251. TALENs Facilitate Targeted Genome Editing in Human Cells With High Specificity and Low Cytotoxicity

ABSTRACTCRISPR technology has opened up many diverse genome editing possibilities in human somatic cells, but has been limited in the therapeutic realm by both potential off-target effects and low genome modification efficiencies. Recent advancements to combat these limitations include delivering Cas9 nucleases directly to cells as highly purified ribonucleoproteins (RNPs) instead of the conventional plasmid DNA and RNA-based approaches. Here, we extend RNP-based delivery in cell culture to a less characterized CRISPR format which implements paired Cas9 nickases. The use of paired nickase Cas9 RNP system, combined with a GMP-compliant non-viral delivery technology, enables editing in human cells with high specificity and high efficiency, a development that opens up the technology for further exploration into a more therapeutic role.

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Exosome/Liposome-like Nanoparticles: New Carriers for CRISPR Genome Editing in Plants

International Journal of Molecular Sciences ◽

10.3390/ijms22147456 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7456

Author(s):

Mousa A. Alghuthaymi ◽

Aftab Ahmad ◽

Zulqurnain Khan ◽

Sultan Habibullah Khan ◽

Farah K. Ahmed ◽

...

Keyword(s):

Genome Editing ◽

Viral Vectors ◽

Polymeric Materials ◽

Inorganic Nanoparticles ◽

Plant Genome ◽

Delivery Methods ◽

Detailed Consideration ◽

Crispr System ◽

Efficient Delivery ◽

Low Cytotoxicity

Rapid developments in the field of plant genome editing using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) systems necessitate more detailed consideration of the delivery of the CRISPR system into plants. Successful and safe editing of plant genomes is partly based on efficient delivery of the CRISPR system. Along with the use of plasmids and viral vectors as cargo material for genome editing, non-viral vectors have also been considered for delivery purposes. These non-viral vectors can be made of a variety of materials, including inorganic nanoparticles, carbon nanotubes, liposomes, and protein- and peptide-based nanoparticles, as well as nanoscale polymeric materials. They have a decreased immune response, an advantage over viral vectors, and offer additional flexibility in their design, allowing them to be functionalized and targeted to specific sites in a biological system with low cytotoxicity. This review is dedicated to describing the delivery methods of CRISPR system into plants with emphasis on the use of non-viral vectors.

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Whole-genome sequencing reveals rare off-target mutations in CRISPR/Cas9-edited grapevine

Horticulture Research ◽

10.1038/s41438-021-00549-4 ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Xianhang Wang ◽

Mingxing Tu ◽

Ya Wang ◽

Wuchen Yin ◽

Yu Zhang ◽

...

Keyword(s):

Whole Genome Sequencing ◽

Genome Editing ◽

Genome Sequencing ◽

Plant Biotechnology ◽

High Specificity ◽

Fruit Trees ◽

Whole Genome ◽

Nucleotide Polymorphisms ◽

Indel Mutation ◽

Target Sites

AbstractThe CRISPR (clustered regularly interspaced short palindromic repeats)-associated protein 9 (Cas9) system is a powerful tool for targeted genome editing, with applications that include plant biotechnology and functional genomics research. However, the specificity of Cas9 targeting is poorly investigated in many plant species, including fruit trees. To assess the off-target mutation rate in grapevine (Vitis vinifera), we performed whole-genome sequencing (WGS) of seven Cas9-edited grapevine plants in which one of two genes was targeted by CRISPR/Cas9 and three wild-type (WT) plants. In total, we identified between 202,008 and 272,397 single nucleotide polymorphisms (SNPs) and between 26,391 and 55,414 insertions/deletions (indels) in the seven Cas9-edited grapevine plants compared with the three WT plants. Subsequently, 3272 potential off-target sites were selected for further analysis. Only one off-target indel mutation was identified from the WGS data and validated by Sanger sequencing. In addition, we found 243 newly generated off-target sites caused by genetic variants between the Thompson Seedless cultivar and the grape reference genome (PN40024) but no true off-target mutations. In conclusion, we observed high specificity of CRISPR/Cas9 for genome editing of grapevine.

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CRISPR-Cpf1-mediated genome editing and gene regulation in human cells

Biotechnology Advances ◽

10.1016/j.biotechadv.2018.10.013 ◽

2019 ◽

Vol 37 (1) ◽

pp. 21-27 ◽

Cited By ~ 6

Author(s):

Tianwen Li ◽

Linwen Zhu ◽

Bingxiu Xiao ◽

Zhaohui Gong ◽

Qi Liao ◽

...

Keyword(s):

Gene Regulation ◽

Genome Editing ◽

Human Cells

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Efficient target cleavage by Type V Cas12a effector programmed with split CRISPR RNA

10.1101/2020.12.21.423781 ◽

2020 ◽

Author(s):

Regina Tkach ◽

Natalia Nikitchina ◽

Nikita Shebanov ◽

Vladimir Mekler ◽

Egor Ulashchik ◽

...

Keyword(s):

Genome Editing ◽

Dna Cleavage ◽

Target Recognition ◽

Human Cells ◽

Stable Complex ◽

Slight Effect ◽

Target Dna ◽

Type V

ABSTRACTCRISPR RNAs (crRNAs) directing target DNA cleavage by type V-A Cas12a nucleases consist of repeat-derived 5’-scaffold moiety and 3’-spacer moiety. We demonstrate that removal of most of the 20-nucleotide scaffold has only a slight effect on in vitro target DNA cleavage by Cas12a ortholog from Acidaminococcus sp (AsCas12a). In fact, residual cleavage was observed even in the presence of a 20-nucleotide crRNA spacer part only, while crRNAs split into two individual moieties (scaffold and spacer RNAs) catalyzed highly specific and efficient cleavage of target DNA. Our data also indicate that AsCas12a combined with split crRNA forms a stable complex with the target. These observations were also confirmed in lysates of human cells expressing AsCas12a. The ability of the AsCas12a nuclease to be programmed with split crRNAs opens new lines of inquiry into the mechanisms of target recognition and cleavage and will further facilitate genome editing techniques based on Cas12a nucleases.

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Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1818461116 ◽

2019 ◽

Vol 116 (42) ◽

pp. 20959-20968 ◽

Cited By ~ 10

Author(s):

Sundaram Acharya ◽

Arpit Mishra ◽

Deepanjan Paul ◽

Asgar Hussain Ansari ◽

Mohd. Azhar ◽

...

Keyword(s):

Genome Editing ◽

Genetic Disorders ◽

High Specificity ◽

Francisella Novicida ◽

Guide Rna ◽

Homology Directed Repair ◽

Multiple Loci ◽

Specificity Of Binding ◽

Induced Pluripotent ◽

Very High

Genome editing using the CRISPR/Cas9 system has been used to make precise heritable changes in the DNA of organisms. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain regarding their putative off-targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher homology-directed repair rates, and very low nonspecific genome editing compared to SpCas9. We demonstrate FnCas9-mediated correction of the sickle cell mutation in patient-derived induced pluripotent stem cells and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.

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