Staudinger reaction on the solid phase between an electronodeficit organic azide, such as sulfonyl azide, and the phosphite triester formed upon phosphoramidite coupling is a convenient method for the chemical modification of oligonucleotides at the internucleotidic phosphate position. In this work, 4-carboxybenzenesulfonyl azide, either with a free carboxy group or in the form of an activated ester such as pentafluorophenyl, 4-nitrophenyl, or pentafluorobenzyl, was used to introduce a carboxylic acid function to the terminal or internal internucleotidic phosphate of an oligonucleotide via the Staudinger reaction. A subsequent treatment with excess primary alkyl amine followed by the usual work-up, after prior activation with a suitable peptide coupling agent such as a uronium salt/1-hydroxybenzotriazole in the case of a free carboxyl, afforded amide-linked oligonucleotide conjugates in good yields including multiple conjugations of up to the exhaustive modification at each phosphate position for a weakly activated pentafluorobenzyl ester, whereas more strongly activated and, thus, more reactive aryl esters provided only single conjugations at the 5′-end. The conjugates synthesized include those with di- and polyamines that introduce a positively charged side chain to potentially assist the intracellular delivery of the oligonucleotide.

The in vivo editing of genetic information necessitates tools of unprecedented accuracy. CRISPR-Cas-based systems have emerged as leading technologies for precisely targeting the genome. The Cas9 endonuclease derived from Streptococcus pyogenes is the most commonly used instrument for targeted DNA cleavage. The development of engineered and chimeric Cas9 variants with enhanced activity and specificity has enabled not only the simple knockout of target genes but also the sophisticated engineering of the epigenome. This advancement has broadened the potential applications of CRISPR-Cas9 technology for the treatment of various disorders characterized by a combination of mutations, deletions, and duplications in the coding and non-coding regions of the genome. The inherent simplicity and predictability of the CRISPR-Cas9 targeting mechanism have led to an explosive growth in the development of prototype gene-editing drugs. However, their therapeutic application is still challenged by potential off-target effects. The erroneous editing of tumor suppressors and oncogenes could lead to adverse outcomes that mitigate the benefits of CRISPR therapy. The evolution of DNA-targeting technologies requires a comprehensive understanding of the mechanisms underlying CRISPR-Cas9 off-target binding and cleavage. The use of massive libraries of DNA targets and guide RNAs, coupled with high-throughput sequencing, contributes significantly to the analysis of mismatch tolerance. Nevertheless, the detection of ultra-low levels of off-target activity is hindered by the sensitivity limitations of current technologies. This review focuses on the mechanisms responsible for off-target interactions during CRISPR-Cas9-mediated gene editing. We discuss the influence of various factors, including nucleotide context, enzyme concentration, guide RNA structure, and the energetics of the RNA–DNA hybrid on mismatch tolerance in vitro and in vivo. Recent advances in the development of technologies for predicting off-target effects are briefly summarized. Particular emphasis is placed on the role of the Cas9 protein structure in the allosteric regulation of the specific and non-specific activity of the Cas9–sgRNA complex.

The non-ideal accuracy and insufficient selectivity of CRISPR/Cas9 systems is a serious problem for their use as a genome editing tool. It is important to select the target sequence correctly so that the CRISPR/Cas9 system does not cut similar sequences. This requires an understanding of how and why mismatches in the target sequence can affect the efficiency of the Cas9/sgRNA complex. In this work, we studied the catalytic activity of the Cas9 enzyme to cleave DNA substrates containing nucleotide mismatch at different positions relative to the PAM in the “seed” sequence. We show that mismatches in the complementarity of the sgRNA/DNA duplex at different positions relative to the protospacer adjacent motif (PAM) sequence tend to decrease the cleavage efficiency and increase the half-maximal reaction time. However, for two mismatches at positions 11 and 20 relative to the PAM, an increase in cleavage efficiency was observed, both with and without an increase in half-reaction time. Thermodynamic parameters were obtained from molecular dynamics results, which showed that mismatches at positions 8, 11, and 20 relative to the PAM thermodynamically stabilize the formed complex, and a mismatch at position 2 of the PAM fragment exerts the greatest stabilization compared to the original DNA sequence. The weak correlation of the thermodynamic binding parameters of the components of the Cas9/sgRNA:dsDNA complex with the cleavage data of DNA substrates containing mismatches indicates that the efficiency of Cas9 operation is mainly affected by the conformational changes in Cas9 and the mutual arrangement of sgRNA and substrates.

Genome-editing systems, being some of the key tools of molecular biologists, represent a reasonable hope for progress in the field of personalized medicine. A major problem with such systems is their nonideal accuracy and insufficient selectivity. The selectivity of CRISPR-Cas9 systems can be improved in several ways. One efficient way is the proper selection of the consensus sequence of the DNA to be cleaved. In the present work, we attempted to evaluate the effect of formed non-Watson–Crick pairs in a DNA duplex on the efficiency of DNA cleavage in terms of the influence of the structure of the formed partially complementary pairs. We also studied the effect of the location of such pairs in DNA relative to the PAM (protospacer-adjacent motif) on the cleavage efficiency. We believe that the stabilization of the Cas9-sgRNA complex with a DNA substrate containing noncomplementary pairs is due to loop reorganization in the RuvC domain of the enzyme. In addition, PAM-proximal mismatches in the DNA substrate lower enzyme efficiency because the “seed” region is involved in binding and cleavage, whereas PAM-distal mismatches have no significant impact on target DNA cleavage. Our data suggest that in the case of short duplexes with mismatches, the stages of recognition and binding of dsDNA substrates by the enzyme determine the reaction rate and time rather than the thermodynamic parameters affected by the “unwinding” of DNA. The results will provide a theoretical basis for predicting the efficiency and accuracy of CRISPR-Cas9 systems at cleaving target DNA.