CRISPR-Cas systems: ushering in the new genome editing era

Abstract

In recent years there has been great progress with the implementation and utilization of Clustered Regularly Interspaced Palindromic Repeats (CRISPR) and CRISPR-associated protein (Cas) systems in the world of genetic engineering. Many forms of CRISPR-Cas9 have been developed as genome editing tools and techniques and, most recently, several non-genome editing CRISPR-Cas systems have emerged. Most of the CRISPR-Cas systems have been classified as either Class I or Class II and are further divided among several subtypes within each class. Research teams and companies are currently in dispute over patents for these CRISPR-Cas systems as numerous powerful applications are concurrently under development. This mini review summarizes the appearance of CRISPR-Cas systems with a focus on the predominant CRISPR-Cas9 system as well as the classifications and subtypes for CRISPR-Cas. Non-genome editing uses of CRISPR-Cas are also highlighted and a brief overview of the commercialization of CRISPR is provided.

Keywords: CRISPR products; CRISPR-Cas figure; Cas classes; Cas types; Cas13; Cas9; HDR; NHEJ; TALEN; ZFN; genome editing.

Figure 1.

a) The crRNA from the CRISPR array combines with a smaller tracrRNA molecule, becoming a gRNA complex. b) The gRNA binds with a Cas9 protein, forming a gRNA:Cas9 complex. c) The gRNA guides the Cas9 protein, targeting a specific DNA sequence, which it first recognizes by the PAM motif. The RuvC and HNC nuclease sites cuts the target sequence, leaving two homologous blunt ends. d) The desired DNA repair template inserts the desired gene and repairs the strands by HDR, the product DNA then undergoes adaptation into the organism’s genome.

Figure 2.

a) CRISPR-Cas13 targets ssRNA with its crRNA, and the twin HEPN nuclease domains cleaves the sequence non-specifically after the first crRNA guided cleavage at the binding site, leaving blunt ends. b) The dCas9 combines with an activator/repressor domain to activate/repress an upstream gene, resulting in transcription of that gene into RNA or blocked transcription. c) dCas9-LSD1 complex targets the genome at the chromatin to repress transcription of the targeted gene by demethylation. d) CRISPR-dCas9-EGFP as a fluorescent marker complex for imaging.