Beyond editing: repurposing CRISPR–Cas9 for precision genome regulation and interrogation

Dominguez, Antonia A.; Lim, Wendell A.; Qi, Lei S.

doi:10.1038/nrm.2015.2

Cited by 729 publications

(579 citation statements)

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“…More studies on improving the fidelity and finding an alternative have been reported 1, 3, 4. To broaden the applicability of our delivery system, we explored other gene editing alternatives.…”

Section: Resultsmentioning

confidence: 99%

“…Gene mutations and allele variations usually contribute to disease heterogeneity, which may result in treatment failure; thus, precision medicine‐based therapeutic approaches have increasingly attracted attention, especially since the CRISPR/Cas9 system was reported 1. The CRISPR/Cas9 system allows targeted genome editing, including frameshift knockout, gene insertion, and alteration, under the guidance of a specific guide RNA (gRNA) 1.…”

Section: Introductionmentioning

confidence: 99%

“…The CRISPR/Cas9 system allows targeted genome editing, including frameshift knockout, gene insertion, and alteration, under the guidance of a specific guide RNA (gRNA) 1. Since the first system engineered from Streptococcus pyogenes Cas9 endonuclease,2 many Cas9 variants (e.g., Staphylococcus aureus Cas93), analogues (e.g., CRISPR/Cpf14 and FEN‐1/FokI fused endonuclease5) have emerged.…”

Section: Introductionmentioning

confidence: 99%

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HPV Oncogene Manipulation Using Nonvirally Delivered CRISPR/Cas9 or Natronobacterium gregoryi Argonaute

Lao

Gao

et al. 2018

Advanced Science

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CRISPR/Cas9 technology enables targeted gene editing; yet, the efficiency and specificity remain unsatisfactory, particularly for the nonvirally delivered, plasmid‐based CRISPR/Cas9 system. To tackle this, a self‐assembled micelle is developed and evaluated for human papillomavirus (HPV) E7 oncogene disruption. The optimized micelle enables effective delivery of Cas9 plasmid with a transient transgene expression profile, benefiting the specificity of Cas9 recognition. Furthermore, the feasibility of using the micelle is explored for another nucleic acid‐guided nuclease system, Natronobacterium gregoryi Argonaute (NgAgo). Both systems are tested in vitro and in vivo to evaluate their therapeutic potential. Cas9‐mediated E7 knockout leads to significant inhibition of HPV‐induced cancerous activity both in vitro and in vivo, while NgAgo does not show significant E7 inhibition on the xenograft mouse model. Collectively, this micelle represents an efficient delivery system for nonviral gene editing, adding to the armamentarium of gene editing tools to advance safe and effective precision medicine‐based therapeutics.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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HPV Oncogene Manipulation Using Nonvirally Delivered CRISPR/Cas9 or Natronobacterium gregoryi Argonaute

Lao

Gao

et al. 2018

Advanced Science

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“…MxB knockout (KO) cell lines were generated by using the clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 gene system (Edit-R CRISPR-Cas9 gene engineering with SMARTCas9 nucleases; GE Healthcare) (32). The CRISPR genomic guide RNA sequences for human MxB in THP-1 and HT-1080 cells were designed to target exon 2 near the start codon.…”

Section: Generation Of Mxb Knockout Human Cellsmentioning

confidence: 99%

MxB Is Not Responsible for the Blocking of HIV-1 Infection Observed in Alpha Interferon-Treated Cells

Opp

Vieira

Schulte

et al. 2016

J Virol

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MxB restricts HIV-1 infection by directly interacting with the HIV-1 core, which is made of viral capsid; however, the contribution of MxB to the HIV-1 restriction observed in alpha interferon (IFN-␣)-treated human cells is unknown. To understand this contribution, we used HIV-1 bearing the G208R capsid mutant (HIV-1-G208R), which overcomes the restriction imposed by cells expressing MxB. Here we showed that the reason why MxB does not block HIV-1-G208R is that MxB does not interact with HIV-1 cores bearing the mutation G208R. M yxovirus resistance proteins represent a family of interferon-inducible factors with a wide range of antiviral activities (1-3). The myxovirus B (MxB) gene was originally cloned from a human glioblastoma cell line treated with alpha interferon (IFN-␣) (4, 5). MxB as well as the related protein MxA belong to the dynamin-like family of proteins, which have diverse functions ranging from vesicle transport to antiviral activity (1, 6-11). The most studied dynamin-like protein that exhibits antiviral activity is MxA (1, 2). Contrary to MxB, the antiviral role of MxA has been extensively studied for viruses including influenza virus (1, 12-15), tick-borne Thogoto virus (16), African swine fever virus (17), hepatitis B virus (18), and La Crosse virus (19,20). The antiviral activity of the long form of MxB was recently described (9, 21-23); these investigations led to the discovery that the IFN-␣-inducible protein MxB blocks HIV-1 infection.Genetic evidence suggested that the HIV-1 capsid is the determinant for the ability of MxB to block HIV-1 infection (9,22,23). In agreement with these findings, we recently demonstrated that MxB binds to the HIV-1 capsid and correlated the ability of MxB to block HIV-1 infection with inhibition of uncoating (24). We also showed that the ability of MxB to block infection requires a capsid binding domain and an oligomerization domain provided by the 90 N-terminal and the 143 C-terminal amino acids of MxB, respectively (24). In addition, the work of others and our work showed that the 90 N-terminal amino acids of MxB are important for its ability to bind capsid and restrict HIV-1 infection (24)(25)(26).MxB contains a previously described putative nuclear localization signal on its N-terminal 25 amino acids (4). Deletion of the N-terminal 25 amino acids annihilates the ability of MxB to block HIV-1 infection and to bind to the HIV-1 core (23,24,27). Mutagenic studies have revealed that the N-terminal 25 amino acids of MxB exhibit a triple-arginine motif ( 11 RRR 13 ) that is important for restriction and the ability of MxB to bind to the HIV-1 core (28, 29). HIV-1 CA-NC expression and purification. The CA-NC proteins of HIV-1 and HIV-1 bearing the G208R capsid mutant (HIV-1-G208R) were expressed, purified, and assembled as previously described (30). MATERIALS AND METHODS CellMxB binding to in vitro-assembled HIV-1 and HIV-1-G208R CA-NC complexes. HEK 293T cells were transfected with a plasmid expressing the wild-type MxB protein. Forty-eight hours after t...

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“…Second, the "overlapping gene problem"-technologies like Cre-lox or CRISPR, which supposedly cleanly alter gene expression, often do not 73 . Roughly 10% of genes in the mouse genome have overlapping reading frames 74 .…”

Section: Reviewmentioning

confidence: 99%

Introducing Therioepistemology: the study of how knowledge is gained from animal research

et al. 2017

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We are honored to write the opening article of this focus issue of Lab Animal. This focus issue could not occur at a more important time for biomedical research and the use of animals in science in general. The progressive worsening of success rates in human trials (currently 1 in 9 drugs entering human trials will succeed) 1-4 , combined with the explosion of interest in the reproducibility crisis 5-8 and the recognition that most drugs fail in human trials due to insufficient efficacy 1,2,4 , has led to a growing suspicion that the failure of translation from animal work to human outcomes may in some way reflect issues in animal research itself 5-19 -after all, every drug that fails in humans "worked" in an animal model. Indeed pharmaceutical companies continue to disinvest in internal animal R&D, a trend begun in the last decade, passing on the cost and risk to academia and startups 5,20 . Even this approach is not foolproof as pharmaceutical companies often cannot replicate the results of published work from academia 5,8,13 . Accordingly, there is a growing trend to focus on human, not animal, work for basic discovery 17 .Discarding animal research entirely is not the answer. When properly used, animal models have incredible value, not least the ability to follow biomarkers from birth to disease onset in a year or less (in the case of mice), which is impossible in humans 17,18 . There are patterns and principles that can help us identify models and results that are more or less likely to translate, and there are also easily realized, simple changes in the execution of animal work that will inherently improve translation [16][17][18] . This isn't a new concept; looking back over the last 10-15 years we can see many authors have been candid about the merits, strengths, weaknesses, reproducibility, and translatability of various animal models 1,2,[4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] . Our goal with this article is to unite the common themes in this broader emerging literature and this special issue.Thus, the central point is that we (i.e., refs. 17,18,26-28,39,41) do not represent a voice in the wilderness, but one voice in a chorus and that this emerging literature 1,2,[4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][21][22][23][24][25][26][27][28][29][30][31][32][33][34][35][36][37][38][39][40]42,43 reflects a nascent discipline which can be codified as the study of how knowledge is gained from animal research. We propose the title "Therioepistemology" This focus issue of Lab Animal coincides with a tipping point in biomedical research. For the first time, the scale of the reproducibility and translatability crisis is widely understood beyond the small cadre of researchers who have been studying it and the pharmaceutical and biotech companies who have been living it. Here we argue that an emerging literature, including the papers in this focus issue, has begun to congeal around a set of recurring themes, which themselves represent a paradigm shift. This paradigm shift can be c...

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Beyond editing: repurposing CRISPR–Cas9 for precision genome regulation and interrogation

Cited by 729 publications

References 89 publications

HPV Oncogene Manipulation Using Nonvirally Delivered CRISPR/Cas9 or Natronobacterium gregoryi Argonaute

HPV Oncogene Manipulation Using Nonvirally Delivered CRISPR/Cas9 or Natronobacterium gregoryi Argonaute

MxB Is Not Responsible for the Blocking of HIV-1 Infection Observed in Alpha Interferon-Treated Cells

Introducing Therioepistemology: the study of how knowledge is gained from animal research

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