleases. Stable sgRNAs are rich in G, thus having the potential to form G-quadruplex motifs [77]. Two synthetic motifs G2UG2UG2UG2 and G3U3G3U3G3U3G3 have been chosen for insertion into the both ends of sgRNA (Table 1) [76]. In vitro tests revealed that adding G-quadruplexes to a 50 end completely suppresses the editing activity while still leading to the formation of the complex with DNA and dCas9, although less effectively in comparison with the unmodified sgRNAs. By contrast, 30 modifications do not alter the editing efficiency and have been shown to increase average lifetime fivefold. A 30 terminus is

57

reported to be more prone to participation in Cas9esgRNA assembly and cleavage suggesting that it enables incorporation of modifications. In vivo experiments have revealed an up to 13% increase in the number of indels. Both G-quadruplex length and the secondary structure determine sgRNA functioning. No toxicity or off-target cleavage have been observed. Similar results have been obtained using a G-rich hairpin as a structural modification. Another modification type is RNA structures and sequences recognized by specific RNA-binding proteins. Such structures have been integrated into the sgRNA backbone thus yielding more productive and diverse effectors (Table 1) [78e80].

6. gRNA modifications lead to a decrease in an innate immune response A variety of effects of sgRNA modifications include a decrease in an immune response and antiproliferative and reduced cytotoxic effects, which are interesting from the perspective of therapeutically useful RNA design [81e86]. It has been demonstrated that a significant part of the sgRNA is protected by the protein from a solvent and consequently from the action of cellular nucleases and of innate-immunity components. Nevertheless, there is still a problem with an induced interferon response as in the case of other classes of synthetic RNA-based regulators [60,87e89]. This issue has been investigated in detail by Schubert et al. who have carried out in vitro research on peripheral blood mononuclear cells (PBMCs) [90]. Alt-R crRNA and tracrRNA products represent chemically modified RNAs containing a set of modifications such as end-blocking groups, 20 -O-methylation, and phosphorothioate linkages. IVT sgRNAs have been synthesized from the templates, with some of them having no 50 -triphosphates. The experiments detected on-target editing as well as toxicity to the cells transfected with IVT sgRNAs either with or without 50 -triphosphates. The analysis of HEK293 culture supernatants has revealed that type I and III interferons are detectable only with cells transfected with IVT sgRNAs containing 50 -triphosphates. IVT sgRNAs without 50 triphosphates did not elicit any immune response. Presumably, HEK293 cells react to sgRNA through the RIG-I pathway depending on the 50 -triphosphate presence. Healthy donors’ PBMCs were transfected with gRNAs, and culture supernatants were analyzed after 24h. An induced type I interferon immune response developed similarly to the pathways described for traditional therapeutic oligonucleotides (e. g., siRNAs). The induction of this interferon response could be suppressed with 20 -modification of the backbone mentioned in chapter 4 [91,92]. The removal of 50 -triphosphate from IVT sgRNAs decreased but did not eliminate their ability to elicit an immune response in human blood leukocytes. In comparison with HEK293 cells, delivery of gRNAs and sgRNAs into PBMCs did not affect the cell viability. A number of modifications that were first discovered in native mRNAs and ncRNAs [93,94] apparently can be also utilized to reduce immunogenicity when using artificial sgRNAs [84,95,96]. Such naturally occurring nucleotide modifications have been often used earlier in the development of artificial expression regulators. For instance, to bypass innate antiviral responses in cells, Luigi et al. have suggested to incorporate such modifications as the 50 -m7G cap, 5-methylcytidine, or pseudouridine into synthetic messenger RNAs used for somatic cell reprogramming [97]. Nevertheless, to date, the use of naturally occurring nucleotide modifications in sgRNA synthesis remains an interesting and poorly studied task. The key issues in these studies that should be addressed are the impact of modifications on RNA folding and on RNA interaction with DNA as well as the possibility of site-specific and statistical incorporation of these monomers into the sgRNA structure.

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7. Conclusions Due to the development of high-throughput analysis methods and improvements in the sensitivity of physicochemical analytical methods, an increasing number of modified nucleotides is discovered in native types of RNA. Furthermore, the chemical synthesis of nucleotide derivatives results in a greater number of possible monomers with well-described features to be applied to the design of RNA-based gene expressioneregulating agents. Noteworthy also is the advancement of knowledge on pathological processes developing in various cell types. The combination of a “proper” cell model and a precise tool for the correction of genomic disorders might become a promising platform for the design of new therapeutic agents and strategies. Modified sgRNAs are believed to have a great potential for “non-standard” genome editing tasks. For example, gene editing in slowly dividing cells should be helpful for neuropathology correction [98]. Besides, an interesting objective is to edit the mitochondrial genome because the high temperature inside this special compartment obstructs the process of DNA engineering [99,100]. Although it is still possible to modify an editing system by altering the nuclease structure, the RNA modifications described above represent a more effective and easy-to-follow strategy. Thus, sgRNA modifications are likely to offer a large amount of additional useful features of RNA as an accurate and highly specific genome engineering tool. Conflicts of interest The authors declare that they have no conflict of interest. Funding

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This work was supported by the Russian Foundation for Basic Research [grants 16-34-60136 and 18-29-07073]; the grant of the President of the Russian Federation for young scientists [grant 6196.2018.4]; and Russian Science Foundation [grant 18-75-10069]. The work was partially supported by the State Budget Program (0245-2019-0001). We thank Academician of the RAS, Prof. Valentin Vlassov and Dr. Alexandra Shintyapina for providing helpful comments about the manuscript. The English language was corrected and certified by shevchuk-editing.com.

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