vv 001 Citation: Sai YRKM (2021) Cytosine extensions optimize case activity for telomere length regulation: Implications for CRISPR-based therapies – A short communication. Ann Mol Genet Med 5(1): 001-003. DOI: https://dx.doi.org/10.17352/amgm.000009 https://dx.doi.org/10.17352/amgm DOI: MEDICAL GROUP Abstract Telomeres are nucleoprotein structures that play a crucial role in maintaining genomic stability, and their length determines cellular lifespan. Telomere shortening is linked to cellular senescence and an increased risk of cancer. The CRISPR-Cas9 system has emerged as a tool for genome engineering and telomere length regulation. However, several factors, including chromatin accessibility, the efficiency of Double-Stranded Break (DSB) repair and the specificity of the sgRNA/Cas9 complex, limit the efficiency of Cas9-mediated telomere length regulation. Recent studies have demonstrated the use of modified Cas9 nucleases, such as Cas9-NG, and the development of modified sgRNAs to improve the efficiency of Cas9-mediated telomere length regulation. In this study, Bhattacharyya, et al. investigated the optimization of Cas9 activity through the addition of cytosine (C) extensions to the 5’ end of sgRNAs. They found that C extensions significantly increased Cas9 activity at telomeres and demonstrated that the optimal length of C extensions was three Cs. The addition of C extensions did not affect the specificity of the sgRNA/Cas9 complex, as assessed by the frequency of off-target DSBs. These findings have important implications for the development of CRISPR-Cas9-based therapies for telomere-related diseases. Further studies are needed to confirm these findings and optimize the use of C extensions in different cell types and disease contexts. Short communication Cytosine extensions optimize case activity for telomere length regulation: Implications for CRISPR-based therapies – A short communication YRKM Sai* Independent Researcher/Unaffiliated, MSc Biochemistry, Former Student of GITAM Institute of Sciences, Gandhi Institute of Technology and Management, Visakhapatnam, Andhra Pradesh, India Received: 15 December, 2021 Accepted: 27 December, 2021 Published: 28 December, 2021 *Corresponding author: YRKM Sai, Independent Researcher/Unaffiliated, MSc Biochemistry, Former Stu- dent of GITAM Institute of Sciences, Gandhi Institute of Technology and Management, Visakhapatnam, Andhra Pradesh, India, Tel: +91 9573300975; E-mail: saiyrkm2454@gmail.com ORCID: https://orcid.org/0000-0002-6151-5687 Keywords: CRISPR-Cas9; Telomere length regulation; Cytosine extensions; Single-guide RNA; Chromatin accessibility; Specificity; Telomere length; CRISPR-Cas9; Genome engineering; Cytosine extensions; Optimization Copyright License: © 2022 Sai YRKM. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited https://www.peertechzpublications.com Introduction Telomeres are nucleoprotein structures that cap the ends of chromosomes, protecting them from degradation and fusion. Telomere length plays a critical role in maintaining genomic stability, as a shortening of telomeres is associated with cellular senescence and an increased risk of cancer [1]. Telomerase, a reverse transcriptase, can lengthen telomeres by adding telomeric repeats to the 3’ end of chromosomes. However, most human somatic cells do not express telomerase, resulting in telomere shortening with each cell division [1]. The CRISPR-Cas9 system has been developed as a versatile tool for genome engineering, including the regulation of telomere length. The Cas9 nuclease, guided by a single-guide RNA (sgRNA), can introduce Double-Stranded Breaks (DSBs) at specific genomic locations. When these DSBs are repaired through Homology-Directed Repair (HDR) or Non-Homologous End Joining (NHEJ), telomere length can be altered [2]. However, the efficiency of Cas9-mediated telomere length regulation can be limited by several factors, including the accessibility of telomeric chromatin, the efficiency of DSB repair, and the specificity of the sgRNA/Cas9 complex. To address these limitations, a recent study by Bhattacharyya, et al. [3] investigated the optimization of Cas9 activity through the addition of Cytosine (C) extensions to the 5’ end of sgRNAs [3].