High Efficiency CRISPR/Cas9-mediated Gene Editing in Primary Human T-cells Using Mutant Adenoviral E4orf6/E1b55k “Helper” Proteins

Many future therapeutic applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 and related RNA-guided nucleases are likely to require their use to promote gene targeting, thus necessitating development of methods that provide for delivery of three components—Cas9, gu...

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Bibliographic Details
Published in:Molecular therapy 2016-09, Vol.24 (9), p.1570-1580
Main Authors: Gwiazda, Kamila S, Grier, Alexandra E, Sahni, Jaya, Burleigh, Stephen M, Martin, Unja, Yang, Julia G, Popp, Nicholas A, Krutein, Michelle C, Khan, Iram F, Jacoby, Kyle, Jensen, Michael C, Rawlings, David J, Scharenberg, Andrew M
Format: Article
Language:English
Subjects:
Adeno-associated virus
CRISPR
CRISPR-Cas Systems
Dependovirus - genetics
Efficiency
Gene Editing
Gene Expression
Gene Knock-In Techniques
Gene Knockout Techniques
Gene Order
Gene Targeting
Gene Transfer Techniques
Genetic Vectors - genetics
Homologous Recombination
Humans
Medical research
Mutant Proteins
Original
Proteins
Research centers
Restrictions
RNA, Guide, CRISPR-Cas Systems - genetics
T-Lymphocytes - metabolism
Transduction, Genetic
Viral Proteins - genetics
Viral Proteins - metabolism
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Summary:Many future therapeutic applications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 and related RNA-guided nucleases are likely to require their use to promote gene targeting, thus necessitating development of methods that provide for delivery of three components—Cas9, guide RNAs and recombination templates—to primary cells rendered proficient for homology-directed repair. Here, we demonstrate an electroporation/transduction codelivery method that utilizes mRNA to express both Cas9 and mutant adenoviral E4orf6 and E1b55k helper proteins in association with adeno-associated virus (AAV) vectors expressing guide RNAs and recombination templates. By transiently enhancing target cell permissiveness to AAV transduction and gene editing efficiency, this novel approach promotes efficient gene disruption and/or gene targeting at multiple loci in primary human T-cells, illustrating its broad potential for application in translational gene editing.
ISSN:1525-0016
1525-0024
DOI:10.1038/mt.2016.105