CRISPR-Cas9: From a bacterial immune system to genome-edited human cells in clinical trials

Abstract

The adaptive bacterial immune system CRISPR-Cas is revolutionizing all fields of life science and has opened up new frontiers toward personalised medicine. Since the elucidation of the molecular mechanism of Cas9 from Streptococcus pyogenes in 2012 and its development as a genomic engineering tool, genetic modifications in more than 40 species have been performed, over 290 patents have been filed worldwide and the first clinical trials using CRISPR-Cas-modified T-cells have recently been started in China and in the US. In this review we summarise current design developments, novel Cas systems and their antagonists, present and potential future applications as well as the ongoing debate on ethical issues, which has arisen through the CRISPR-Cas technology.

Keywords: CRISPR-Cas; bacterial immune system; clinical trials; genome editing.

Figure 1.

Function and classification of CRISPR-systems. (A) The 3 stages of CRISPR-Cas mediated immunity. In the adaptation step, selected sequences from the invading DNA, so-called protospacers, are integrated into the CRISPR loci. Following transcription of the CRISPR loci, the resulting pre-crRNAs are processed to the mature crRNAs, each consisting of a single spacer and repeat sequence. These crRNAs are bound by the Cas endonuclease forming the active effector nuclease complex, which recognizes the foreign nucleic acid by base paring during a subsequent infection, and degrades it (= interference). (B) Classification and characteristics of CRISPR systems. Type I is shown in green, type II in blue, type III in red, type IV in orange, type V in brown and type VI in gray. To date, the cleavage specificity of type IV systems is not known.

Figure 2.

CRISPR-Cas9 genome editing. Cas9 (gray) binds the target DNA (yellow) via a protospacer-sgRNA (blue) duplex. The depicted sgRNA consists of a crRNA part (in bright blue) for sequence specification and a tracrRNA part (in cyan) for Cas9 binding. The 2 catalytic subunits (HNH domain and RuvC-like domain) introduce a blunt-ended double-strand break (DSB) 3 nucleotides upstream of the protospacer adjacent motif (PAM, in pink). These DSBs can be repaired either via non-homologous end-joining (NHEJ) or homology-directed repair (HDR). NHEJ introduces smaller insertions and/or deletions (indels), which results in a disruption of the gene sequence. In contrast, whole gene sequences can be replaced or introduced with HDR, where at the provided donor DNA serves as template for DNA repair (green). HDR also allows the introduction of specific point mutations (in red).