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Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.

Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA - Nucleic Acids Res. (2014)

Bottom Line: To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method.Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells.This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA.

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Golden Gate assembly of single lentiviral vectors encoding CRISPR/Cas9 and multiple sgRNA expression cassettes. In the first step, individual sgRNAs are cloned into separate expression vectors and sequence verified. Fragments containing each sgRNA expression cassette are then transferred into a Cas9-expressing lentivirus in step 2 by Golden Gate assembly as shown. Black triangles shown in the lentiviral plasmid represent loxP sites flanking the entire sgRNA and Cas9 expression cassette.
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Figure 2: Golden Gate assembly of single lentiviral vectors encoding CRISPR/Cas9 and multiple sgRNA expression cassettes. In the first step, individual sgRNAs are cloned into separate expression vectors and sequence verified. Fragments containing each sgRNA expression cassette are then transferred into a Cas9-expressing lentivirus in step 2 by Golden Gate assembly as shown. Black triangles shown in the lentiviral plasmid represent loxP sites flanking the entire sgRNA and Cas9 expression cassette.

Mentions: The gene encoding human codon optimized Cas9 (hCas9) nuclease was obtained from Addgene (Plasmid #41815) (25), and we reported the expression cassette for the Streptococcus pyogenes sgRNA previously (Addgene Plasmid #47108) (18). Additional promoters for mU6 (38), H1 (39) and 7SK (40) Pol III promoters were synthesized using GeneBlocks (IDT) and cloned in place of the hU6 sgRNA expression cassette. A GeneBlock (IDT) was inserted into the 3′ of the hCas9 coding sequence to fuse a T2A skipping peptide and eGFP gene to monitor Cas9 expression. The coding sequence for hCas9-T2A-GFP was then transferred into the FUGW lentiviral expression vector (Addgene Plasmid #14883) containing the human ubiquitin C (hUbC) promoter to drive expression of hCas9-T2A-GFP, as well as restriction sites to facilitate Golden Gate cloning of sgRNA expression cassettes immediately upstream of the hUbC promoter (Figure 2). All plasmids have been made publicly available through the Addgene non-profit plasmid repository (plasmids #53186–53192).


Multiplex CRISPR/Cas9-based genome engineering from a single lentiviral vector.

Kabadi AM, Ousterout DG, Hilton IB, Gersbach CA - Nucleic Acids Res. (2014)

Golden Gate assembly of single lentiviral vectors encoding CRISPR/Cas9 and multiple sgRNA expression cassettes. In the first step, individual sgRNAs are cloned into separate expression vectors and sequence verified. Fragments containing each sgRNA expression cassette are then transferred into a Cas9-expressing lentivirus in step 2 by Golden Gate assembly as shown. Black triangles shown in the lentiviral plasmid represent loxP sites flanking the entire sgRNA and Cas9 expression cassette.
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Related In: Results  -  Collection

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Figure 2: Golden Gate assembly of single lentiviral vectors encoding CRISPR/Cas9 and multiple sgRNA expression cassettes. In the first step, individual sgRNAs are cloned into separate expression vectors and sequence verified. Fragments containing each sgRNA expression cassette are then transferred into a Cas9-expressing lentivirus in step 2 by Golden Gate assembly as shown. Black triangles shown in the lentiviral plasmid represent loxP sites flanking the entire sgRNA and Cas9 expression cassette.
Mentions: The gene encoding human codon optimized Cas9 (hCas9) nuclease was obtained from Addgene (Plasmid #41815) (25), and we reported the expression cassette for the Streptococcus pyogenes sgRNA previously (Addgene Plasmid #47108) (18). Additional promoters for mU6 (38), H1 (39) and 7SK (40) Pol III promoters were synthesized using GeneBlocks (IDT) and cloned in place of the hU6 sgRNA expression cassette. A GeneBlock (IDT) was inserted into the 3′ of the hCas9 coding sequence to fuse a T2A skipping peptide and eGFP gene to monitor Cas9 expression. The coding sequence for hCas9-T2A-GFP was then transferred into the FUGW lentiviral expression vector (Addgene Plasmid #14883) containing the human ubiquitin C (hUbC) promoter to drive expression of hCas9-T2A-GFP, as well as restriction sites to facilitate Golden Gate cloning of sgRNA expression cassettes immediately upstream of the hUbC promoter (Figure 2). All plasmids have been made publicly available through the Addgene non-profit plasmid repository (plasmids #53186–53192).

Bottom Line: To address the need for uniform and sustained delivery of multiplex CRISPR/Cas9-based genome engineering tools, we developed a single lentiviral system to express a Cas9 variant, a reporter gene and up to four sgRNAs from independent RNA polymerase III promoters that are incorporated into the vector by a convenient Golden Gate cloning method.Each sgRNA is efficiently expressed and can mediate multiplex gene editing and sustained transcriptional activation in immortalized and primary human cells.This delivery system will be significant to enabling the potential of CRISPR/Cas9-based multiplex genome engineering in diverse cell types.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Engineering, Duke University, Durham, NC 27708, USA.

Show MeSH