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CRISPR/Cas: RNA-guided gene targeting

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Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) are the hallmark of a bacterial defense system that forms the basis for CRISPR/Cas sequence-specific gene targeting technology [1-5].

CRISPR/Cas systems typically consists of two components:

There are several ways to introduce Cas endonucleases and sequence-specific guide RNA into cells (Fig. 1) however, delivery as ribonucleoprotein (RNP) complex (Fig. 1A) or RNA molecules (Fig. 1B) shows the highest efficiency in most cases.[6-7]

Component 1Component 2
Cas endonucleases (s)gRNA Synthesis

Figure 1: Cas protein and sequence-specific guide RNA (e.g. sgRNA/Cas9) can be delivered as ribonucleoprotein (RNP) complex consisting of in vitro transcribed or chemically synthesized (s)gRNA and recombinant Cas protein (A), in vitro transcribed or chemically synthesized (s)gRNA and Cas protein-encoding mRNA (B) or Cas protein and sgRNA-encoding expression plasmids (C).

CRISPR/Cas principle:
Complexation of guide RNA and Cas endonuclease is required to exert their function: The guide RNA directs the binding of this ribonucleoprotein (RNP) complex to the complementary gene region of interest that is site-specifically cleaved prior to gene editing by a Cas endonuclease upstream of a Cas endonuclease-specific recognition sequence[1].

Cas endonucleases can conveniently be programmed to target different gene sites by altering the guide RNA sequence only. Due to this simplicity, CRISPR/Cas systems are the most flexible and efficient site-directed gene targeting tools currently available [2,3].
Dr. Barbara Zschoernig

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Selected References:

[1] Jinek et al. (2012) A programmable dual-RNA-guided, DNA endonuclease in adaptive bacterial immunity. Science 337(6096):816.
[2] Wang et al. (2016) CRISPR/Cas9 in Genome Editing and Beyond. Annu. Rev. Biochem. 85:227.
[3] Gaj et al. (2013) ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 31(7):397.
[4] Aldi et al. (2018) The CRISPR tool kit for genome editing and beyond. Nature Communications 9:1911.
[5] Pickar-Oliver et al. (2018) The next generation of CRISPR–Cas technologies and applications. Nature Reviews Molecular Cell Biology 20:507.
[6] DeWitt et al. (2017) Genome editing via delivery of Cas9 ribonucleoprotein. Methods 121:9.
[7] Kim et al. (2014) Highly efficient RNA-guided genome editing in humancells via delivery of purified Cas9 ribonucleoproteins. Genome Research 24:1012.