Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins

Kim, Sojung; Kim, Daesik; Cho, Seung Woo; Kim, Jung-Eun; Kim, Jin-Soo

doi:10.1101/gr.171322.113

Cited by 1,621 publications

(1,503 citation statements)

References 56 publications

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“…As direct introduction of Cas9 protein is more efficient than using a plasmid 9,19 , recombinant Cas9 protein microinjection was adopted, injecting a mixture of sgRNA, Cas9 protein and ssODN DNA into the cytoplasm of pronuclear stage zygotes 18 h after fertilization. Injected zygotes and intact controls were cultured for 3 days before each embryonic blastomere was isolated and individually analysed by sequencing (Fig.…”

Section: Hdr Efficiency In Heterozygous Mybpc3 ∆Gagt Zygotesmentioning

confidence: 99%

“…, Jianhui gong 5,6,7,8 , Ying gu 5,6,7 , Xun Xu 5,6,7 , David Battaglia 1,9 , sacha a. Krieg 9 , David M. Lee 9 , Diana H. Wu 9 , Don P. Wolf 1 , stephen B. Heitner 10 , Juan carlos izpisua Belmonte 3 §, Paula amato 1,9 §, Jin-soo Kim 2,4 §, sanjiv Kaul 10 § & shoukhrat Mitalipov 1,10 § More than 10,000 monogenic inherited disorders have been identified, affecting millions of people worldwide. Among these are autosomal dominant mutations, where inheritance of a single copy of a defective gene can result in clinical symptoms.…”

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confidence: 99%

“…In particular, CRISPR-Cas9 is a versatile tool for recognizing specific genomic sequences and inducing DSBs [7][8][9][10] . DSBs are then resolved by endogenous DNA repair mechanisms, preferentially using a non-homologous end-joining (NHEJ) pathway.…”

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Correction of a pathogenic gene mutation in human embryos

Martí-Gutiérrez

Park

et al. 2017

Nature

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More than 10,000 monogenic inherited disorders have been identified, affecting millions of people worldwide. Among these are autosomal dominant mutations, where inheritance of a single copy of a defective gene can result in clinical symptoms. Genes in which dominant mutations manifest as late-onset adult disorders include BRCA1 and BRCA2, which are associated with a high risk of breast and ovarian cancers 1 , and MYBPC3, mutation of which causes hypertrophic cardiomyopathy (HCM) 2 . Because of their delayed manifestation, these mutations escape natural selection and are often transmitted to the next generation. Consequently, the frequency of some of these founder mutations in particular human populations is very high. For example, the MYBPC3 mutation is found at frequencies ranging from 2% to 8% 3 in major Indian populations, and the estimated frequency of both BRCA1 and BRCA2 mutations among Ashkenazi Jews exceeds 2% 4 .HCM is a myocardial disease characterized by left ventricular hypertrophy, myofibrillar disarray and myocardial stiffness; it has an estimated prevalence of 1:500 in adults 5 and manifests clinically with heart failure. HCM is the commonest cause of sudden death in otherwise healthy young athletes. HCM, while not a uniformly fatal condition, has a tremendous impact on the lives of individuals, including physiological (heart failure and arrhythmias), psychological (limited activity and fear of sudden death), and genealogical concerns. MYBPC3 mutations account for approximately 40% of all genetic defects causing HCM and are also responsible for a large fraction of other inherited cardiomyopathies, including dilated cardiomyopathy and left ventricular non-compaction 6 . MYBPC3 encodes the thick filament-associated cardiac myosin-binding protein C (cMyBP-C), a signalling node in cardiac myocytes that contributes to the maintenance of sarcomeric structure and regulation of both contraction and relaxation 2 .Current treatment options for HCM provide mostly symptomatic relief without addressing the genetic cause of the disease. Thus, the development of novel strategies to prevent germline transmission of founder mutations is desirable. One approach for preventing second-generation transmission is preimplantation genetic diagnosis (PGD) followed by selection of non-mutant embryos for transfer in the context of an in vitro fertilization (IVF) cycle. When only one parent carries a heterozygous mutation, 50% of the embryos should be mutationfree and available for transfer, while the remaining carrier embryos are discarded. Gene correction would rescue mutant embryos, increase the number of embryos available for transfer and ultimately improve pregnancy rates.Recent developments in precise genome-editing techniques and their successful applications in animal models have provided an option for correcting human germline mutations. In particular, CRISPR-Cas9 is a versatile tool for recognizing specific genomic sequences and inducing DSBs 7-10 . DSBs are then resolved by endogenous DNA repair mechanisms, prefer...

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Section: Hdr Efficiency In Heterozygous Mybpc3 ∆Gagt Zygotesmentioning

confidence: 99%

mentioning

confidence: 99%

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Correction of a pathogenic gene mutation in human embryos

Martí-Gutiérrez

Park

et al. 2017

Nature

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818

540

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“…The delivery of the Cas9 protein and sgRNA plasmid directly, which can not only reduce the size of the plasmid, but also avoid the instability of the RNA, is a promising strategy for CRISPR‐mediated gene editing. Furthermore, this strategy can accurately manipulate the intracellular concentration of the Cas9 protein/sgRNA plasmid complex and the editing timeframe, thus, it could potentially allow for optimization of gene editing 13, 14, 16, 17. However, the delivery of proteins also encounters difficulties: the proteins are usually not stable, and the plasmids cannot penetrate cell membrane without appropriate encapsulations 18.…”

Section: Introductionmentioning

confidence: 99%

Genome Editing for Cancer Therapy: Delivery of Cas9 Protein/sgRNA Plasmid via a Gold Nanocluster/Lipid Core–Shell Nanocarrier

et al. 2017

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The type II bacterial clustered, regularly interspaced, short palindromic repeats (CRISPR)‐Cas9 (CRISPR‐associated protein) system (CRISPR‐Cas9) is a powerful toolbox for gene‐editing, however, the nonviral delivery of CRISPR‐Cas9 to cells or tissues remains a key challenge. This paper reports a strategy to deliver Cas9 protein and single guide RNA (sgRNA) plasmid by a nanocarrier with a core of gold nanoclusters (GNs) and a shell of lipids. By modifying the GNs with HIV‐1‐transactivator of transcription peptide, the cargo (Cas9/sgRNA) can be delivered into cell nuclei. This strategy is utilized to treat melanoma by designing sgRNA targeting Polo‐like kinase‐1 (Plk1) of the tumor. The nanoparticle (polyethylene glycol‐lipid/GNs/Cas9 protein/sgPlk1 plasmid, LGCP) leads to >70% down‐regulation of Plk1 protein expression of A375 cells in vitro. Moreover, the LGCP suppresses melanoma progress by 75% on mice. Thus, this strategy can deliver protein‐nucleic acid hybrid agents for gene therapy.

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“…For applications that seek a one-time, permanent modification of genomic DNA, we and others have shown that the delivery of non-replicable, transient Cas9:single guide RNA (sgRNA) ribonucleotide protein (RNPs) complexes in vivo offer improved DNA specificity and potentially greater safety and applicability 14,15 compared with methods that introduce DNA expressing these agents. Approximately 20% of alleles associated with genetic deafness are dominantly inherited 1 .…”

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confidence: 99%

Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents

Gao

Tao

Lamas

et al. 2017

Nature

446

346

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Although genetic factors contribute to almost half of all deafness cases, treatment options for genetic deafness are limited1–5. We developed a genome editing approach to target a dominantly inherited form of genetic deafness. Here we show that cationic lipid-mediated in vivo delivery of Cas9:guide RNA complexes can ameliorate hearing loss in a mouse model of human genetic deafness. We designed and validated in vitro and in primary fibroblasts genome editing agents that preferentially disrupt the dominant deafness-associated allele in the Tmc1 (transmembrane channel-like 1) Beethoven (Bth) mouse model, even though the mutant Bth allele differs from the wild-type allele at only a single base pair. Injection of Cas9:guide RNA:lipid complexes targeting the Bth allele into the cochlea of neonatal Bth/+ mice substantially reduced progressive hearing loss. We observed higher hair cell survival rates and lower auditory brainstem response (ABR) thresholds in injected ears compared with uninjected ears or ears injected with complexes that target an unrelated gene. Enhanced acoustic reflex responses were observed among injected compared to uninjected Bth/+ animals. These findings suggest protein:RNA complex delivery of target gene-disrupting agents in vivo as a potential strategy for the treatment of some autosomal dominant hearing loss diseases.

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Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins

Cited by 1,621 publications

References 56 publications

Correction of a pathogenic gene mutation in human embryos

Correction of a pathogenic gene mutation in human embryos

Genome Editing for Cancer Therapy: Delivery of Cas9 Protein/sgRNA Plasmid via a Gold Nanocluster/Lipid Core–Shell Nanocarrier

Treatment of autosomal dominant hearing loss by in vivo delivery of genome editing agents

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