[{"data":1,"prerenderedAt":-1},["ShallowReactive",2],{"detail-sidebar-cat-0-en-105":3,"doc-seo-435332-105":59,"doc-detail-435332-en":131},{"code":4,"msg":5,"data":6},0,"success",[7,13,18,23,28,33,38,43,48,51,55],{"id":8,"doc_module":4,"doc_module_name":9,"category_name":10,"show_sort_weight":11,"slug":12},1,"Document","Story & Novel",90,"story-novel",{"id":14,"doc_module":4,"doc_module_name":9,"category_name":15,"show_sort_weight":16,"slug":17},2,"Literature",80,"literature",{"id":19,"doc_module":4,"doc_module_name":9,"category_name":20,"show_sort_weight":21,"slug":22},4,"Exam",70,"exam",{"id":24,"doc_module":4,"doc_module_name":9,"category_name":25,"show_sort_weight":26,"slug":27},5,"Comic",60,"comic",{"id":29,"doc_module":4,"doc_module_name":9,"category_name":30,"show_sort_weight":31,"slug":32},6,"Technology",50,"technology",{"id":34,"doc_module":4,"doc_module_name":9,"category_name":35,"show_sort_weight":36,"slug":37},7,"Healthcare",40,"healthcare",{"id":39,"doc_module":4,"doc_module_name":9,"category_name":40,"show_sort_weight":41,"slug":42},8,"Research & Report",30,"research-report",{"id":44,"doc_module":4,"doc_module_name":9,"category_name":45,"show_sort_weight":46,"slug":47},9,"Religion & Spirituality",20,"religion-spirituality",{"id":46,"doc_module":4,"doc_module_name":9,"category_name":49,"show_sort_weight":46,"slug":50},"World Cup","world-cup",{"id":52,"doc_module":4,"doc_module_name":9,"category_name":53,"show_sort_weight":52,"slug":54},10,"Lifestyle","lifestyle",{"id":56,"doc_module":4,"doc_module_name":9,"category_name":57,"show_sort_weight":24,"slug":58},19,"General","general",{"code":4,"msg":60,"data":61},"ok",{"site_id":62,"language":63,"slug":64,"title":65,"keywords":66,"description":67,"schema_data":68,"social_meta":124,"head_meta":126,"extra_data":128,"updated_unix":130},105,"en","in-situ-gene-editing-of-hematopoietic-stem-cells-via-aav-delivered-crispr-guide-rnas","In situ gene editing of hematopoietic stem cells via AAV-delivered CRISPR guide RNAs","","In situ genome editing of hematopoietic stem cells (HSCs) is pursued to leverage their self-renewal and engraftment while correcting endogenous disease-causing variants without ex vivo manipulation. The study uses adeno-associated virus (AAV) delivery of CRISPR guide RNAs to edit HSC genomes in vivo, evaluating systemic AAV design and dosing parameters. Self-complementary AAVs and a higher guide-to-Cas9 ratio enhance editing efficiency while preserving multilineage output and engraftability, and a dual AAV-CRISPR application supports robust modification with therapeutic fetal hemoglobin induction in a sickle cell mouse model.",{"@graph":69,"@context":123},[70,84,106],{"@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":35,"@type":76,"position":81},"https://docshare.wps.com/document/healthcare/",3,{"item":83,"name":65,"@type":76,"position":19},"https://docshare.wps.com/document/in-situ-gene-editing-of-hematopoietic-stem-cells-via-aav-delivered-crispr-guide-rnas/435332/",{"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-situ-gene-editing-of-hematopoietic-stem-cells-via-aav-delivered-crispr-guide-rnas/435332.png","ImageObject",300,407,{"name":92,"@type":93},"Rainbow Cat","Person",{"url":74,"name":95,"@type":96},"DocShare","Organization","application/pdf","2026-11-08","2026-09-29",true,{"@type":102,"interactionType":103,"userInteractionCount":105},"InteractionCounter",{"@type":104},"ViewAction",14,{"@type":107,"mainEntity":108},"FAQPage",[109,115,119],{"name":110,"@type":111,"acceptedAnswer":112},"How does AAV-delivered CRISPR guide RNA enable in situ editing of HSCs?","Question",{"text":113,"@type":114},"AAV delivers CRISPR gRNAs systemically to HSCs in vivo, directing cleavage around target loci so that reporter expression and genome modification can be induced within the body.","Answer",{"name":116,"@type":111,"acceptedAnswer":117},"What factors improved editing efficiency in the study?",{"text":118,"@type":114},"Higher editing efficiency was associated with self-complementary AAVs and an increased guide-to-Cas9 ratio, indicating that vector format and guide abundance influence outcome.",{"name":120,"@type":111,"acceptedAnswer":121},"Did the editing affect HSC potency after engraftment?",{"text":122,"@type":114},"The study reports preserved multilineage output and engraftability of edited HSCs, consistent with retention of full stem cell potency following in situ modification.","https://schema.org",{"og:url":83,"og:type":125,"og:title":65,"og:site_name":95,"og:description":67},"article",{"robots":127,"canonical":83},"index,follow",{"doc_id":129,"site_id":62},435332,1790864560,{"code":4,"msg":5,"data":132},{"doc_id":129,"user_id":133,"nickname":92,"user_avatar":134,"doc_module":4,"category_id":34,"category_name":35,"doc_title":65,"doc_description":67,"doc_content":135,"file_id":136,"file_url":137,"file_type":138,"file_size":139,"view_count":105,"is_deleted":4,"is_public":8,"is_downloadable":8,"audit_status":8,"page_count":140,"language":141,"language_code":63,"site_id":62,"html_lang":63,"table_of_contents":142,"faqs":143,"seo_title":144,"seo_description":67,"update_tm":145,"read_time":41,"preview_page_count":4},962090769181,"https://ap-avatar.wpscdn.com/davatar_6f874abed73319feea01a86fa6f0fab8","REGULAR ARTICLE  \nIn situ gene editing of hematopoietic stem cells via AAV-delivered CRISPR guide RNAs  \nAlborz Karimzadeh, 1, 2 Rebekah Kim, 1, 2 Vivian Garcia,2 Michael Florea,2 Bryan L. Peacker,2, 3 Shio Kobayashi, 1 Drake Watkins,1 Kathleen Messemer,2 Jing Zeng,4 Daniel E. Bauer,4 Thomas Serwold,1 and Amy J. Wagers 1-3,5  \n1 Section on Islet Cell and Regenerative Biology, Joslin Diabetes Center, Boston, MA; 2 Department of Stem Cell and Regenerative Biology, Harvard University, Cambridge, MA; 3 Harvard Medical School, Boston, MA; 4 Division of Hematology/Oncology, Boston Children’s Hospital, Boston, MA; and 5 Harvard Stem Cell Institute, Cambridge, MA  \nKey Points  \n• AAV serotype 8– mediated delivery of CRISPR gRNAs enables in situ modification of reporter alleles and diseaserelevant loci in HSCs.  \n• In situ HSC modification efficiency varies with age, vector configuration, and guide copy number.  \nHematopoietic stem cells (HSCs) are self-renewing, multipotent, and engraftable precursors of all blood cells. Efficient delivery of therapeutic gene products and gene editing machinery to correct disease-causing gene variants in endogenous HSCs while they remain in the body holds exciting potential to leverage HSC potency for the treatment of monogenic blood disorders. Toward this goal, we used adeno-associated virus (AAV) to deliver CRISPR guide RNAs (gRNAs) to edit HSC genomes in situ in Ai9;SpCas9-EGFP transgenic mice carrying a Cas9-activatable Lox-STOP-Lox-tdTomato reporter cassette together with a constitutive SpCas9-2A-EGFP. Using a variety of conditions and vector designs, we tested whether systemic administration to these mice ofAAVs carrying SpCas9-compatible gRNAs designed to cut DNA upstream and downstream of the STOP cassette would induce tdTomato expression in HSCs. Our findings identify self-complementary AAVs (scAAVs) and increased ratio of guide to Cas9 as parameters facilitating higher editing efficiency. Of note, we find preserved multilineage output and engraftability of HSCs upon scAAV-gRNA editing. In an example application of this technology, we explore the potential for in situ HSC gene editing by dual AAV-CRISPR delivery and demonstrate robust gene modification, concurrent with induction of therapeutic fetal hemoglobin, in a sickle cell disease mouse model modified to express SpCas9 . In summary, this work offers a sensitive and adaptable platform that allows robust modification of HSC genomes in situ.  \nIntroduction  \nSelf-renewing hematopoietic stem cells (HSCs) can efficiently reconstitute all adult blood cell compartments upon transplantation, making HSC transplantation a curative intervention for monogenic blood disorders.1 Ex vivo methods for HSC gene modification, such as Casgevy (exagamglogene autotemcel) for the treatment of sickle cell disease (SCD), successfully use HSC’s DNA repair machinery to rescue genetic abnormalities.2-6 However, the ex vivo isolation and manipulation of HSCscan diminish their subsequent engraftment efficiency and multilineage potency and may introduce unwanted selective pressures with potential long-term negative consequences.7 HSC transplantation itself also requires complicated procedures and extensive infrastructure, posing a challenge to global access.8-10 Approaches that target HSCs while they remain in the body hold promise to overcome these difficulties.  \nSubmitted 11 April 2025; accepted 7 September 2025; prepublished online on Blood Advances First Edition 24 September 2025. [https://doi.org/10.1182/](https://doi.org/10.1182/)[ ](https://doi.org/10.1182/)[bloodadvances.2025016775](bloodadvances.2025016775.)[.](bloodadvances.2025016775.)  \nOriginal data are available on request from the corresponding author, Amy J. Wagers ([amy_wagers@harvard.edu](amy_wagers@harvard.edu)).  \nThe full-text version of this article contains a data supplement.  \n© 2025 American Society of Hematology. Published by Elsevier Inc. Licensed under Creative Commons Attribution-NonComme","cbCaihJs12RIMYvL","https://ap.wps.com/l/cbCaihJs12RIMYvL","pdf",648613,12,"English","# Key Points\n## In situ AAV-CRISPR strategy and design criteria\n## Introduction: rationale for targeting HSCs in vivo\n## Prior work and study aims\n## Delivery advantages and study contribution","[{\"question\":\"How does AAV-delivered CRISPR guide RNA enable in situ editing of HSCs?\",\"answer\":\"AAV delivers CRISPR gRNAs systemically to HSCs in vivo, directing cleavage around target loci so that reporter expression and genome modification can be induced within the body.\"},{\"question\":\"What factors improved editing efficiency in the study?\",\"answer\":\"Higher editing efficiency was associated with self-complementary AAVs and an increased guide-to-Cas9 ratio, indicating that vector format and guide abundance influence outcome.\"},{\"question\":\"Did the editing affect HSC potency after engraftment?\",\"answer\":\"The study reports preserved multilineage output and engraftability of edited HSCs, consistent with retention of full stem cell potency following in situ modification.\"}]","In situ gene editing of hematopoietic stem cells via AAV-delivered CRISPR guide RNAs | PDF",1790673018]