DnaQ mediates directional spacer acquisition in the CRISPR-Cas system by a time-dependent mechanism.

In the spacer acquisition stage of CRISPR-Cas immunity, spacer orientation and protospacer adjacent motif (PAM) removal are two prerequisites for functional spacer integration. Cas4 has been implicated in both processing the prespacer and determining the spacer orientation. In Cas4-lacking systems, host 3'-5' DnaQ family exonucleases were recently reported to play a Cas4-like role. However, the molecular details of DnaQ functions remain elusive. Here, we characterized the spacer acquisition of the adaptation module of the Streptococcus thermophilus type I-E system, in which a DnaQ domain naturally fuses with Cas2. We presented X-ray crystal structures and cryo-electron microscopy structures of this adaptation module. Our biochemical data showed that DnaQ trimmed PAM-containing and PAM-deficient overhangs with different efficiencies. Based on these results, we proposed a time-dependent model for DnaQ-mediated spacer acquisition to elucidate PAM removal and spacer orientation determination in Cas4-lacking CRISPR-Cas systems.

Discovery of a potent inhibitor targeting the cap-binding domain of the PB2 subunit of influenza RNA-dependent RNA polymerase.

Influenza pandemics have emerged as a significant global public health and security concern. PB2, a crucial subunit of the influenza RNA-dependent RNA polymerase (RdRP), has been identified as a promising target for influenza treatment. We herein report the discovery of a potent novel PB2 inhibitor, 7-51A, with a KD value of 1.64 nM as determined by ITC. The high activity of 7-51A was elucidated by the co-crystal structure of the PB2-7-51A complex, and comparative analysis revealed unique interactions that had never been observed before. The preliminary pharmacological evaluation indicated that 7-51A exhibited commendable cellular safety, hepatic microsomal metabolic safety and stability. Collectively, 7-51A was found to be an effective PB2 inhibitor and could be used as a lead compound for further studies.

Targeted, programmable, and precise tandem duplication in the mammalian genome.

Tandem duplications are frequent structural variations of the genome and play important roles in genetic disease and cancer. However, interpreting the phenotypic consequences of tandem duplications remains challenging, in part owing to the lack of genetic tools to model such variations. Here, we developed a strategy, tandem duplication via prime editing (TD-PE), to create targeted, programmable, and precise tandem duplication in the mammalian genome. In this strategy, we design a pair of in trans prime editing guide RNAs (pegRNAs) for each targeted tandem duplication, which encode the same edits but prime the single-stranded DNA (ssDNA) extension in opposite directions. The reverse transcriptase (RT) template of each extension is designed homologous to the target region of the other single guide RNA (sgRNA) to promote the reannealing of the edited DNA strands and the duplication of the fragment in between. We showed that TD-PE produced robust and precise in situ tandem duplications of genomic fragments ranging from ∼50 bp to ∼10 kb, with a maximal efficiency up to 28.33%. By fine-tuning the pegRNAs, we achieved simultaneous targeted duplication and fragment insertion. Finally, we successfully produced multiple disease-relevant tandem duplications, showing the general utility of TD-PE in genetic research.

Recent advances in therapeutic CRISPR-Cas9 genome editing: mechanisms and applications.

Recently, clustered regularly interspaced palindromic repeats (CRISPR)-Cas9 derived editing tools had significantly improved our ability to make desired changes in the genome. Wild-type Cas9 protein recognizes the target genomic loci and induced local double strand breaks (DSBs) in the guidance of small RNA molecule. In mammalian cells, the DSBs are mainly repaired by endogenous non-homologous end joining (NHEJ) pathway, which is error prone and results in the formation of indels. The indels can be harnessed to interrupt gene coding sequences or regulation elements. The DSBs can also be fixed by homology directed repair (HDR) pathway to introduce desired changes, such as base substitution and fragment insertion, when proper donor templates are provided, albeit in a less efficient manner. Besides making DSBs, Cas9 protein can be mutated to serve as a DNA binding platform to recruit functional modulators to the target loci, performing local transcriptional regulation, epigenetic remolding, base editing or prime editing. These Cas9 derived editing tools, especially base editors and prime editors, can introduce precise changes into the target loci at a single-base resolution and in an efficient and irreversible manner. Such features make these editing tools very promising for therapeutic applications. This review focuses on the evolution and mechanisms of CRISPR-Cas9 derived editing tools and their applications in the field of gene therapy.

Case report: A case of delayed cutaneous metastases from signet-ring cell mixed-type gastric cancer.

Background: Signet-ring cell gastric carcinoma is a highly malignant tumor, with the characteristics of strong invasiveness, rapid progression, a high degree of malignancy, and generally poor prognosis. The most common site of metastases is the abdominal organs, especially the liver, while delayed cutaneous metastases are rare. Case presentation: We report a case of cutaneous metastases on the head, groin, and thigh, which recurred 7 years after signet-ring cell gastric carcinoma surgery. The patient was diagnosed with a 2.0×1.5×1.0cm tumor at the angle of stomach, and treated with Billroth II distal gastrectomy accompanied with D2 lymph node dissection. According the pathology, the stage was pT1N3M0. Then the patient received two cycles of oxaliplatin and tegafur chemotherapy, which was discontinued due to the inability to tolerate the side effects of chemotherapy. Seven years after the surgery, the patient initially presented with a fleshy mass on the head and beaded nodules in the groin; then, the mass gradually became larger, along with the thighs turning red, swollen, and crusty. Firstly, the patient was diagnosed with "lower extremity lymphangitis" and treated mostly with anti-inflammatory, promote lymphatic return, detumescence and elastic force cannula in vascular surgery department. However, the symptoms relieved insufficient. Finally, the skin biopsy indicates a signet-ring cell gastric carcinoma cutaneous metastasis. The whole-body PET-CT examination showed multiple nodules with increased metabolism. Then the patient was transferred to The Department of Oncology for further chemotherapy. Conclusion: Our case highlights that gastric tumor recurrence and metastasis should be highly suspected when skin lesions appear in patients with signet-ring cell gastric carcinoma. At the same time, multidisciplinary consultation and close cooperation between surgeons, oncologists, and dermatologists are of great significance to the diagnosis and treatment of this disease.

Dual-AAV split prime editor corrects the mutation and phenotype in mice with inherited retinal degeneration.

The prime editor (PE) can edit genomes with almost any intended changes, including all 12 possible types of base substitutions, small insertions and deletions, and their combinations, without the requirement for double strand breaks or exogenous donor templates. PE demonstrates the possibility of correcting a variety of disease-causing mutations and might expand the therapeutic application of gene editing. In this study, PE was optimized based on a dual-adeno-associated virus (AAV) split-intein system in vitro by screening different split sites and split inteins. We found that splitting PE before amino acid 1105(Ser) of SpCas9 with Rma intein resulted in the highest on-target editing. The orientations of pegRNA and nicking sgRNA in the AAV vector were further optimized. To test the in vivo performance of the optimized dual-AAV split-PE3, it was delivered by subretinal injection in rd12 mice with inherited retinal disease Leber congenital amaurosis. The prime editors corrected the pathogenic mutation with up to 16% efficiency in a precise way, with no detectable off-target edits, restored RPE65 expression, rescued retinal and visual function, and preserved photoceptors. Our findings establish a framework for the preclinical development of PE and motivate further testing of PE for the treatment of inherited retinal diseases caused by various mutations.

Erratum: HDAC inhibitors improve CRISPR-Cas9 mediated prime editing and base editing.

[This corrects the article DOI: 10.1016/j.omtn.2022.05.036.].

Erratum: Long-Term Metabolic Correction of Phenylketonuria by AAV-Delivered Phenylalanine Amino Lyase.

[This corrects the article DOI: 10.1016/j.omtm.2019.12.014.].

HDAC inhibitors improve CRISPR-Cas9 mediated prime editing and base editing.

Recent advances in CRISPR-Cas9 techniques, especially the discovery of base and prime editing, have significantly improved our ability to make precise changes in the genome. We hypothesized that modulating certain endogenous pathway cells could improve the action of those editing tools in mammalian cells. We established a reporter system in which a small fragment was integrated into the genome by prime editing (PE). With this system, we screened an in-house small-molecule library and identified a group of histone deacetylase inhibitors (HDACi) increasing prime editing. We also found that HDACi increased the efficiency of both cytosine base editing (CBE) and adenine base editing (ABE). Moreover, HDACi increased the purity of cytosine base editor products, which was accompanied by an upregulation of the acetylation of uracil DNA glycosylase (UNG) and UNG inhibitor (UGI) and an enhancement of their interaction. In summary, our work demonstrated that HDACi improves Cas9-mediated prime editing and base editing.

Internally inlaid SaCas9 base editors enable window specific base editing.

Rationale: Base editors composed of catalytic defective Cas9 and cytosine or adenosine deaminase are powerful tools to convert bases in a genome. However, the fixed and narrow editing window of current base editors has impeded their utility. To increase the scope and diversify the editing patterns is quite necessary. Methods and Results: We designed a subset of base editors derived from SaCas9 in which deaminase was inlaid into various locations of the SaCas9 protein. The resulting base editors were characterized with multiple genomic sites and were found to have distinct editing features to the N-terminal SaCas9 CBE (Sa-CBE-N). Among them, Sa-CBE-693, in which a cytosine deaminase was inserted between amino acids 693 and 694, showed an increased editing efficiency and a significantly expanded editing window ranging from bases 2-18. This feature enhanced the editing efficiency of BCL11A enhancer that contains multiple consensus bases in a 15-bp fragment. Another variant, Sa-CBE-125, displayed backward-shifted editing window, which we showed was particularly powerful in editing cytosines that were accompanied with unintended bystander cytosines at their 5' side. Additionally, these editors showed reduced Cas9 independent DNA off-target editing compared with Sa-CBE-N. Conclusion: Our inlaid base editors improved the targeting scope and diversified the editing pattern.

Bi-PE: bi-directional priming improves CRISPR/Cas9 prime editing in mammalian cells.

Prime editors consisting of Cas9-nickase and reverse transcriptase enable targeted precise editing of small DNA pieces, including all 12 kinds of base substitutions, insertions and deletions, while without requiring double-strand breaks or donor templates. Current optimized prime editing strategy (PE3) uses two guide RNAs to guide the performance of prime editor. One guide RNA carrying both spacer and templating sequences (pegRNA) guides prime editor to produce ssDNA break and subsequent extension, and the other one produces a nick in the complementary strand. Here, we demonstrated that positioning the nick sgRNA nearby the templating sequences of the pegRNA facilitated targeted large fragment deletion and that engineering both guide RNAs to be pegRNAs to achieve bi-direction prime editing (Bi-PE) further increase the efficiency by up to 16 times and improved the accuracy of editing products by 60 times. In addition, we showed that Bi-PE strategy also increased the efficiency of simultaneous conversion of multiple bases but not single base conversion over PE3. In conclusion, Bi-PE strategy expanded the editing scope and improved the efficiency and the accuracy of prime editing system, which might have a wide range of potential applications.

WT-PE: Prime editing with nuclease wild-type Cas9 enables versatile large-scale genome editing.

Large scale genomic aberrations including duplication, deletion, translocation, and other structural changes are the cause of a subtype of hereditary genetic disorders and contribute to onset or progress of cancer. The current prime editor, PE2, consisting of Cas9-nickase and reverse transcriptase enables efficient editing of genomic deletion and insertion, however, at small scale. Here, we designed a novel prime editor by fusing reverse transcriptase (RT) to nuclease wild-type Cas9 (WT-PE) to edit large genomic fragment. WT-PE system simultaneously introduced a double strand break (DSB) and a single 3' extended flap in the target site. Coupled with paired prime editing guide RNAs (pegRNAs) that have complementary sequences in their 3' terminus while target different genomic regions, WT-PE produced bi-directional prime editing, which enabled efficient and versatile large-scale genome editing, including large fragment deletion up to 16.8 megabase (Mb) pairs and chromosomal translocation. Therefore, our WT-PE system has great potential to model or treat diseases related to large-fragment aberrations.

Expanding PAM recognition and enhancing base editing activity of Cas9 variants with non-PI domain mutations derived from xCas9.

The recognition of protospacer adjacent motif (PAM) is a key factor for the CRISPR (i.e. clustered regularly interspaced short palindromic repeats)/CRISPR-associated 9 (Cas9) system to distinguish foreign DNAs from the host genome, and also significantly restricts the targeting scope of the system during genome-editing applications. Structurally, the PAM interacting (PI) domain, which usually is located in the C-terminus of Cas9 proteins, directly binds to PAM and plays a key role in determining the recognition specificity. However, several lines of evidence showed that other regions of Cas9 protein beyond the PI domain might also play roles in PAM interaction. Here, we constructed a mosaic SpCas9 protein (xCas9-NG) by fusing the PI domain of SpCas9 PAM variant, Cas9-NG with the non-PI fragment of xCas9 protein that contains multiple amino acid substitutions. We found that non-PI fragment of xCas9 expanded PAM recognition of the Cas9-NG PI domain. In addition, xCas9-NG showed an improved editing efficiency in the majority of targets harboring xCas9 and Cas9-NG PAMs. Importantly, this finding was also successfully extended to other Cas9 variants, including SpRY and the non-G SpCas9 series. Together, our work expands the target scope of SpCas9 editing system and demonstrates the notion that the non-PI domain fragment plays an important role in PAM restriction.

Erratum: Long-Term Metabolic Correction of Phenylketonuria by AAV-Delivered Phenylalanine Amino Lyase.

[This corrects the article DOI: 10.1016/j.omtm.2019.12.014.].

A universal strategy for AAV delivery of base editors to correct genetic point mutations in neonatal PKU mice.

Base editing tools enabled efficient conversion of C:G or A:T base pairs to T:A or G:C, which are especially powerful for targeting monogenic lesions. However, in vivo correction of disease-causing mutations is still less efficient because of the large size of base editors. Here, we designed a dual adeno-associated virus (AAV) strategy for in vivo delivery of base editors, in which deaminases were linked to Cas9 through the interaction of GCN4 peptide and its single chain variable fragment (scFv) antibody. We found that one or two copies of GCN4 peptide were enough for the assembly of base editors and produced robust targeted editing. By optimization of single-guide RNAs (sgRNAs) that target phenylketonuria (PKU) mutation, we were able to achieve up to 27.7% correction in vitro. In vivo delivery of this dual AAV base editing system resulted in efficient correction of PKU-related mutation in neonatal mice and subsequent rescue of hyperphenylalaninemia-associated syndromes. Considering the similarity between Cas9 proteins from different organisms, our delivery strategy will be compatible with other Cas9-derived base editors.

A split cytosine deaminase architecture enables robust inducible base editing.

Directed base substitution with base editing technology enables efficient and programmable conversion of C:G or A:T base pairs to T:A or G:C in the genome. Although this technology has shown great potentials in a variety of basic research, off-target editing is among one of the biggest challenges toward its way to clinical application. Base editing tools, especially the tools converting C to T, caused unpredictable off-target editing throughout the genome, which raise the concern that long-term application of these tools would induce genomic instability or even tumorigenesis. To overcome this challenge, we designed an inducible base editing tool that was active only in the presence of a clinically safe chemical, rapamycin. In the guidance of structural information, we designed four split-human APOBEC3A (A3A) -BE3 base editors in which these A3A deaminase enzymes were split at sites that were opposite to the protein-nucleotide interface. We showed that by inducible deaminase reconstruction with a rapamycin responsible interaction system (FRB and FKBP); three out of four split-A3A-derived base editors showed robust inducible base editing. However, in the absence of rapamycin, their editing ability was dramatically inhibited. Among these split editors, splicing at Aa85 of A3A generated the most efficient inducible editing. In addition, compared to the full-length base editor, the splitting did not obviously alter the editing window and motif preference, but slightly increased the product purity. We also expanded this strategy to another frequently used cytosine deaminase, rat APOBEC1 (rA1), and observed a similar induction response. In summary, these results demonstrated the concept that splitting deaminases is a practicable method for timely controlling of base editing tools.

Design, synthesis and biological evaluation of tyrosinase-targeting PROTACs.

The human tyrosinase is the most prominent therapeutic target for pigmentary skin disorders. However, the overwhelming majority efforts have been devoted to search mushroom tyrosinase inhibitors, which show poor inhibitory activity on human tyrosinase and certain side effects that cause skin damage in practical application. Herein, a series of degraders that directly targeted human tyrosinase was firstly designed and synthesized based on newly developed PROTAC technology. The best PROTAC TD9 induced human tyrosinase degradation obviously in dose and time-dependent manner, and its mechanism of inducing tyrosinase degradation has also been clearly demonstrated. Besides, encouraging results that low-toxicity PROTAC TD9 was applied to reduce zebrafish melanin synthesis have been obtained, highlighting the potential to treatment of tyrosinase-related disorders. Moreover, this work has innovatively expanded the application scope of PROTAC technology and laid a solid foundation for further development of novel drugs treating pigmentary skin disorders.

Recent Advances in CRISPR/Cas9 Directed Base Editing.

Recently, CRISPR-based techniques have significantly improved our ability to make desired changes and regulations in various genomes. Among them, targeted base editing is one of the most powerful techniques in making precise genomic editing. Base editing enabled the irreversible conversion of a specific single DNA base, from C to T or and from A to G, in desired genomic loci. This technique has important implications in the study of human genetic diseases, considering that many of them resulted from point mutations. More importantly, the high efficiency of these editing tools also provided great promise in clinical applications. In this review, we discuss the recent progress and challenges of base editing tools.

A distinct structure of Cas1-Cas2 complex provides insights into the mechanism for the longer spacer acquisition in Pyrococcus furiosus.

In the adaptation stage of CRISPR-Cas systems, the Cas1-Cas2 integrase captures and integrates new invader-derived spacers into the CRISPR locus, serving as a molecular memory of prior infection. As of yet, the structural information of Cas1-Cas2 complex is available only for two species. Here we present the crystal structure of Cas1-Cas2 complex of Pyrococcus furiosus, which showed a distinct architecture from the known Cas1-Cas2 complexes. The shorter C-terminal tail of Pfu Cas2 directs the Cas1 dimers go in the opposite direction, resulting in a different prespacer binding mode. Based on our structural and mutagenesis results, we modeled a prespacer with a shorter duplex and longer 3' overhangs to bind Pfu Cas1-Cas2 complex. The prespacer preference was confirmed by EMSA, fluorescence polarization, and in vitro integration assays. This model provides a potential explanation for the longer spacer acquisition observed in P. furiosus when deleting both cas4 genes. Our study highlights the diversity of the CRISPR adaptation module.