It is revealed that a wide variety of mutation types are possible with the base editors in this VLP collection, revealing that a wide variety of mutation types are possible with the base editors in this collection.
Asfar Lathif Salaudeen, Trevor Shyiak, Carl G. de Boer· bioRxiv· 0 citations
ABSTRACT Despite the promise of DNA base editors for diverse genome editing applications, their utility remains constrained by off‐target effects, which are exacerbated by short spacers in miniature systems and the large size of Cas9‐derived editors, which impedes adeno‐associated virus (AAV) delivery. Here, guided by structural insights into a compact Cas9d nuclease from Deltaproteobacteria, we developed an efficient Cas9d system (Cas9dUltra) through gRNA and protein engineering, and further developed its base editors (9dBEs). Cas9dUltra and 9dBEs enabled efficient and precise genome editing in human cells. Notably, 9dCBE induced premature termination codons in 89% of mouse pups by microinjection, facilitating robust disease modeling. Furthermore, a single AAV delivering 9dCBE achieved efficient Pcsk9 editing and a concomitant reduction in serum LDL‐C levels in mice. Collectively, this study establishes a series of compact, potent genome editing tools poised to advance biological and biomedical translational research.
Qingquan Xiao, Zhijin Tian, Luqi Weng et al.· Advancement of science· 0 citations
Base editing enables precise genome modifications without introducing DNA double-strand breaks. Using Streptococcus pyogenes Cas9 as a prototype, we previously developed a modular base editing platform in which the deaminase is recruited by an RNA aptamer engineered into the gRNA, thereby separating sequence recognition from base modification. Here, we expanded this modular base editor toolbox by engineering Staphylococcus aureus Cas9 (SaCas9) in combination with various vertebrate effectors derived from activation induced cytidine deaminase (AID) and apolipoprotein B mRNA editing enzyme, catalytic subunit 1 (APOBEC1) orthologs, from bat, lizard, human, and rat. Moreover, we adopted the SaCas9 variants with different protospacer adjacent motif requirements. These base editors generally showed high editing efficiency with low on-target indel formation and low-to-undetectable off-target activities. Quantitative and qualitative differences in editing occur among the base editors when applied to diverse loci, allowing sequence-specific optimization. Together, our study demonstrates the effectiveness of the SaCas9 modular base editors, the robustness of the platform’s modularity, and its feasibility for convenient screening of target-specific base editors.
J. Collantes, Kellen Xu, M. Ruiz-Urigüen et al.· The CRISPR Journal· 0 citations
Base editors (BEs) are transformative genome engineering tools that enable precise nucleotide substitutions without inducing double-strand breaks (DSBs) or requiring donor DNA templates. Since the first cytosine base editor (CBE) was developed in 2016, the field has advanced rapidly, with the creation of diverse BE variants that incorporate distinct deaminases and glycosylases. These engineered editors have significantly expanded the scope of genome editing by generating deaminated bases or apurinic/apyrimidinic (AP) site lesions, thereby harnessing endogenous DNA repair or replication mechanisms to produce base transitions and transversions. Among these endogenous pathways, trans-lesion synthesis (TLS) plays a particularly critical role in converting AP sites into specific base substitutions. TLS polymerases insert nucleotides opposite AP lesions, and the final editing outcome is dictated by the unique nucleotide preferences of individual TLS polymerases. This review focuses on the action mode of different base editors, highlights their interplays with the TLS, summarizes their potential therapeutic applications and discusses perspective strategies to improve precision and expand targeting scope.
Rui Tao, Min Li, Tongyun Luo et al.· Biotechnology Advances· 0 citations
Adenine base editors (ABEs), which enable A•T-to-G•C base editing, have emerged as a powerful tool with potential therapeutic applications. However, conventional ABEs suffer from bystander nucleotide conversions, limiting their utility for precise editing. Here we present a single-nucleotide resolution ABE (snuABE) created by fusing a nickase Cas9, nCas9-H840A, with the deaminase domain of ADAR (adenosine deaminase acting on RNA), which acts on DNA:RNA hybrids, instead of TadA, which acts on single-stranded DNA in conventional ABEs. snuABE requires a target-adenine guide RNA (tagRNA) that introduces a mismatch at the target adenine, enabling highly specific A-to-G editing by ADAR. Engineering ADAR from Pediculus humanus using the in silico protein evolution algorithm EvolvePro, along with 3'-end protection of the tagRNA, enhanced snuABE activity, yielding a median efficiency of 5.4% and a maximum efficiency of 50.0% across 32 targets in HEK293T cells. snuABE exhibits no detectable DNA off-target editing at predicted off-target or orthogonal R-loop sites, highlighting its potential as a precise and safe base-editing technology.
Hyeon Woo Im, Bada Jeong, Yeji Lee et al.· Nature Biotechnology· 0 citations
Base editing (BE), the precise installation of single-nucleotide changes in DNA or RNA without inducing double-strand breaks, holds substantial therapeutic promise for correcting single-nucleotide variants, which constitute more than half of the known pathogenic genetic variants. Recent advances have improved base editor specificity, efficiency, and delivery, enabling clinically oriented procedures. Clinically, BE has shown early success or strong translational promise in sickle cell disease, β-thalassemia, leukemia (via CAR T and epitope engineering), hypercholesterolemia (PCSK9 and ANGPTL3), alpha-1-antitrypsin deficiency, and glycogen storage disease type Ia. Key remaining challenges include bystander editing within the activity window, residual off-target DNA and RNA editing, delivery constraints (payload size, tissue targeting, and redosing limits), immunogenicity, and the need for durable long-term safety evidence across relevant cell types and disease contexts. Continued technological refinements, careful preclinical validation, and rigorous clinical assessment will be essential to fully realize BE's transformative potential in precision medicine.
Moksada Regmi, K. Ma, C. Bi et al.· Cell Genomics· 0 citations