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 1 | Component 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 (s)gRNA and recombinant Cas protein (A), in vitro transcribed (s)gRNA and Cas protein-encoding mRNA (B) or Cas protein and sgRNA-encoding expression plasmids (C).
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[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.
