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Cas9 limitations include large size and stringent targeting requirements. Cas12j2, a CasΦ-2 variant, offers potential advantages, but its effectiveness in mammalian cells is less studied. This work optimizes a CRISPR-Cas12j2 system for targeted knockout of the HPV E6 oncogene using computational tools to assess structural integrity and gRNA binding.",{"@graph":69,"@context":122},[70,84,105],{"@type":71,"itemListElement":72},"BreadcrumbList",[73,77,79,82],{"item":74,"name":75,"@type":76,"position":8},"https://docshare.wps.com","Home","ListItem",{"item":78,"name":9,"@type":76,"position":14},"https://docshare.wps.com/document/",{"item":80,"name":40,"@type":76,"position":81},"https://docshare.wps.com/document/research-report/",3,{"item":83,"name":65,"@type":76,"position":19},"https://docshare.wps.com/document/in-silico-design-and-characterization-of-a-rationally-engineered-cas12j2-gene-editing-system-for-the-treatment-of-hpv-associated-cancers/344669/",{"url":83,"name":65,"@type":85,"image":86,"author":91,"headline":65,"publisher":94,"fileFormat":97,"inLanguage":63,"description":67,"dateModified":98,"datePublished":99,"encodingFormat":97,"isAccessibleForFree":100,"interactionStatistic":101},"DigitalDocument",{"url":87,"@type":88,"width":89,"height":90},"https://docshare.wps.com/thumbnails/in-silico-design-and-characterization-of-a-rationally-engineered-cas12j2-gene-editing-system-for-the-treatment-of-hpv-associated-cancers/344669.png","ImageObject",300,407,{"name":92,"@type":93},"Himbo","Person",{"url":74,"name":95,"@type":96},"DocShare","Organization","application/pdf","2026-09-23","2026-09-22",true,{"@type":102,"interactionType":103,"userInteractionCount":14},"InteractionCounter",{"@type":104},"ViewAction",{"@type":106,"mainEntity":107},"FAQPage",[108,114,118],{"name":109,"@type":110,"acceptedAnswer":111},"What problem does this study address for gene editing against HPV-associated cancers?","Question",{"text":112,"@type":113},"It targets the E6 oncogene in HPV-associated cancers while addressing limitations of existing systems, especially the size and strict targeting requirements of Cas9.","Answer",{"name":115,"@type":110,"acceptedAnswer":116},"How was the Cas12j2 system optimized in this research?",{"text":117,"@type":113},"The study used multiple computational tools (e.g., ColabFold, CCTop, Cas-OFFinder, HADDOCK2.4, and molecular dynamics with Amber) to evaluate engineered modifications affecting structure and gRNA binding.",{"name":119,"@type":110,"acceptedAnswer":120},"What key computational findings support the Cas12j2 variant used here?",{"text":121,"@type":113},"The Cas12j2_F2 variant showed structural similarity to wild-type Cas12j2 in RMSD/RMSF profiles and compact Rg values, with minimal electrostatic perturbation and validated gRNA binding behavior.","https://schema.org",{"og:url":83,"og:type":124,"og:title":65,"og:site_name":95,"og:description":67},"article",{"robots":126,"canonical":83},"index,follow",{"doc_id":128,"site_id":62},344669,1790148865,{"code":4,"msg":5,"data":131},{"doc_id":128,"user_id":132,"nickname":92,"user_avatar":133,"doc_module":4,"category_id":39,"category_name":40,"doc_title":65,"doc_description":67,"doc_content":134,"file_id":135,"file_url":136,"file_type":137,"file_size":138,"view_count":14,"is_deleted":4,"is_public":8,"is_downloadable":8,"audit_status":8,"page_count":139,"language":140,"language_code":63,"site_id":62,"html_lang":63,"table_of_contents":141,"faqs":142,"seo_title":143,"seo_description":67,"update_tm":144,"read_time":145},687197100911,"https://ap-avatar.wpscdn.com/avatar/a000239b6f1da00475?x-image-process=image/resize,m_fixed,w_180,h_180&k=1785132997149421697","Article  \nIn Silico Design and Characterization of a Rationally Engineered Cas12j2 Gene Editing System for the Treatment of  \nHPV-Associated Cancers  \nCaleb Boren , Rahul Kumar and Lauren Gollahon *  \nAcademic Editor: Marco Ciotti  \nReceived: 11 December 2025  \nRevised: 14 January 2026  \nAccepted: 16 January 2026  \nPublished: 21 January 2026  \nCopyright: © 2026 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license.  \nDepartment of Biological Sciences, Texas Tech University, 2500 Broadway, Lubbock, TX 79409, USA;  \n[caleb.boren@ttu.edu](caleb.boren@ttu.edu) (C.B.); [rahulkum@ttu.edu](rahulkum@ttu.edu) (R.K.)  \n* Correspondence: [lauren.gollahon@ttu.edu](lauren.gollahon@ttu.edu)  \nAbstract  \nCRISPR-Cas9 systems have enabled unprecedented advances in genome engineering, particularly in developing treatments for human diseases, like cancer. Despite potential applications, limitations of Cas9 include its relatively large size and strict targeting requirements. Cas12j2, a variant ofCasΦ-2, shows promise for overcoming these limitations. However, its effectiveness in mammalian cells remains relatively unexplored. This study sought to develop an optimized CRISPR-Cas12j2 system for targeted knockout of the E6 oncogene in HPV-associated cancers. A combination of computational tools (ColabFold, CCTop, Cas-OFFinder, HADDOCK2.4, and Amber for Molecular Dynamics) was utilized to investigate the impact of engineered modifications on structural integrity and gRNA binding of Cas12j2 fusion constructs, in potential intracellular conditions. Cas12j2_F2, a Cas12j2 variant designed and evaluated in this study, behaves similarly to the wild-type Cas12j2 structure in terms of RMSD/RMSF profiles, compact Rg values, and minimal electrostatic perturbation. The computationally validated Cas12j2 variant was incorporated into a custom expression vector, co-expressing the engineered construct along with a dual gRNA for packaging into a viral vector for targeted knockout of HPV-associated cancers. This study provides a structural and computational foundation for the rational design of Cas12j2 fusion constructs with enhanced stability and functionality, supporting their potential application for precise genome editing in mammalian cells.  \nKeywords: Cas12j2; CasΦ-2; CRISPR-Cas; HPV16/18; HPV-associated cancers; molecular dynamics simulations (AMBER); protein-RNA docking; gene therapy; off-target analysis; in-silico modeling  \n1. Introduction  \nThree different vaccines are currently approved in the United States for preventative use against HPV infection—Gardasil, Gardasil-9, and Cervarix. Upon completion of the vaccination series, individuals without prior HPV infection experience a nearly 100% reduction in their risk of developing HPV-associated cancers [1] . As the HPV infection progresses to the formation of pre-cancerous lesions, early medical intervention involves the local treatment with the removal and application of topical creams [2,3] . If left untreated, these pre-cancerous lesions can progress into invasive HPV-associated cancers with high metastatic potential [4] . In the advanced stages, the current standard of treatment involves the surgical resection of the primary tumor and a combination of interventions such as radiotherapy, chemotherapy, and immunotherapy [5] .  \nDespite available vaccines and current treatment standards, HPV-associated cancers remain a significant global burden. Furthermore, studies have shown a shift from cervical cancer formation to tonsillar and rectal cancer incidence [6] . One study investigating trends in the global impact of HPV and the development of HPV-associated cancers found that in 2022 alone, there were over 1.5 million new diagnoses and >750,000 deaths [7] . Because the integration of E6/E7 drives carcinogenesis, various studies have investigated the therapeutic potential of CRI","cbCaieLQEKdCNJwa","https://ap.wps.com/l/cbCaieLQEKdCNJwa","pdf",6662148,32,"English","# Abstract\n# Keywords\n# 1. Introduction\n## Vaccines and clinical management of HPV\n## Role of E6/E7 in carcinogenesis\n## CRISPR-Cas targeting and system limitations\n## Motivation for engineered Cas12j2 systems","[{\"question\":\"What problem does this study address for gene editing against HPV-associated cancers?\",\"answer\":\"It targets the E6 oncogene in HPV-associated cancers while addressing limitations of existing systems, especially the size and strict targeting requirements of Cas9.\"},{\"question\":\"How was the Cas12j2 system optimized in this research?\",\"answer\":\"The study used multiple computational tools (e.g., ColabFold, CCTop, Cas-OFFinder, HADDOCK2.4, and molecular dynamics with Amber) to evaluate engineered modifications affecting structure and gRNA binding.\"},{\"question\":\"What key computational findings support the Cas12j2 variant used here?\",\"answer\":\"The Cas12j2_F2 variant showed structural similarity to wild-type Cas12j2 in RMSD/RMSF profiles and compact Rg values, with minimal electrostatic perturbation and validated gRNA binding behavior.\"}]","In Silico Design and Characterization of a Rationally Engineered Cas12j2 Gene Editing System for the Treatment of HPV-Associated Cancers | PDF",1790054800,81]