Engineering TALE-linked deaminases to facilitate precision adenine base editing in mitochondrial DNA.

DddA-derived cytosine base editors (DdCBEs) and transcription activator-like effector (TALE)-linked deaminases (TALEDs) catalyze targeted base editing of mitochondrial DNA (mtDNA) in eukaryotic cells, a method useful for modeling of mitochondrial genetic disorders and developing novel therapeutic modalities. Here, we report that A-to-G-editing TALEDs but not C-to-T-editing DdCBEs induce tens of thousands of transcriptome-wide off-target edits in human cells. To avoid these unwanted RNA edits, we engineered the substrate-binding site in TadA8e, the deoxy-adenine deaminase in TALEDs, and created TALED variants with fine-tuned deaminase activity. Our engineered TALED variants not only reduced RNA off-target edits by >99% but also minimized off-target mtDNA mutations and bystander edits at a target site. Unlike wild-type versions, our TALED variants were not cytotoxic and did not cause developmental arrest of mouse embryos. As a result, we obtained mice with pathogenic mtDNA mutations, associated with Leigh syndrome, which showed reduced heart rates.

Prime editing with genuine Cas9 nickases minimizes unwanted indels.

Unlike CRISPR-Cas9 nucleases, which yield DNA double-strand breaks (DSBs), Cas9 nickases (nCas9s), which are created by replacing key catalytic amino-acid residues in one of the two nuclease domains of S. pyogenesis Cas9 (SpCas9), produce nicks or single-strand breaks. Two SpCas9 variants, namely, nCas9 (D10A) and nCas9 (H840A), which cleave target (guide RNA-pairing) and non-target DNA strands, respectively, are widely used for various purposes, including paired nicking, homology-directed repair, base editing, and prime editing. In an effort to define the off-target nicks caused by these nickases, we perform Digenome-seq, a method based on whole genome sequencing of genomic DNA treated with a nuclease or nickase of interest, and find that nCas9 (H840A) but not nCas9 (D10A) can cleave both strands, producing unwanted DSBs, albeit less efficiently than wild-type Cas9. To inactivate the HNH nuclease domain further, we incorporate additional mutations into nCas9 (H840A). Double-mutant nCas9 (H840A + N863A) does not exhibit the DSB-inducing behavior in vitro and, either alone or in fusion with the M-MLV reverse transcriptase (prime editor, PE2 or PE3), induces a lower frequency of unwanted indels, compared to nCas9 (H840A), caused by error-prone repair of DSBs. When incorporated into prime editor and used with engineered pegRNAs (ePE3), we find that the nCas9 variant (H840A + N854A) dramatically increases the frequency of correct edits, but not unwanted indels, yielding the highest purity of editing outcomes compared to nCas9 (H840A).

Targeted A-to-G base editing of chloroplast DNA in plants.

Chloroplast DNA (cpDNA) encodes up to 315 (typically, 120-130) genes1, including those for essential components in photosystems I and II and the large subunit of RuBisCo, which catalyses CO2 fixation in plants. Targeted mutagenesis in cpDNA will be broadly useful for studying the functions of these genes in molecular detail and for developing crops and other plants with desired traits. Unfortunately, CRISPR-Cas9 and CRISPR-derived base editors, which enable targeted genetic modifications in nuclear DNA, are not suitable for organellar DNA editing2, owing to the difficulty of delivering guide RNA into organelles. CRISPR-free, protein-only base editors (including DddA-derived cytosine base editors3-8 and zinc finger deaminases9), originally developed for mitochondrial DNA editing in mammalian cells, can be used for C-to-T, rather than A-to-G, editing in cpDNA10-12. Here we show that heritable homoplasmic A-to-G edits can be induced in cpDNA, leading to phenotypic changes, using transcription activator-like effector-linked deaminases13.

Base editing in human cells with monomeric DddA-TALE fusion deaminases.

Inter-bacterial toxin DddA-derived cytosine base editors (DdCBEs) enable targeted C-to-T conversions in nuclear and organellar DNA. DddAtox, the deaminase catalytic domain derived from Burkholderia cenocepacia, is split into two inactive halves to avoid its cytotoxicity in eukaryotic cells, when fused to transcription activator-like effector (TALE) DNA-binding proteins to make DdCBEs. As a result, DdCBEs function as pairs, which hampers gene delivery via viral vectors with a small cargo size. Here, we present non-toxic, full-length DddAtox variants to make monomeric DdCBEs (mDdCBEs), enabling mitochondrial DNA editing with high efficiencies of up to 50%, when transiently expressed in human cells. We demonstrate that mDdCBEs expressed via AAV in cultured human cells can achieve nearly homoplasmic C-to-T editing in mitochondrial DNA. Interestingly, mDdCBEs often produce mutation patterns different from those obtained with conventional dimeric DdCBEs. Furthermore, mDdCBEs allow base editing at sites for which only one TALE protein can be designed. We also show that transfection of mDdCBE-encoding mRNA, rather than plasmid, can reduce off-target editing in human mitochondrial DNA.

Targeted A-to-G base editing in human mitochondrial DNA with programmable deaminases.

Mitochondrial DNA (mtDNA) editing paves the way for disease modeling of mitochondrial genetic disorders in cell lines and animals and also for the treatment of these diseases in the future. Bacterial cytidine deaminase DddA-derived cytosine base editors (DdCBEs) enabling mtDNA editing, however, are largely limited to C-to-T conversions in the 5'-TC context (e.g., TC-to-TT conversions), suitable for generating merely 1/8 of all possible transition (purine-to-purine and pyrimidine-to-pyrimidine) mutations. Here, we present transcription-activator-like effector (TALE)-linked deaminases (TALEDs), composed of custom-designed TALE DNA-binding arrays, a catalytically impaired, full-length DddA variant or split DddA originated from Burkholderia cenocepacia, and an engineered deoxyadenosine deaminase derived from the E. coli TadA protein, which induce targeted A-to-G editing in human mitochondria. Custom-designed TALEDs were highly efficient in human cells, catalyzing A-to-G conversions at a total of 17 target sites in various mitochondrial genes with editing frequencies of up to 49%.

Nuclear and mitochondrial DNA editing in human cells with zinc finger deaminases.

Base editing in nuclear DNA and mitochondrial DNA (mtDNA) is broadly useful for biomedical research, medicine, and biotechnology. Here, we present a base editing platform, termed zinc finger deaminases (ZFDs), composed of custom-designed zinc-finger DNA-binding proteins, the split interbacterial toxin deaminase DddAtox, and a uracil glycosylase inhibitor (UGI), which catalyze targeted C-to-T base conversions without inducing unwanted small insertions and deletions (indels) in human cells. We assemble plasmids encoding ZFDs using publicly available zinc finger resources to achieve base editing at frequencies of up to 60% in nuclear DNA and 30% in mtDNA. Because ZFDs, unlike CRISPR-derived base editors, do not cleave DNA to yield single- or double-strand breaks, no unwanted indels caused by error-prone non-homologous end joining are produced at target sites. Furthermore, recombinant ZFD proteins, expressed in and purified from E. coli, penetrate cultured human cells spontaneously to induce targeted base conversions, demonstrating the proof-of-principle of gene-free gene therapy.

Pupil responses associated with the perception of gravitational vertical under directional optic flows.

This study assessed the pupil responses in the sensory integration of various directional optic flows during the perception of gravitational vertical. A total of 30 healthy participants were enrolled with normal responses to conventional subjective visual vertical (SVV) which was determined by measuring the difference (error angles) between the luminous line adjusted by the participants and the true vertical. SVV was performed under various types of rotational (5°/s, 10°/s, and 50°/s) and straight (5°/s and 10°/s) optic flows presented via a head-mounted display. Error angles (°) of the SVV and changes in pupil diameters (mm) were measured to evaluate the changes in the visually assessed subjective verticality and related cognitive demands. Significantly larger error angles were measured under rotational optic flows than under straight flows (p < 0.001). The error angles also significantly increased as the velocity of the rotational optic flow increased. The pupil diameter increased after starting the test, demonstrating the largest diameter during the final fine-tuning around the vertical. Significantly larger pupil changes were identified under rotational flows than in straight flows. Pupil changes were significantly correlated with error angles and the visual analog scale representing subjective difficulties during each test. These results suggest increased pupil changes for integrating more challenging visual sensory inputs in the process of gravity perception.

Adenine Base Editor Ribonucleoproteins Delivered by Lentivirus-Like Particles Show High On-Target Base Editing and Undetectable RNA Off-Target Activities.

Adenine base editors (ABEs) can correct gene mutations without creating double-strand breaks. However, in recent reports, these editors showed guide-independent RNA off-target activities. This work describes our development of a delivery method to minimize ABEs' RNA off-target activity. After discovering a RNA off-target hot spot for sensitive detection of RNA off-target activities, we found that delivering ribonucleoproteins (RNPs) by electroporation generated undetectable non-specific RNA editing, but on-target base editing activity was also relatively low. We then explored a lentivirus capsid-based delivery strategy to deliver ABE. We used aptamer/aptamer-binding protein (ABP) interactions to package ABE RNPs into lentiviral capsids. Capsid RNPs were delivered to human cells for highly efficient guided base editing. Importantly, RNA off-target activities from the capsid RNPs were undetectable. Our new lentiviral capsid-based ABE RNP delivery method with minimal RNA off-target activities makes ABE one step closer to possible therapeutic applications.

Genome-wide target specificity of CRISPR RNA-guided adenine base editors.

Adenine base editors1 enable efficient targeted adenine-to-guanine single nucleotide conversions to induce or correct point mutations in human cells, animals, and plants1-4. Here we present a modified version of Digenome-seq, an in vitro method for identifying CRISPR (clustered regularly interspaced short palindromic repeats)-induced double-strand breaks using whole-genome sequencing5-8, to assess genome-wide target specificity of adenine base editors. To produce double-strand breaks at sites containing inosines, the products of adenine deamination, we treat human genomic DNA with an adenine base editor 7.10 protein-guide RNA complex and either endonuclease V or a combination of human alkyladenine DNA glycosylase and endonuclease VIII in vitro. Digenome-seq detects adenine base editor off-target sites with a substitution frequency of 0.1% or more. We show that adenine base editor 7.10, the cytosine base editor BE3, and unmodified CRISPR-associated protein 9 (Cas9) often recognize different off-target sites, highlighting the need for independent assessments of their genome-wide specificities6. Using targeted sequencing, we also show that use of preassembled adenine base editor ribonucleoproteins, modified guide RNAs5,8-11, and Sniper/Cas9 (ref. 12) reduces adenine base editor off-target activity in human cells.

Microsurgical Foraminotomy via Wiltse Paraspinal Approach for Foraminal or Extraforaminal Stenosis at L5-S1 Level : Risk Factor Analysis for Poor Outcome.

OBJECTIVE: The purpose of this study was to present the outcome of the microsurgical foraminotomy via Wiltse paraspinal approach for foraminal or extraforaminal (FEF) stenosis at L5-S1 level. We investigated risk factors associated with poor outcome of microsurgical foraminotomy at L5-S1 level. METHODS: We analyzed 21 patients who underwent the microsurgical foraminotomy for FEF stenosis at L5-S1 level. To investigate risk factors associated with poor outcome, patients were classified into two groups (success and failure in foraminotomy). Clinical outcomes were assessed by the visual analogue scale (VAS) scores of back and leg pain and Oswestry disability index (ODI). Radiographic parameters including existence of spondylolisthesis, existence and degree of coronal wedging, disc height, foramen height, segmental lordotic angle (SLA) on neutral and dynamic view, segmental range of motion, and global lumbar lordotic angle were investigated. RESULTS: Postoperative VAS score and ODI improved after foraminotomy. However, there were 7 patients (33%) who had persistent or recurrent leg pain. SLA on neutral and extension radiographic films were significantly associated with the failure in foraminotomy (p<0.05). Receiver-operating characteristics curve analysis revealed the optimal cut-off values of SLA on neutral and extension radiographic films for predicting failure in foraminotomy were 17.3° and 24°s, respectively. CONCLUSION: Microsurgical foraminotomy for FEF stenosis at L5-S1 level can provide good clinical outcomes in selected patients. Poor outcomes were associated with large SLA on preoperative neutral (>17.3°) and extension radiographic films (>24°).

Postlaminectomy Bilateral Lumbar Intraspinal Synovial Cysts.

Lumbar intraspinal synovial cysts are included in the difference diagnosis of lumbar radiculopathy. Developing imaging modalities has result in increased reporting about these lesions. However, the case of bilateral new lumbar intraspinal synovial cysts after laminectomy has been rarely reported. We report of a rare case with bilateral lumbar intraspinal synovial cysts after laminectomy, requiring surgical excision.