Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Feb 15;339(6121):819-23.
doi: 10.1126/science.1231143. Epub 2013 Jan 3.

Multiplex genome engineering using CRISPR/Cas systems

Le Cong  1 , F Ann Ran, David Cox, Shuailiang Lin, Robert Barretto, Naomi Habib, Patrick D Hsu, Xuebing Wu, Wenyan Jiang, Luciano A Marraffini, Feng Zhang
Affiliations
Free PMC article

Multiplex genome engineering using CRISPR/Cas systems

Le Cong et al. Science. .
Free PMC article
Favorites

Abstract

Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.

Figures

Fig. 1
The type II CRISPR locus from S. pyogenes SF370 can be reconstituted in mammalian cells to facilitate targeted DSBs of DNA. (A) Engineering of SpCas9 and SpRNase III with NLSs enables import into the mammalian nucleus. GFP indicates green fluorescent protein; scale bars, 10 μm. (B) Mammalian expression of human codon–optimized SpCas9 (hSpCas9) and SpRNase III (hSpRNase III) genes were driven by the elongation factor 1α (EF1 α) promoter, whereas tracrRNA and pre-crRNA array (DR-Spacer-DR) were driven by the U6 promoter. A protospacer (blue highlight) from the human EMX1 locus with PAM was used as template for the spacer in the pre-crRNA array. (C) Schematic representation of base pairing between target locus and EMX1-targeting crRNA. Red arrow indicates putative cleavage site. (D) SURVEYOR assay for SpCas9-mediated indels. (E) An example chromatogram showing a microdeletion, as well as representative sequences of mutated alleles identified from 187 clonal amplicons. Red dashes, deleted bases; red bases, insertions or mutations.
Fig. 2
SpCas9 can be reprogrammed to target multiple genomic loci in mammalian cells. (A) Schematic of the human EMX1 locus showing the location of five protospacers indicated by blue lines with corresponding PAM in magenta. (B) Schematic of the pre-crRNA:tracrRNA complex (top) showing hybridization between the direct repeat (gray) region of the pre-crRNA and tracrRNA. Schematic of a chimeric RNA design (12) (bottom). tracrRNA sequence is shown in red and the 20-bp spacer sequence in blue. (C) SURVEYOR assay comparing the efficacy of Cas9-mediated cleavage at five protospacers in the human EMX1 locus. Each protospacer was targeted by using either processed pre-crRNA:tracrRNA complex (crRNA) or chimeric RNA (chiRNA). Arrowheads indicate cleavage products for each protospacer target.
Fig. 3
Evaluation of the SpCas9 specificity and comparison of efficiency with TALENs. (A) EMX1-targeting chimeric crRNAs with single point mutations were generated to evaluate the effects of spacer-protospacer mismatches. (B) SURVEYOR assay comparing the cleavage efficiency of different mutant chimeric RNAs. (C) Schematic showing the design of TALENs that target EMX1. (D) SURVEYOR gel comparing the efficiency of TALEN and SpCas9 (N = 3).
Fig. 4
Applications of Cas9 for homologous recombination and multiplex genome engineering. (A) Mutation of the RuvC I domain converts Cas9 into a nicking enzyme (SpCas9n). HNH, histidine-asparagine-histidine endonuclease domain. (B) Coexpression of EMX1-targeting chimeric RNA with SpCas9 leads to indels, whereas SpCas9n does not (N = 3). (C) Schematic representation of the recombination strategy. A homology repair (HR) template is designed to insert restriction sites into EMX1 locus. Primers used to amplify the modified region are shown as red arrows. (D) Restriction fragment length polymorphism gel analysis. Arrows indicate fragments generated by HindIII digestion. (E) Example chromatogram showing successful recombination. (F) SpCas9 can facilitate multiplex genome modification by using a crRNA array that contains two spacers targeting EMX1 and PVALB. Schematic showing the design of the crRNA array (top). Both spacers mediate efficient protospacer cleavage (bottom). (G) SpCas9 can be used to achieve precise genomic deletion. Two spacers targeting EMX1 (top) mediated a 118-bp genomic deletion (bottom).
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by 4,403 articles

See all "Cited by" articles

Publication types

Feedback