Gene and genome editing最新文献

This article reviews some of the early events leading to the development of the first genome editing platform, zinc-finger nucleases. It describes some of the earliest uses of ZFNs and the advent of the more user-friendly platforms, TALENs and CRISPR. Some current applications and their implications are reviewed, followed by a brief look into the future.

Base editors are an innovative addition to the genome editing toolbox that introduced a new genome editing strategy to the field. Instead of using double-stranded DNA breaks, base editors use nucleobase modification chemistry to efficiently and precisely incorporate single nucleotide variants (SNVs) into the genome of living cells. Two classes of DNA base editors currently exist: deoxycytidine deamination-derived editors (CBEs, which facilitate C•G to T•A mutations) and deoxyadenosine deamination-derived base editors (ABEs, which facilitate A•T to G•C mutations). More recently, the development of mitochondrial base editors allowed the introduction of C•G to T•A mutations into mitochondrial DNA as well. Base editors show great potential as therapeutic agents and research tools, and extensive studies have been carried out to improve upon the original base editor constructs to aid researchers in a variety of disciplines. Despite their widespread use, there are few publications that focus on elucidating the biological pathways involved during the processing of base editor intermediates. Because base editors introduce unique types of DNA damage products (a U•G mismatch with a DNA backbone nick for CBEs, and an I•T mismatch with a DNA backbone nick for ABEs) to facilitate genome editing, a deep understanding of the DNA damage repair pathways that facilitate or impede base editing represents an important aspect for the further expansion and improvement of the technologies. Here, we first review canonical deoxyuridine, deoxyinosine, and single-stranded break repair. Then, we discuss how interactions among these different repair processes can lead to different base editing outcomes. Through this review, we hope to promote thoughtful discussions on the DNA repair mechanisms of base editing, as well as help researchers in the improvement of current base editors and the development of new base editors.

Recent studies showed that CRISPR nucleases can boost plant immunity against infected virus by inducing the cleavage of viral dsDNA intermediate in a host plant. Here, we demonstrate that CRISPR-Cas9 can also improve plant resistance against a bacterial pathogen, Pseudomonas syringae, when sgRNAs that selectively target the bacterial genome are either transiently or constitutively expressed in plants. Our findings indicate that plant-expressed CRISPR-Cas9 components can transport into bacterial cells and disrupt the bacterial genome, suggesting a novel defense strategy against pathogens in plants, which could be widely applied regardless of the bacterial species.

The 2018 announcement regarding safe childbirths via germline genome-editing (GGE) with parental consent shocked the world. This minireview examines the predictable risks, burdens, and potential harms of human GGE and explores the question of responsibility for using GGE in human reproduction. Although there is currently no international consensus on proving the absence of harmful off-target mutations in the genome, preclinical GGE study can demonstrate the non-existence under specific conditions. Initially, the clinical application will be limited to small studies without controls. In any case, individuals born via GGE should be followed up for long period. However, such persons can decline follow-up. Due to limited screening, an overlooked off-target mutation may harm the entire body. Some persons suffering such harm might claim damages on the ground that their life is less valuable. However, most jurisdictions will reject such claims. Practitioners are responsible for proving there are no harmful off-target mutations in each GGE case, although the appropriateness of proof is currently difficult to accept. Parents who consented to GGE, as well as practitioners, assume responsibility for the safety of genome-edited offspring; however, the fulfillment of responsibility ultimately depends on the offspring's autonomy. Meanwhile, practitioners and parents may be exempt from some damage claims by offspring harmed by unsafe GGE. The uncertainty of assigning responsibility may underpin GGE's prohibition in light of the unacceptable risks, burdens and potential harms for persons born via GGE; or it may oppositely underpin its permission if an acceptable risk-benefit balance is reached for parents and society.

CRISPR-Cas nucleases, base editors (BEs), and prime editors (PEs) are efficient genome editing tools used widely in diverse research fields, including biology, biotechnology, and medicine. While the genome editing mechanism is different for each tool, the target specificity is conferred in common by binding of guide RNAs (gRNAs) and their complementary target sequences. However, gRNAs can bind to off-target sequences with a few mismatches, provoking off-target editing, and the editing activities/outcomes vary depending on the gRNAs. Therefore, selection of gRNAs as well as analysis of the outcomes is crucial to improve the editing strategies. In this review, we introduce various programs currently used in selection of gRNAs and analysis of results for each genome editing tool and briefly describe the purpose and features of each program, which will be informative to researchers when planning genome editing.