The Right Tools for the Right Job: CRISPR-pass Could Offer Safe Gene Correction for Many Disorders
Authors: David J. Segal
Journal: Molecular Therapy (2019)

The ability to correct genetic mutations by gene editing at first sounds wonderful, but, in fact, it is a nearly foreboding proposition. The seemingly simple term "mutated gene" actually encompasses hundreds of different genetic variants, and current methods for the correction of each of these variants by homology-directed repair (HDR) is generally neither efficient nor error-free. However, in this issue of Molecular Therapy, Lee et al.1Lee C. Hyun Jo D. Hwang G.H. Yu J. Kim J.H. Park S.E. Kim J.S. Kim J.H. Bae S. CRISPR-pass: Gene rescue of nonsense mutations using adenine base editors.Mol. Ther. 2019; 27 (this issue): 1364-1371Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar present a practical strategy for ameliorating a subset of these variants in any mutated gene. CRISPR-pass combines a highly active version of an adenosine deaminase base editor with a target-site expanded xCas9 to allow read-through of 95.5% of known premature termination codons (PTCs) in a manner that is both efficient and virtually error-free. This combination of "the right tools for the right job" seems unusually poised to realize the promise of gene editing for the treatment of many genetic disorders. PTCs or nonsense mutations introduce an inappropriate stop codon in an open reading frame, resulting in truncated proteins or mRNAs degraded by nonsense-mediated decay. Although PTCs constitute only 16.2% of the 50,376 mutations causing pathological phenotypes in the ClinVar database, they represent a special therapeutic opportunity. Unlike frameshifts, splice-variants, and missense (amino acid substitution) mutations, it may not be necessary to correct nonsense mutations back to the original amino acid codon as long as translation is allowed to "read through" and complete the full-length protein. This special property allows many more ways to ameliorate the problem than having only to restore the genetic sequence. The clinical interest in read-through therapy is perhaps best evidenced by the recent clinical trials of aminoglycoside small-molecule drugs such as gentamicin2Malik V. Rodino-Klapac L.R. Viollet L. Wall C. King W. Al-Dahhak R. Lewis S. Shilling C.J. Kota J. Serrano-Munuera C. et al.Gentamicin-induced readthrough of stop codons in Duchenne muscular dystrophy.Ann. Neurol. 2010; 67: 771-780PubMed Google Scholar and the safer, orally administered, alternative ataluren,3McDonald C.M. Campbell C. Torricelli R.E. Finkel R.S. Flanigan K.M. Goemans N. Heydemann P. Kaminska A. Kirschner J. Muntoni F. et al.Clinical Evaluator Training Group; ACT DMD Study GroupAtaluren in patients with nonsense mutation Duchenne muscular dystrophy (ACT DMD): a multicentre, randomised, double-blind, placebo-controlled, phase 3 trial.Lancet. 2017; 390: 1489-1498Abstract Full Text Full Text PDF PubMed Scopus (272) Google Scholar which bypass nonsense mutations by recruiting near-cognate tRNAs at the site of the PTC. Approximately 15% of patients with Duchenne muscular dystrophy (DMD) have nonsense mutations that terminate translation of dystrophin. Ataluren was shown to have clinical efficacy in a subgroup of these patients and is now approved for use by the European Medicines Agency (EMA). In the US, clinical trials are ongoing for DMD, aniridia (absence of the iris), and the epilepsy syndromes Dravet and CDKL5 deficiency. Bypass of PTCs could be a useful therapeutic approach for many genetic conditions, including many autism spectrum disorders.4Iossifov I. Ronemus M. Levy D. Wang Z. Hakker I. Rosenbaum J. Yamrom B. Lee Y.H. Narzisi G. Leotta A. et al.De novo gene disruptions in children on the autistic spectrum.Neuron. 2012; 74: 285-299Abstract Full Text Full Text PDF PubMed Scopus (1047) Google Scholar Gene editing is also being investigated as a method for long-term amelioration of PTCs in DMD. In some of the earliest in vivo uses of adeno-associated virus (AAV)-Cas9, three groups reported that excision of PTC-containing exon 23 in a mouse model of DMD enabled the remaining exons to splice into a nearly full-length dystrophin mRNA, providing a partial rescue of muscle function.5Long C. Amoasii L. Mireault A.A. McAnally J.R. Li H. Sanchez-Ortiz E. Bhattacharyya S. Shelton J.M. Bassel-Duby R. Olson E.N. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy.Science. 2016; 351: 400-403Crossref PubMed Scopus (673) Google Scholar, 6Nelson C.E. Hakim C.H. Ousterout D.G. Thakore P.I. Moreb E.A. Castellanos Rivera R.M. Madhavan S. Pan X. Ran F.A. Yan W.X. et al.In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy.Science. 2016; 351: 403-407Crossref PubMed Scopus (809) Google Scholar, 7Tabebordbar M. Zhu K. Cheng J.K.W. Chew W.L. Widrick J.J. Yan W.X. Maesner C. Wu E.Y. Xiao R. Ran F.A. et al.In vivo gene editing in dystrophic mouse muscle and muscle stem cells.Science. 2016; 351: 407-411Crossref PubMed Scopus (743) Google Scholar This type of deletion-based exon skipping relies on the non-homologous end joining (NHEJ) DNA repair pathway, which functions at high efficiency in the non-dividing myofibrils. However, not all PTCs will be amendable to deletion-based exon skipping. Targeted correction of the nonsense mutation by HDR is inefficient in most cells, especially non-dividing cells. An additional donor DNA repair template would be required but frequently ignored by the cell in favor of the more efficient NHEJ repair pathway, leading to new indel mutations of PTCs that would likely result in frameshifts. Even the NHEJ-type repair mechanisms used in the DMD studies, which tolerated indels because the cleavages occurred in flanking introns, was not without consequences. A 1-year follow-up of the treated mice revealed significant unintended on-target events, including inversions rather than excision of exon 23, long deletions spanning up to 5,000 base pairs from the cleavage site, and frequent insertions of the AAV vector genome at the breakpoint.8Nelson C.E. Wu Y. Gemberling M.P. Oliver M.L. Waller M.A. Bohning J.D. Robinson-Hamm J.N. Bulaklak K. Castellanos Rivera R.M. Collier J.H. et al.Long-term evaluation of AAV-CRISPR genome editing for Duchenne muscular dystrophy.Nat. Med. 2019; 25: 427-432Crossref PubMed Scopus (222) Google Scholar Indeed, a growing number of studies in the past year have reported similar unintended events, suggesting that in vivo correction of PTCs by nuclease-based gene editing may not yet be ready for clinical use. In contrast, the CRISPR-pass method described by Lee et al.1Lee C. Hyun Jo D. Hwang G.H. Yu J. Kim J.H. Park S.E. Kim J.S. Kim J.H. Bae S. CRISPR-pass: Gene rescue of nonsense mutations using adenine base editors.Mol. Ther. 2019; 27 (this issue): 1364-1371Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar presents an enticing solution based on several recent technological advances by David Liu's group at the Broad Institute. Base editors do not create double-strand breaks but instead rely on enzymes that deaminate cytosine (cytosine base editors, CBEs) or adenosine (ABEs). The deaminated bases become fixed to thymine and guanine, respectively, after replication. ABEs are particularly appropriate for altering stop codons, which are comprised of TAA, TAG, and TGA (or TTA, CTA, and TCA on the opposite strand). Moreover, two recent studies observed substantial off-target editing by the current CBEs, but ABEs had high on-target fidelity.9Zuo E. Sun Y. Wei W. Yuan T. Ying W. Sun H. Yuan L. Steinmetz L.M. Li Y. Yang H. Cytosine base editor generates substantial off-target single-nucleotide variants in mouse embryos.Science. 2019; 364: 289-292Crossref PubMed Scopus (401) Google Scholar, 10Jin S. Zong Y. Gao Q. Zhu Z. Wang Y. Qin P. Liang C. Wang D. Qiu J.L. Zhang F. Gao C. Cytosine, but not adenine, base editors induce genome-wide off-target mutations in rice.Science. 2019; 364: 292-295Crossref PubMed Scopus (350) Google Scholar Evolved versions of ABEs such as ABEmax have even greater editing efficiency.11Koblan L.W. Doman J.L. Wilson C. Levy J.M. Tay T. Newby G.A. Maianti J.P. Raguram A. Liu D.R. Improving cytidine and adenine base editors by expression optimization and ancestral reconstruction.Nat. Biotechnol. 2018; 36: 843-846Crossref PubMed Scopus (392) Google Scholar However, base editors constructed on the commonly used deactivated Cas9 from Streptococcus pyogenes (dSpCas9) must bind near NGG protospacer adjacent motif (PAM) sites, which imposes a severe limitation on the number of nonsense mutations that can be targeted. However, evolved variants such as xCas9, which utilizes NG and NAR PAM site, have far less restrictions.12Hu J.H. Miller S.M. Geurts M.H. Tang W. Chen L. Sun N. Zeina C.M. Gao X. Rees H.A. Lin Z. Liu D.R. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity.Nature. 2018; 556: 57-63Crossref PubMed Scopus (869) Google Scholar These are the right tools. Lee et al.1Lee C. Hyun Jo D. Hwang G.H. Yu J. Kim J.H. Park S.E. Kim J.S. Kim J.H. Bae S. CRISPR-pass: Gene rescue of nonsense mutations using adenine base editors.Mol. Ther. 2019; 27 (this issue): 1364-1371Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar did not create the tools, nor were they the first to combine them.12Hu J.H. Miller S.M. Geurts M.H. Tang W. Chen L. Sun N. Zeina C.M. Gao X. Rees H.A. Lin Z. Liu D.R. Evolved Cas9 variants with broad PAM compatibility and high DNA specificity.Nature. 2018; 556: 57-63Crossref PubMed Scopus (869) Google Scholar, 13Zafra M.P. Schatoff E.M. Katti A. Foronda M. Breinig M. Schweitzer A.Y. Simon A. Han T. Goswami S. Montgomery E. et al.Optimized base editors enable efficient editing in cells, organoids and mice.Nat. Biotechnol. 2018; 36: 888-893Crossref PubMed Scopus (179) Google Scholar What their study did show was how the right tools could be used for the right job. The most effective combination was dxCas9 and ABEmax, which allowed read-through of more than 10% of PTC alleles in the XPC gene of GM14867 fibroblasts. That amount of correction was sufficient to rescue resistance to 254 nm UV light. Functional XPC protein was expressed at least 4 weeks after treatment, while cells treated with ataluren or gentamicin showed only transient XPC expression. Importantly, analysis of 12 in silico predicted off-target sites found no evidence of off-target editing. Future studies will need to examine if similar editing rates and fidelity can be observed at different loci. However, these data suggest CRISPR-pass could be one of the safest approaches to gene editing. The potential to restore some functionality to the roughly 16% of proteins impaired by PTC mutations across many different genetic diseases with high-fidelity editing should be vigorously investigated. CRISPR-Pass: Gene Rescue of Nonsense Mutations Using Adenine Base EditorsLee et al.Molecular TherapyMay 23, 2019In BriefLee et al. showed that CRISPR-pass, based on adenine base editors, would be a targeted tool for bypassing premature termination codons. This system could be applicable to ∼95% of clinically significant nonsense mutations, related to genetic diseases, in the ClinVar database. Full-Text PDF Open Access