“…In the context of the CRISPR/Cas9 genome editing system, sgRNA plays a crucial role. It is a synthetic RNA molecule designed to guide the Cas9 nuclease enzyme for precise, site-specific DNA cuts, facilitating accurate gene editing [72,73]. Notably, the sequences of sgRNA are often chemically modified, at the phosphate backbones and sugar moieties, to enhance stability and improve genome editing efficiency.…”
Section: Application Of Md-lc To Emerging Molecule Formatsmentioning
This review provides an overview of the latest advancements and applications in multi‐dimensional liquid chromatography coupled with mass spectrometry (mD‐LC‐MS), covering aspects such as inter‐laboratory studies, digestion strategy, trapping column, and multi‐level analysis. The shift from an offline to an online workflow reduces sample processing artifacts, analytical variability, analysis time, and the labor required for data acquisition. Over the past few years, this technique has demonstrated sufficient maturity for application across a diverse range of complex products. Moreover, there is potential for this strategy to evolve into an integrated process analytical technology tool for the real‐time monitoring of monoclonal antibody quality. This review also identifies emerging trends, including its application to new modalities, the possibility of evaluating biological activity within the mD‐LC set‐up, and the consideration of multi‐dimensional capillary electrophoresis as an alternative to mD‐LC. As mD‐LC‐MS continues to evolve and integrate emerging trends, it holds the potential to shape the next generation of analytical tools, offering exciting possibilities for enhanced characterization and monitoring of complex biopharmaceutical products.
“…In the context of the CRISPR/Cas9 genome editing system, sgRNA plays a crucial role. It is a synthetic RNA molecule designed to guide the Cas9 nuclease enzyme for precise, site-specific DNA cuts, facilitating accurate gene editing [72,73]. Notably, the sequences of sgRNA are often chemically modified, at the phosphate backbones and sugar moieties, to enhance stability and improve genome editing efficiency.…”
Section: Application Of Md-lc To Emerging Molecule Formatsmentioning
This review provides an overview of the latest advancements and applications in multi‐dimensional liquid chromatography coupled with mass spectrometry (mD‐LC‐MS), covering aspects such as inter‐laboratory studies, digestion strategy, trapping column, and multi‐level analysis. The shift from an offline to an online workflow reduces sample processing artifacts, analytical variability, analysis time, and the labor required for data acquisition. Over the past few years, this technique has demonstrated sufficient maturity for application across a diverse range of complex products. Moreover, there is potential for this strategy to evolve into an integrated process analytical technology tool for the real‐time monitoring of monoclonal antibody quality. This review also identifies emerging trends, including its application to new modalities, the possibility of evaluating biological activity within the mD‐LC set‐up, and the consideration of multi‐dimensional capillary electrophoresis as an alternative to mD‐LC. As mD‐LC‐MS continues to evolve and integrate emerging trends, it holds the potential to shape the next generation of analytical tools, offering exciting possibilities for enhanced characterization and monitoring of complex biopharmaceutical products.
“…These factors are obstacles to recognizing and cutting the desired DNA and may eventually reduce the editing efficiency of the CRISPR-Cas9 system. Many researchers have attempted to optimize this tool by engineering Cas9 and sgRNA to overcome their limitations while increasing the editing efficiency (Kleinstiver et al 2015 ; Kleinstiver et al 2016 ; Park et al 2021 ; Dong et al 2022 ; Riesenberg et al 2022 ). Herein, we address considerations that affect the cleavage efficiency for optimizing the CRISPR-Cas9 system and provide directions for future improvements as well as applications to genetic diseases, therapeutics, and generation of animal models (Jin et al 2021 ; Xie et al 2022 ).…”
Section: Introductionmentioning
confidence: 99%
“…Scaffolds can also be engineered to increase the efficiency of the CRISPR-Cas system. The bulk and nexus parts are essential for sgRNA activity, so the lower stem, upper stem, and hairpin parts can be engineered (Dong et al 2022 ). Figure 3.…”
Section: Introductionmentioning
confidence: 99%
“…This is assumed to be because RNA pol III transcription termination occurs when there are three or more T sequences, and the poly T termination signal does not cause termination by itself but may cause backtracking and catalytic inactivation of RNA pol III (Nielsen et al 2013 ; Xu et al 2015 ; Zarate et al 2022 ). Additionally, RNA aptamers with hairpin structures can be added to engineer the upper stem or hairpin of the scaffold to increase sgRNA activity (Dong et al 2022 ). In this case, divalent modification of the aptamer in the scaffold increases sgRNA activity but trivalent modification would reduce such activity (Dong et al 2022 ).…”
Section: Introductionmentioning
confidence: 99%
“…Additionally, RNA aptamers with hairpin structures can be added to engineer the upper stem or hairpin of the scaffold to increase sgRNA activity (Dong et al 2022 ). In this case, divalent modification of the aptamer in the scaffold increases sgRNA activity but trivalent modification would reduce such activity (Dong et al 2022 ). Therefore, excessive hairpin formation may adversely affect the cleavage efficiency of CRISPR-Cas9 (Dong et al 2022 ).…”
The CRISPR-Cas system stands out as a promising genome editing tool due to its cost-effectiveness and time efficiency compared to other methods. This system has tremendous potential for treating various diseases, including genetic disorders and cancer, and promotes therapeutic research for a wide range of genetic diseases. Additionally, the CRISPR-Cas system simplifies the generation of animal models, offering a more accessible alternative to traditional methods. The CRISPR-Cas9 system can be used to cleave target DNA strands that need to be corrected, causing double-strand breaks (DSBs). DNA with DSBs can then be recovered by the DNA repair pathway that the CRISPR-Cas9 system uses to edit target gene sequences. High cleavage efficiency of the CRISPR-Cas9 system is thus imperative for effective gene editing. Herein, we explore several factors affecting the cleavage efficiency of the CRISPR-Cas9 system. These factors include the GC content of the protospacer-adjacent motif (PAM) proximal and distal regions, single-guide RNA (sgRNA) properties, and chromatin state. These considerations contribute to the efficiency of genome editing.
The Cas9 endonuclease of the CRISPR/Cas type IIA system from Streptococcus pyogenes is the heart of genome editing technology that can be used to treat human genetic and viral diseases. Despite its large size and other drawbacks, S. pyogenes Cas9 remains the most widely used genome editor. A vast amount of research is aimed at improving Cas9 as a promising genetic therapy. Strategies include directed evolution of the Cas9 protein, rational design, and domain swapping. The first generation of Cas9 editors comes directly from the wild-type protein. The next generation is obtained by combining mutations from the first-generation variants, adding new mutations to them, or refining mutations. This review summarizes and discusses recent advances and ways in the creation of next-generation genomic editors derived from S. pyogenes Cas9.
Key points
• The next-generation Cas9-based editors are more active than in the first one.
• PAM-relaxed variants of Cas9 are improved by increased specificity and activity.
• Less mutagenic and immunogenic variants of Cas9 are created.
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