Development and Applications of CRISPR-Cas9 for Genome Engineering

Hsu, Patrick; Lander, Eric S.; Zhang, Feng

doi:10.1016/j.cell.2014.05.010

Cited by 4,906 publications

(3,947 citation statements)

References 99 publications

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“…With the advent of the CRISPR/Cas9 technology, any gene can be deleted efficiently and rapidly in many different cell lines, as long as the deletion is compatible with the survival of the cells (Hsu et al , 2014). This revolutionary technology is a game changer for the study of oncogenic signaling of human tumor cells that previously relied on knock‐down and overexpression approaches.…”

Section: Discussionmentioning

confidence: 99%

Continuous signaling of CD 79b and CD 19 is required for the fitness of Burkitt lymphoma B cells

Kläsener

Iype

et al. 2018

The EMBO Journal

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Expression of the B‐cell antigen receptor (BCR) is essential not only for the development but also for the maintenance of mature B cells. Similarly, many B‐cell lymphomas, including Burkitt lymphoma (BL), require continuous BCR signaling for their tumor growth. This growth is driven by immunoreceptor tyrosine‐based activation motif (ITAM) and PI3 kinase (PI3K) signaling. Here, we employ CRISPR/Cas9 to delete BCR and B‐cell co‐receptor genes in the human BL cell line Ramos. We find that Ramos B cells require the expression of the BCR signaling component Igβ (CD79b), and the co‐receptor CD19, for their fitness and competitive growth in culture. Furthermore, we show that in the absence of any other BCR component, Igβ can be expressed on the B‐cell surface, where it is found in close proximity to CD19 and signals in an ITAM‐dependent manner. These data suggest that Igβ and CD19 are part of an alternative B‐cell signaling module that use continuous ITAM/PI3K signaling to promote the survival of B lymphoma and normal B cells.

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Section: Discussionmentioning

confidence: 99%

Continuous signaling of CD 79b and CD 19 is required for the fitness of Burkitt lymphoma B cells

Kläsener

Iype

et al. 2018

The EMBO Journal

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“…Imperfect repair of the DNA break leads, at a variable frequency, to nucleotide addition or deletion, thereby creating a stable mutation. The creation of specific DNA breaks can be obtained by the expression of nucleases such as zinc‐finger nucleases (ZFNs), transcription activator‐like effector nucleases (TALENs) and, more recently, the Cas9 protein associated with type II clustered regulatory interspaced short palindromic repeats (CRISPR) (Hsu et al ., 2014). CRISPR‐Cas9 relies on the presence of a 20‐nucleotide guide RNA (sgRNA) that targets specifically the Cas9 nuclease to the complementary genomic sequence (Jinek et al ., 2012).…”

Section: Introductionmentioning

confidence: 99%

Selective gene dosage by CRISPR‐Cas9 genome editing in hexaploid Camelina sativa

Morineau

Bellec

Tellier

et al. 2017

Plant Biotechnology Journal

244

166

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In many plant species, gene dosage is an important cause of phenotype variation. Engineering gene dosage, particularly in polyploid genomes, would provide an efficient tool for plant breeding. The hexaploid oilseed crop Camelina sativa, which has three closely related expressed subgenomes, is an ideal species for investigation of the possibility of creating a large collection of combinatorial mutants. Selective, targeted mutagenesis of the three delta‐12‐desaturase (FAD2) genes was achieved by CRISPR‐Cas9 gene editing, leading to reduced levels of polyunsaturated fatty acids and increased accumulation of oleic acid in the oil. Analysis of mutations over four generations demonstrated the presence of a large variety of heritable mutations in the three isologous CsFAD2 genes. The different combinations of single, double and triple mutants in the T3 generation were isolated, and the complete loss‐of‐function mutants revealed the importance of delta‐12‐desaturation for Camelina development. Combinatorial association of different alleles for the three FAD2 loci provided a large diversity of Camelina lines with various lipid profiles, ranging from 10% to 62% oleic acid accumulation in the oil. The different allelic combinations allowed an unbiased analysis of gene dosage and function in this hexaploid species, but also provided a unique source of genetic variability for plant breeding.

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“…In the CRISPR‐Cas9 system, the Cas9, an endonuclease, is directed by a single guide RNA (sgRNA) to cause double‐strand break of target DNA sequences with high specificity,2 allowing for much greater ease of construction of knockout reagents than other methods for genetic modification 3, 4, 5. However, it is very challenging for the delivery of the CRISPR‐Cas9 system into cells or tissues because the plasmid encoding both Cas9 and sgRNA has strong negative charges and large size (usually exceeds 10 000 bp).…”

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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Development and Applications of CRISPR-Cas9 for Genome Engineering

Cited by 4,906 publications

References 99 publications

Continuous signaling of CD 79b and CD 19 is required for the fitness of Burkitt lymphoma B cells

Continuous signaling of CD 79b and CD 19 is required for the fitness of Burkitt lymphoma B cells

Selective gene dosage by CRISPR‐Cas9 genome editing in hexaploid Camelina sativa

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

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