Challenges in CRISPR/CAS9Delivery:

Potential Roles of Nonviral Vectors

Ling Li,1Zhi-Yao He,1Xia-Wei Wei,1,*Guang-Ping Gao,2,3and Yu-Quan Wei1

1Lab for Aging Research,State Key Laboratory of Biotherapy,West China Hospital,Sichuan University,Chengdu,Sichuan,PR China;2Gene Therapy Center,

3Department of Microbiology and Physiology Systems,University of Massachusetts Medical School,Worcester,Massachusetts.

CRISPR/Cas9genome editing platforms are widely applied as powerful tools in basic research and potential therapeutics for genome regulation.The appropriate alternative of delivery system is critical if genome editing systems are to be effectively performed in the targeted cells or organisms.To date, the in vivo delivery of the Cas9system remains challenging.Both physical methods and viral vectors are adopted in the delivery of the Cas9-based gene editing platform.However,physical methods are more applicable for in vitro delivery,while viral vectors are generally concerned with safety issues, limited packing capacities,and so on.With the robust development of nonviral drug delivery systems, lipid-or polymer-based nanocarriers might be potent vectors for the delivery of CRISPR/Cas9systems. In this review,we look back at the delivery approaches that have been used for the delivery of the Cas9system and outline the recent development of nonviral vectors that might be potential carriers for the genome editing platform in the future.The efforts in optimizing cationic nanocarriers with structural modi?cation are described and promising nonviral vectors under clinical investigations are highlighted.

INTRODUCTION

T he concept of‘‘genome editing’’allows the targeted alteration of genomic sequences in cells and organisms,which could both contribute as a powerful tool in basic research in biological?elds,and has the potential in the application of genetic disorders therapy.The induction of a DNA double-stranded break(DSB)by nuclease at the targeted site is the ?rst step in the process of precise genome editing. The early genome editing adopted DSB-inducing nucleases such as meganucleases,zinc?nger nucle-ases,and transcription activator-like effector nucle-ases(TALENs),which target the speci?c sites in the genome following protein-based systems with cus-tomization of DNA-binding domains.Recently,a clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated(CRISPR/ Cas)system,an RNA-guided nuclease,has revolu-tionized the way that genome editing is performed.

CRISPR systems are adaptable immune mech-anisms used by many bacteria to protect them-selves from foreign nucleic acids,such as viruses or plasmids.1–4Different CRISPR/Cas system types(I,II,and III)vary in the molecular mecha-nisms to achieve nucleic acid recognition.The type II CRISPR/Cas system relies on only a single pro-tein for speci?c DNA recognition and cleavage guided by RNA,which was an adaptive property for genome editing application.5,6Also,the Cas9pro-tein was identi?ed as a multifunctional protein essential for defense against viral invasion.7In 2013,the targeted genome editing accomplished by the type II CRISPR system from Streptococcus pyogenes was reported.8–13Since then,CRISPR/ Cas9-based systems has been an emerging?eld for genome engineering and genome regulation.

Two critical components must be introduced for the application of the CRISPR/Cas9system, including the Cas9nuclease and a guide RNA (gRNA)that consists of a fusion of a crRNA and a constant tracrRNA.Cas9uses gRNA to form base pairs with site-speci?c DNA sequences and intro-duces a DSB.Thus,Cas9nuclease could be directed to a speci?c site simply by altering the?rst20nts of

*Correspondence:Dr.Xia-Wei Wei,Lab for Aging Research,State Key Laboratory of Biotherapy,West China Hospital,Sichuan University,No.17,Section3,Renmin South Road,Chengdu,Sichuan610041,PR China,E-mail:xiawei_wei@http://www.doczj.com/doc/429bd3bea45177232e60a28d.html

452j HUMAN GENE THERAPY,VOLUME26NUMBER7DOI:10.1089/hum.2015.069a2015by Mary Ann Liebert,Inc.