Multiplex genome engineering using CRISPR/Cas systems

Broad Institute of MIT and Harvard, 7 Cambridge Center, Cambridge, MA 02142, USA and McGovern Institute for Brain Research, Department of Brain and Cognitive Sciences, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Science (Impact Factor: 33.61). 01/2013; 339(6121). DOI: 10.1126/science.1231143
Source: PubMed


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.

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Available from: Le Cong, Mar 24, 2015
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    • "Other rice EALs might possess functions similar to EAL1 and/or EAL2 (see below). Thus although the structures of predicted mature EALs appear very similar, they show very diverse expression pattern, highly specific binding pattern (Uebler et al. 2013), and therefore, functional studies are necessary to ultimately demonstrate similar/orthologous functions in diverse species. "
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    • "Targeting by NmeCas9 Has Relaxed Stringency for Seed Complementarity In Vitro and in Bacteria SpyCas9 requires near-perfect complementarity in the seed region for CRISPR interference and efficient genome editing (Jinek et al., 2012, 2013; Cho et al., 2013; Cong et al., 2013; Hwang et al., 2013; Jiang et al., 2013; Mali et al., 2013). Our previous work showed that a 2 nt mismatch at the cleavage site of a ps abrogated interference, while a 2 nt mismatch further from the PAM (outside the seed) did not (Zhang et al., 2013). "
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    ABSTRACT: Type II CRISPR systems defend against invasive DNA by using Cas9 as an RNA-guided nuclease that creates double-stranded DNA breaks. Dual RNAs (CRISPR RNA [crRNA] and tracrRNA) are required for Cas9's targeting activities observed to date. Targeting requires a protospacer adjacent motif (PAM) and crRNA-DNA complementarity. Cas9 orthologs (including Neisseria meningitidis Cas9 [NmeCas9]) have also been adopted for genome engineering. Here we examine the DNA cleavage activities and substrate requirements of NmeCas9, including a set of unusually complex PAM recognition patterns. Unexpectedly, NmeCas9 cleaves single-stranded DNAs in a manner that is RNA guided but PAM and tracrRNA independent. Beyond the need for guide-target pairing, this "DNase H" activity has no apparent sequence requirements, and the cleavage sites are measured from the 5' end of the DNA substrate's RNA-paired region. These results indicate that tracrRNA is not strictly required for NmeCas9 enzymatic activation, and expand the list of targeting activities of Cas9 endonucleases.
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    • "The effect of alteration in the enzyme concentration is varied at different PAM positions. Under normal enzymatic concentrations, the proximal region to PAM is regarded as highly specific (Cong et al., 2013; Jinek et al., 2012; Sapranauskas et al., 2011). Reduction in the enzyme concentration achieve most specificity (≤80%) at the PAM proximal end compared to the PAM distal end (30%) (Pattanayak et al., 2013). "
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    ABSTRACT: CRISPR/Cas, a microbial adaptive immune system, has recently been reshaped as a versatile genome editing approach, endowing genome engineering with high efficiency and robustness. The DNA endonuclease Cas, a component of CRISPR system, is directed to specific target within genomes by guide RNA (gRNA) and performs gene editing function. However, the system is still in its infancy and facing enormous challenges such as off-target mutation. Lots of attempts have been made to overcome such off-targeting and proven to be effective. In this review we focused on recent progress of increasing the CRISPR specificity realized by rational design of gRNA and modification of Cas9 endonuclease. Meanwhile the methods to screen off-target mutation and their effects are also discussed. Comprehensive consideration and rational design to reduce off-target mutation and selection of effective screening assay will greatly facilitate to achieve successful CRISPR/Cas system mediated gene editing.
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