» Sign in / Register

RNA-guided gene targeting

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 endonuclease and sequence-specific guide RNA (gRNA) can be delivered as ribonucleoprotein (RNP) complex consisting of synthetic gRNA and recombinant Cas endonuclease (A), synthetic gRNA and Cas endonuclease-encoding mRNA (B) or Cas protein and gRNA-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].

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.