Abstract
The CRISPR
One of the overarching lessons from the CRISPR discovery is the salience of the approach of harnessing powerful prokaryotic molecular tools to accomplish therapeutic genetic alterations in human cells. After this paradigm, gene therapy researchers have engineered more efficient and versatile versions of CRISPR technologies. The special focus of this issue is to highlight some of these newer adaptations.
First up in this issue, we are pleased to offer an interview of Dr. David Liu of Harvard Medical School, an incredibly creative pioneer in the field who has opened two completely new subfields within gene editing, base editing, and prime editing (See page 237). Applying protein engineering, he has created fusions of the enzymes cytosine deaminase and adenine deaminase with an altered version Cas9. This fusion combines the RNA-directed DNA sequence specificity of Cas9 with the ability of deaminase enzymes to convert C-to-T or G-to-A within the host genomes. Not resting on his laurels, Dr. Liu followed up this invention with an even more versatile invention, the fusion of Cas9 with reverse transcriptase, a combination that allows one to effectively rewrite any sequence within the host genome. His interview provides an incredibly insightful look into these developments as well as the future of the field.
The so-called CRISPR-transactivators (CRISPRa) and CRISPR-inhibitors (CRISPRi) represent bold new advances in Cas-based RNA-guided gene altering enzymes. The review by Lek et al. describes these ingeniously designed enzymes. 2 The original CRISPRa technology they describe contains the Streptococcus pyogenes Cas9 RuvC and HNH domains with point mutations to deactivate the endonuclease activity fused to either a transcriptional transactivator (in the case of CRISPRa) or a transcriptional repressor (in the case of CRIPSRi). As their review describes, there have been many variations of these multidomain fusions using other RNA guidance domains, domains that either directly activate or repress genes, or domains that mediate epigenome modifications. As the Lek review indicates, the CRISPRa approach has been used successfully both as a research tool for genome screening and as a potential therapeutic. Of particular interest is the application of CRISPRa to single gene disorders caused by haploinsufficiency, such as Dravet syndrome. 2
The focus of the other two reviews in this issue is direct in vivo gene editing. As the review by Dasgupta et al. points out, ex vivo manipulation of autologous cells by gene editing is certainly a powerful technology, but the direct delivery of gene editing tools to target organs in situ could fit a much broader range of potential applications. 3 At least some level of feasibility has been demonstrated for this approach in several different organ systems in animal models, as with recombinant adeno-associated virus (rAAV)-based delivery of gene editing machinery to the liver and muscle. The ability to move in vivo gene editing forward for cell types that are not good candidates for long-term conventional in vivo rAAV gene therapy, such as hematopoietic stem and progenitor cells, may form an important niche for advanced CRISPR applications in the future.
The most advanced programs for in vivo gene editing are directed at retinal diseases, which is the focus of the review by Quinn et al. 4 As this review indicates, the eye provides an ideal target for in vivo gene editing because it is small, accessible, and a self-contained target. It is also relatively immune privileged, thus avoiding immune responses to the bacterial derived Cas9 protein. 4 The intrinsic advantages of doing in vivo gene editing in the eye are reflected by the fact that it has progressed the furthest of all of the in vivo gene editing applications, having reached the clinical trial stage in the EDIT-101 trial from Editas Medicine. The authors also point out conditions for which prime editing may be needed as a next-generation CRISPR process to treat retinal diseases caused by haploinsufficiency.
This issue also features two interesting original research articles. The article by Zhang et al. from the Oregon Health Sciences Center studied a number of variables that might affect in vivo gene editing in their mouse model of hereditary tyrosinemia type 1, a model for which in vivo editing had previously been demonstrated. 5 Their final conclusions indicate that a dual rAAV approach to provide Cas9, sgRNA, and homology template can achieve correction in 7 − 20% of neonatal mice and a lower number of adult mice.
The article by Li et al. from Wenzou Medical University and their nationally leading Wenzhou Eye Hospital describe a new system for optimization of conditions for homology-directed repair (HDR) by different editing enzymes. They utilized a mutant green fluorescent protein (GFP) reporter strategy in which only precise HDR editing of a mutant GFP (embedded within the host cell AAVS1 site) would restore GFP expression. 6 This allowed for direct comparison of the efficiency of HDR in cells between different Cas enzymes and optimization of conditions for HDR.
This special issue provides important new insights into how CRISPR technology has evolved over the past several years, yielding ever more powerful tools for gene therapy. The original concept of CRISPR-Cas9-mediated gene editing targeted by short guide RNAs was to induce sequence-specific double strand breaks into DNA to knock genes out through nonhomologous end joining or repair them by HDR. The next-generation CRISPR technologies presented here expand that repertoire to include base editing, prime editing, CRISPRa, and CRISPRi. These are now being applied both in vivo and ex vivo. In the future, it seems inevitable that these tools and approaches will make up an ever-increasing share of human gene therapy trials, and thus open the door to successful therapy for patients whose diseases might otherwise never be treatable.