Efficient ssODN-Mediated Targeting by Avoiding Cellular Inhibitory RNAs through Precomplexed CRISPR-Cas9/sgRNA Ribonucleoprotein.
Alleles
Anti-Bacterial Agents
/ pharmacology
Base Sequence
CRISPR-Associated Protein 9
/ metabolism
CRISPR-Cas Systems
/ genetics
Distal Myopathies
/ genetics
Dysferlin
/ genetics
Exons
/ genetics
Gene Editing
HEK293 Cells
Haplotypes
/ genetics
Homozygote
Humans
Induced Pluripotent Stem Cells
/ metabolism
Muscular Atrophy
/ genetics
Muscular Dystrophy, Duchenne
/ genetics
Mutagenesis, Insertional
/ genetics
Mutation
/ genetics
Oligodeoxyribonucleotides
/ metabolism
RNA
/ metabolism
RNA Splicing
/ genetics
RNA, Guide, Kinetoplastida
/ metabolism
Ribonucleases
/ metabolism
Ribonucleoproteins
/ metabolism
CRISPR-Cas9
Cas9 inhibition
Cre-loxP recombination
SNP
genome editing
homozygous correction
iPSC
single nucleotide alteration
Journal
Stem cell reports
ISSN: 2213-6711
Titre abrégé: Stem Cell Reports
Pays: United States
ID NLM: 101611300
Informations de publication
Date de publication:
13 04 2021
13 04 2021
Historique:
received:
14
08
2020
revised:
12
02
2021
accepted:
12
02
2021
pubmed:
13
3
2021
medline:
3
3
2022
entrez:
12
3
2021
Statut:
ppublish
Résumé
Combined with CRISPR-Cas9 technology and single-stranded oligodeoxynucleotides (ssODNs), specific single-nucleotide alterations can be introduced into a targeted genomic locus in induced pluripotent stem cells (iPSCs); however, ssODN knockin frequency is low compared with deletion induction. Although several Cas9 transduction methods have been reported, the biochemical behavior of CRISPR-Cas9 nuclease in mammalian cells is yet to be explored. Here, we investigated intrinsic cellular factors that affect Cas9 cleavage activity in vitro. We found that intracellular RNA, but not DNA or protein fractions, inhibits Cas9 from binding to single guide RNA (sgRNA) and reduces the enzymatic activity. To prevent this, precomplexing Cas9 and sgRNA before delivery into cells can lead to higher genome editing activity compared with Cas9 overexpression approaches. By optimizing electroporation parameters of precomplexed ribonucleoprotein and ssODN, we achieved efficiencies of single-nucleotide correction as high as 70% and loxP insertion up to 40%. Finally, we could replace the HLA-C1 allele with the C2 allele to generate histocompatibility leukocyte antigen custom-edited iPSCs.
Identifiants
pubmed: 33711268
pii: S2213-6711(21)00093-X
doi: 10.1016/j.stemcr.2021.02.013
pmc: PMC8072016
pii:
doi:
Substances chimiques
Anti-Bacterial Agents
0
DYSF protein, human
0
Dysferlin
0
Oligodeoxyribonucleotides
0
RNA, Guide
0
Ribonucleoproteins
0
RNA
63231-63-0
CRISPR-Associated Protein 9
EC 3.1.-
Ribonucleases
EC 3.1.-
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
985-996Informations de copyright
Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.
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