I-Silence, Please! An Alternative for Gene Disruption via Adenine Base Editors.

Abstract

The CRISPR-based cytosine and adenine base editors (CBEs and ABEs), which consist of Cas9 nickase fused to a cytidine or adenosine deaminase, respectively, enable precise base substitutions at targeted regions without generating DNA double-strand breaks (DSBs).1Komor A.C. Kim Y.B. Packer M.S. Zuris J.A. Liu D.R. Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature. 2016; 533: 420-424Crossref PubMed Scopus (2441) Google Scholar,2Gaudelli N.M. Komor A.C. Rees H.A. Packer M.S. Badran A.H. Bryson D.I. Liu D.R. Programmable base editing of A⋅T to G⋅C in genomic DNA without DNA cleavage.Nature. 2017; 551: 464-471Crossref PubMed Scopus (1815) Google Scholar To date, researchers have generally used CBEs to convert cytosine to thymine to induce premature termination codons (PTCs) within protein coding sequence (CDS) regions for gene disruption (e.g., CRISPR-STOP and i-STOP),3Kuscu C. Parlak M. Tufan T. Yang J. Szlachta K. Wei X. Mammadov R. Adli M. CRISPR-STOP: gene silencing through base-editing-induced nonsense mutations.Nat. Methods. 2017; 14: 710-712Crossref PubMed Scopus (200) Google Scholar,4Billon P. Bryant E.E. Joseph S.A. Nambiar T.S. Hayward S.B. Rothstein R. Ciccia A. CRISPR-Mediated Base Editing Enables Efficient Disruption of Eukaryotic Genes through Induction of STOP Codons.Mol. Cell. 2017; 67: 1068-1079.e4Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar whereas ABEs have been used in the reverse manner, to convert an adenine to a guanine in a PTC for gene rescue.5Lee 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: 1364-1371Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar In this issue of Molecular Therapy, Wang et al.6Wang X. Liu Z. Li G. Dang L. Huang S. He L. Ma Y. Li C. Liu M. Yang G. et al.Efficient Gene Silencing by Adenine Base Editor-Mediated Start Codon Mutation.Mol. Ther. 2020; 28: 431-440Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar present an alternative means of ABE-mediated gene disruption, named i-Silence, which involves the conversion of an adenine to a guanine in a start codon in the coding strand (ATG-to–GTG) or noncoding strand (ATG-to-ACG) (Figure 1). Gene editing tools, including CRISPR-Cas9 nucleases, enable targeted gene disruption or rescue at desired sites. Initially, such tools were developed to target one specific site to generate DNA DSBs. After DNA cleavage, gene disruption or recovery occurs through the cell’s two mechanisms of DNA repair: non-homologous end joining (NHEJ) or homology-directed repair (HDR). However, recent studies have found that DNA DSBs can lead to a p53-mediated DNA damage response, which results in cell death7Haapaniemi E. Botla S. Persson J. Schmierer B. Taipale J. CRISPR-Cas9 genome editing induces a p53-mediated DNA damage response.Nat. Med. 2018; 24: 927-930Crossref PubMed Scopus (602) Google Scholar,8Ihry R.J. Worringer K.A. Salick M.R. Frias E. Ho D. Theriault K. Kommineni S. Chen J. Sondey M. Ye C. et al.p53 inhibits CRISPR-Cas9 engineering in human pluripotent stem cells.Nat. Med. 2018; 24: 939-946Crossref PubMed Scopus (512) Google Scholar and can frequently cause large deletions, which results in genomic rearrangements and/or chromosomal truncations.9Kosicki M. Tomberg K. Bradley A. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements.Nat. Biotechnol. 2018; 36: 765-771Crossref PubMed Scopus (48) Google Scholar,10Shin H.Y. Wang C. Lee H.K. Yoo K.H. Zeng X. Kuhns T. Yang C.M. Mohr T. Liu C. Hennighausen L. CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome.Nat. Commun. 2017; 8: 15464Crossref PubMed Scopus (170) Google Scholar In this regard, gene editing technologies that do not generate DNA DSBs have attracted a great deal of attention. In the new study, Wang et al.6Wang X. Liu Z. Li G. Dang L. Huang S. He L. Ma Y. Li C. Liu M. Yang G. et al.Efficient Gene Silencing by Adenine Base Editor-Mediated Start Codon Mutation.Mol. Ther. 2020; 28: 431-440Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar first tested this strategy in an enhanced green fluorescent protein (EGFP)-based reporter system as a proof-of-concept. Similar to negative control experiments that use start codon-mutated reporter plasmids (CDS1GTG-GFP or CDS2ACG-GFP), the authors showed that i-Silence can efficiently disrupt the EGFP gene by mutating the start codon. Next, the authors applied i-Silence at four endogenous genes (HDAC1, SEC61B, PIGH, and FTL) in HEK293T cells. High-throughput sequencing data from bulk populations of cells revealed efficient editing frequencies (from 60% to 80%) for all genes. In addition, in the case of the HDAC1 target, Sanger sequencing data and western blot results verified the complete gene knockout (KO) at the level of single transformants. The authors ultimately applied the i-Silence approach to knock out a gene in vivo in mice. They injected mRNAs that encoded ABEs targeting the PD-1 gene into ten mouse zygotes and found that the average editing efficiency ranged from 31.8% to 73.6% in different embryos, enabling the generation of PD-1 KO mice. Furthermore, the authors verified the precision of ABE-mediated i-Silence by both whole genome sequencing in mice and amplicon-based deep sequencing of predicted off-target sites in cells. One potential concern with the i-Silence strategy is its genome-wide utility, because its target sites are strictly confined to start codon sequences (ATG); in contrast, i-STOP has a much broader target range, typically within the first 20% of CDS regions (limited to avoid incomplete KOs caused by the expression of partially functional, truncated proteins). Use of wild-type (WT) Cas9 from Streptococcus pyogenes (SpCas9), the most popular CRISPR effector in the genome editing field, allows targeting of 8,423 genes by i-Silence, about half of the number that can be targeted by i-STOP (15,122), among the 19,267 human genes registered in the National Center for Biotechnology Information (NCBI) database. However, when SpCas9 variants and Cas9 orthologs, which recognize an expanded pool of protospacer adjacent motifs (PAMs), are considered, both i-Silence (17,804) and i-STOP (18,075) can target most genes, indicating that the target gene ranges of the two methods are comparable. In addition, because some genes are targetable by i-STOP but not by i-Silence and vice versa, the two methods could be used as complementary strategies. Compared to CBE-mediated gene KO strategies, i-Silence has several potential benefits. First, conventional WT Cas9- or CBE-mediated gene KOs are generated by the creation of PTCs within the CDS region of mRNAs. mRNA transcripts containing PTCs are frequently degraded via the nonsense-mediated decay (NMD) pathway in cells, which can trigger a genetic compensation response, resulting in the expression of alternative transcripts that have a similar function as the initial transcripts.11Ma Z. Zhu P. Shi H. Guo L. Zhang Q. Chen Y. Chen S. Zhang Z. Peng J. Chen J. PTC-bearing mRNA elicits a genetic compensation response via Upf3a and COMPASS components.Nature. 2019; 568: 259-263Crossref PubMed Scopus (228) Google Scholar,12El-Brolosy M.A. Kontarakis Z. Rossi A. Kuenne C. Günther S. Fukuda N. Kikhi K. Boezio G.L.M. Takacs C.M. Lai S.-L. et al.Genetic compensation triggered by mutant mRNA degradation.Nature. 2019; 568: 193-197Crossref PubMed Scopus (468) Google Scholar However, because i-Silence induces gene KO by eliminating the start codon, genetic compensation responses may be avoided. Second, recent studies have reported that CBEs, but not ABEs, induce substantial DNA genomic off-target C-to-T conversions in mammalian cells, regardless of the existence of a guide RNA.13Jin 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,14Zuo 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 (404) Google Scholar These results may push i-Silence to the forefront of gene KO strategies. In addition to providing an alternative gene KO strategy, i-Silence could be useful in basic cell science for examining the response to initiation codon disruption in various targets, including disease-associated genes. Recently, genes edited to contain PTCs by frameshift have been reported to produce aberrant proteins (detected by western blot).15Tuladhar R. Yeu Y. Tyler Piazza J. Tan Z. Rene Clemenceau J. Wu X. Barrett Q. Herbert J. Mathews D.H. Kim J. et al.CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation.Nat. Commun. 2019; 10: 4056Crossref PubMed Scopus (94) Google Scholar The mechanisms involved alternative translation initiation (ATI) or mRNA transcription skipping of the PTC-bearing exon. ATI might occur during the use of i-Silence, although the authors did not detect any products from ATI in the tested targets. Further tests of i-Silence in an expanded group of targets might show unexpected outcomes, which would enhance our understanding of the gene expression process. i-Silence represents a novel and robust means of gene disruption, but a few concerns about its use still remain. Although ABEs are used to mutate the initial start codon, some genes have another downstream ATG codon within the initial CDS region. As described above, it could act as a supplementary start codon, resulting in functional protein production via an ATI mechanism. Furthermore, another recent study revealed that, in addition to adenine conversion, the current version of ABE can induce unexpected cytosine conversions by catalyzing cytosine deamination,16Kim H.S. Jeong Y.K. Hur J.K. Kim J.-S. Bae S. Adenine base editors catalyze cytosine conversions in human cells.Nat. Biotechnol. 2019; 37: 1145-1148Crossref PubMed Scopus (54) Google Scholar which may result in unwanted effects mediated by i-Silence. If these potential concerns are successfully overcome, i-Silence might be widely used for gene disruption in organisms from bacteria to humans. Efficient Gene Silencing by Adenine Base Editor-Mediated Start Codon MutationWang et al.Molecular TherapyNovember 28, 2019In BriefWang et al. describe a new strategy to induce gene silencing (i-Silence) with CRISPR-based adenine base editor (ABE)-mediated start codon mutation. i-Silence is efficient and precise for producing gene knockout, which can be employed on a genome-wide scale and to model human diseases with start codon mutations. Full-Text PDF Open Access