Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection

Liang, Xiquan; Potter, Jason; Kumar, Shantanu; Zou, Yanfei; Quintanilla, Rene H.; Sridharan, Mahalakshmi; Carte, Jason; Chen, Wen; Roark, Natasha; Ranganathan, Sridhar; Ravinder, Namritha; Chesnut, Jonathan D.

doi:10.1016/j.jbiotec.2015.04.024

Cited by 627 publications

(561 citation statements)

References 20 publications

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“…To quantify the Cas9 expression level, we normalized the mean fluorescent intensity of the GFP + cells at each time point to that at 4 h. The Cas9 expression peaked at 24 h with 32.1% transfection efficiency (Figure 2A,B). Unlike our observation, previous work has shown that Cas9 was cumulatively expressed in HEK293T cells when using the plasmid‐loaded liposomal carrier 9. This suggests that this micellar approach may have lower off‐target effects on CRISPR/Cas9 editing due to its faster turnover rate.…”

Section: Resultscontrasting

confidence: 85%

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: Resultscontrasting

confidence: 85%

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

Lao

Gao

et al. 2018

Advanced Science

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“…The prophage-encoded inhibitor proteins AcrIIA2 and AcrIIA4, which allow phages to evade the bacterial host's CRISPR/Cas immune system, were found to inhibit Cas9-based targeting in their native host Listeria monocytogenes, as well as Cas9 of Streptococcus pyogenes in bacteria and human cells. Another approach for minimizing off-target effects employs the delivery of Cas9 protein/gRNA ribonucleoprotein complexes (37). When delivering Cas9 protein directly, cleavage occurs only temporarily, as the Cas9 protein is rapidly degraded in cells (38).…”

Section: Discussionmentioning

confidence: 99%

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells

et al. 2017

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Abstract. Genomic editing using the CRISPR/Cas9 technology allows selective interference with gene expression. With this method, a multitude of haploid and diploid cells from different organisms have been employed to successfully generate knockouts of genes coding for proteins or small RNAs. Yet, cancer cells exhibiting an aberrant ploidy are considered to be less accessible to CRISPR/Cas9-mediated genomic editing, as amplifications of the targeted gene locus could hamper its effectiveness. Here we examined the suitability of CRISPR/Cas9 to knockout the receptor tyrosine kinase Axl in the human hepatoma cell lines HLF and SNU449. The genomic editing events were validated in two single cell clones each from putative HLF and SNU449 knockout cells (HLF-Axl --1, HLF-Axl --2, SNU449-Axl --1, SNU449-Axl --2). Sequence analysis of respective AXL loci revealed one to six editing events in each individual Axl -clone. The majority of insertions and deletions in the AXL gene at exon 7/8 resulted in a frameshift and thus a premature stop in the coding region.However, one genomic editing event led to an insertion of two amino acids resulting in an altered protein sequence rather than in a frameshift in the AXL locus of the SNU449-Axl --1 cells. Notably, while no Axl protein expression could be detected by immunoblotting in all four cell clones, both expression of total Axl as well as release of soluble Axl into the supernatant was observed by ELISA in incompletely edited SNU449-Axl --1 cells. Importantly, a comparative genomic hybridization array revealed comparable genomic changes in Axl knockout cells as well as in cells expressing Cas9 nickase without guide RNAs in SNU449 and HLF cells, indicating vast alterations in genomic DNA triggered by nickase. Together, these data show that the dynamics of CRISPR/Cas9 may cause incomplete editing events in cancer cell lines, as gene copy numbers vary based on genomic heterogeneity.

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“…Two limitations for the use of the Cas9:sgRNA complex for genome editing are delivery into target cells and modification at off-target DNA sites (22)(23)(24). We and others have shown that delivery of the Cas9:sgRNA ribonucleoprotein complex results in comparable efficacy and reduced off-target cleavage events compared with traditional plasmid-based delivery methods (4,25). The ribonucleoprotein complex is anionic, facilitating complexation with cationic nanoparticles without the need for fusion with a supernegatively charged protein.…”

Section: Protein Charge Determines Gene Recombination Efficiencymentioning

confidence: 99%

Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles

Wang

Zuris

Meng

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

527

473

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A central challenge to the development of protein-based therapeutics is the inefficiency of delivery of protein cargo across the mammalian cell membrane, including escape from endosomes. Here we report that combining bioreducible lipid nanoparticles with negatively supercharged Cre recombinase or anionic Cas9:single-guide (sg)RNA complexes drives the electrostatic assembly of nanoparticles that mediate potent protein delivery and genome editing. These bioreducible lipids efficiently deliver protein cargo into cells, facilitate the escape of protein from endosomes in response to the reductive intracellular environment, and direct protein to its intracellular target sites. The delivery of supercharged Cre protein and Cas9:sgRNA complexed with bioreducible lipids into cultured human cells enables gene recombination and genome editing with efficiencies greater than 70%. In addition, we demonstrate that these lipids are effective for functional protein delivery into mouse brain for gene recombination in vivo. Therefore, the integration of this bioreducible lipid platform with protein engineering has the potential to advance the therapeutic relevance of protein-based genome editing.

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Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection

Cited by 627 publications

References 20 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

Dynamics of CRISPR/Cas9-mediated genomic editing of the AXL locus in hepatocellular carcinoma cells

Efficient delivery of genome-editing proteins using bioreducible lipid nanoparticles

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