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Zhang Lab CRISPR Plasmids Available from Addgene

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Click HERE to browse the latest Zhang lab LentiCRISPRv2 plasmids and human GeCKO libraries.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a microbial nuclease system involved in defense against invading phages and plasmids. CRISPR loci in microbial hosts contain a combination of CRISPR-associated (Cas) genes as well as non-coding RNA elements capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage. We have recently harnessed the type II CRISPR nuclease system to facilitate genome editing in mammalian cells (Cong et al., Science 2013).

The CRISPR/Cas system can be implemented in mammalian cells by co-expressing the bacterial Cas9 nuclease along with the guide RNA. Two forms of guide RNAs can be used to facilitate Cas9-mediated genome cleavage, using a CRISPR RNA array and tracrRNA or a synthetic guide RNA fusing the CRISPR RNA with the tracrRNA. These two systems are described below.

1. S. pyogenes Cas9 (or Cas9 D10A nickase) + CRISPR RNA array + tracrRNA: This plasmid contains three expression cassettes. In order to target a given site, the plasmid can be digested using BbsI, and a pair of annealed oligos (design is indicated below) can be cloned into the CRISPR array. The oligos is designed based on the target site sequence (30bp) and needs to be flanked on the 3' end by a 3bp NGG PAM sequence.

2. S. pyogenes Cas9 (or Cas9 D10A nickase) + chimeric guide RNA containing +85nt of tracrRNA: This plasmid contains two expression cassettes, hSpCas9 and the chimeric guide RNA. The vector can be digested using BbsI, and a pair of annealed oligos (design is indicated below) can be cloned into the guide RNA. The oligos is designed based on the target site sequence (20bp) and needs to be flanked on the 3' end by a 3bp NGG PAM sequence. We have found that increasing the length of the chimeric guide RNA can increase targeting efficiency; therefore this version of the backbone contains a longer fragment of the tracrRNA (+85nt).

 

Application Notes: For application of Cas9 for site-specific genome editing in eukaryotic cells and organisms, we have computationally identified suitable target sites for the S. pyogenes Cas9. These sites are viewable as UCSC Genome Browser tracks for the human, mouse, rat, zebrafish, C. elegans, and D. melanogaster genomes. Sites are selected such that the seed sequence for each SpCas9 target site, 5’-NNNNNNNNNNNN-NGG-3’, is specific to the relevant genome. A protocol for oligo cloning is included here and the plasmid sequences and additional information can be found at Zhang Lab's website (www.genome-engineering.org) as well as in the following publication:

Multiplex Genome Engineering using CRISPR/Cas Systems
Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F.
Science. 2013 Jan 3.
DOI: 10.1126/science.1231143

For additional tips and information for using the Zhang Lab CRISPRs, click here.

ID Plasmid
42230 pX330: A human codon-optimized SpCas9 and chimeric guide RNA expression plasmid. Add to Cart
42335 pX335: A human codon-optimized SpCas9 nickase and chimeric guide RNA expression plasmid. Add to Cart
42337 pX261: Dual expression plasmid of human codon-optimized SpCas9 and a gRNA to the human Emx1 locus, can be used to test SpCas9 cleavage in cell lines of choice. Add to Cart
41863 pX097: This plasmid is used to reconstitute the complete Type II CRISPR system from S. pyogenes and contains the host factor RNaseIII and tracrRNA. Add to Cart
42229 pX260: This plasmid separately encodes a human codon-optimized SpCas9, a tracrRNA and customizable crRNA. Add to Cart
42333 pX334: This plasmid separately encodes a human codon-optimized SpCas9 nickase, a tracrRNA and customizable crRNA. Add to Cart

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