RNA in Therapeutics: CRISPR in the Clinic

Abstract

The advent of the CRISPR-Cas genome editing platform has greatly enhanced the capabilities of researchers in many areas of biology. Its use has also been turned to the development of therapies for genetic diseases and to the enhancement of cell therapies. This review describes some recent advances in these areas.

Keywords: CRISPR; Cas9; base editing; genome editing; prime editing; sickle cell disease.

Fig. 1. Illustration of CRISPR components bound to a DNA target site.

The target DNA is shown as blue lines, the guide RNA as orange lines, and the Cas9 protein as a pale orange shape. Base pairing between target DNA and guide RNA (gRNA) is shown with short, vertical blue and orange lines. The PAM (protospacer adjacent motif) sequence, which is required for target recognition by Cas9, is indicated (purple). PAM is nGG (where n is any base) in the top strand for the commonly used Streptococcus pyogenes Cas9. Base pairing between gRNA and DNA is responsible for sequence-specific recognition; Cas9 has the nuclease activity that cuts both strands of the target DNA (black arrowheads).

Fig. 2. Illustration of the cellular repair activities and outcomes following a unique, targeted double-strand break induced by CRISPR.

Nonhomologous end joining (NHEJ) often leads to insertions and/or deletions (indels) at the target. Homology-dependent repair (HDR) uses a template to copy sequences into the break site; the template can be one provided by the researcher. gRNA, guide RNA. Adapted from the article of Carroll and Charo (2015) (Genome Biol. 16, 242) under the terms of the Creative Commons Attribution 4.0 International (CC BY 4.0) license.

Fig. 3. Additional CRISPR tools.

(A) Generalized picture of the cytosine and adenine base editors. A version of Cas9 that cuts only the RNA-bound strand (nCas9) is fused to a relevant deaminase that modifies one or a few bases on the displaced DNA strand (red star) at some distance from the PAM (protospacer adjacent motif). The cytosine base editor is also linked to two copies of UNGi, an inhibitor of the cellular uracil N-glycosylase enzyme that would normally remove uracil from DNA (not shown). The nicking activity of Cas9 directs the cellular mismatch repair system to use the modified DNA strand preferentially as the template for repair. (B) Schematic illustration of the prime editor. Cas9 is modified so it only cuts the displaced strand at the target, and it is fused to a viral reverse transcriptase (RTase). The guide RNA (gRNA) is extended at its 3’ end (orange line) with a sequence that has homology to the displaced DNA strand and carries a desired sequence modification to the right (in this picture) of the break in that strand. The RNA-DNA hybrid (orange-blue) is treated as a primer-template complex by RTase, which extends the 3’ end of the DNA across the modification in the RNA. Additional steps lead to the incorporation of this modification into the target. As in the base editors, a second gRNA is provided to create a nick nearby in the bottom strand of the DNA to favor the modified strand as the ultimate template for repair (not shown).