Improving the efficiency of CRISPR-Cas12a-based genome editing with site-specific covalent Cas12a-crRNA conjugates.

The CRISPR-Cas12a system shows unique features compared with widely used Cas9, making it an attractive and potentially more precise alternative. However, the adoption of this system has been hindered by its relatively low editing efficiency. Guided by physical chemical principles, we covalently conjugated 5' terminal modified CRISPR RNA (crRNA) to a site-specifically modified Cas12a through biorthogonal chemical reaction. The genome editing efficiency of the resulting conjugated Cas12a complex (cCas12a) was substantially higher than that of the wild-type complex. We also demonstrated that cCas12a could be used for precise gene knockin and multiplex gene editing in a chimeric antigen receptor T cell preparation with efficiency much higher than that of the wild-type system. Overall, our findings indicate that covalently linking Cas nuclease and crRNA is an effective approach to improve the Cas12a-based genome editing system and could potentially provide an insight into engineering other Cas family members with low efficiency as well.

In-depth assessment of the PAM compatibility and editing activities of Cas9 variants.

A series of Cas9 variants have been developed to improve the editing fidelity or targeting range of CRISPR-Cas9. Here, we employ a high-throughput sequencing approach primer-extension-mediated sequencing to analyze the editing efficiency, specificity and protospacer adjacent motif (PAM) compatibility of a dozen of SpCas9 variants at multiple target sites in depth, and our findings validate the high fidelity or broad editing range of these SpCas9 variants. With regard to the PAM-flexible SpCas9 variants, we detect significantly increased levels of off-target activity and propose a trade-off between targeting range and editing specificity for them, especially for the near-PAM-less SpRY. Moreover, we use a deep learning model to verify the consistency and predictability of SpRY off-target sites. Furthermore, we combine high-fidelity SpCas9 variants with SpRY to generate three new SpCas9 variants with both high fidelity and broad editing range. Finally, we also find that the existing SpCas9 variants are not effective in suppressing genome instability elicited by CRISPR-Cas9 editing, raising an urgent issue to be addressed.

Global detection of DNA repair outcomes induced by CRISPR-Cas9.

CRISPR-Cas9 generates double-stranded DNA breaks (DSBs) to activate cellular DNA repair pathways for genome editing. The repair of DSBs leads to small insertions or deletions (indels) and other complex byproducts, including large deletions and chromosomal translocations. Indels are well understood to disrupt target genes, while the other deleterious byproducts remain elusive. We developed a new in silico analysis pipeline for the previously described primer-extension-mediated sequencing assay to comprehensively characterize CRISPR-Cas9-induced DSB repair outcomes in human or mouse cells. We identified tremendous deleterious DSB repair byproducts of CRISPR-Cas9 editing, including large deletions, vector integrations, and chromosomal translocations. We further elucidated the important roles of microhomology, chromosomal interaction, recurrent DSBs, and DSB repair pathways in the generation of these byproducts. Our findings provide an extra dimension for genome editing safety besides off-targets. And caution should be exercised to avoid not only off-target damages but also deleterious DSB repair byproducts during genome editing.

The origin of unwanted editing byproducts in gene editing.

The rapid development of CRISPR-Cas genome editing tools has greatly changed the way to conduct research and holds tremendous promise for clinical applications. During genome editing, CRISPR-Cas enzymes induce DNA breaks at the target sites and subsequently the DNA repair pathways are recruited to generate diverse editing outcomes. Besides off-target cleavage, unwanted editing outcomes including chromosomal structural variations and exogenous DNA integrations have recently raised concerns for clinical safety. To eliminate these unwanted editing byproducts, we need to explore the underlying mechanisms for the formation of diverse editing outcomes from the perspective of DNA repair. Here, we describe the involved DNA repair pathways in sealing Cas enzyme-induced DNA double-stranded breaks and discuss the origins and effects of unwanted editing byproducts on genome stability. Furthermore, we propose the potential risk of inhibiting DNA repair pathways to enhance gene editing. The recent combined studies of DNA repair and CRISPR-Cas editing provide a framework for further optimizing genome editing to enhance editing safety.

Supramolecule self-assembly synthesis of amyloid phenylalanine-Cu fibrils with laccase-like activity and their application for dopamine determination.

Laccases are multicopper proteins for dioxygen-involved oxidation of a broad spectrum of organic compounds. I Novel amyloid-like phenylalanine-Cu (F-Cu(II)) fibrils were developed, which were obtained via supramolecular self-assembly of Cu2+ and phenylalanine (F) under basic condition. The obtained amyloid-like fibrils represented highly periodic structure, of which the lattice unit was constructed via alternating hydrophobic (aromatic environment) and hydrophilic (both hydrogen bonding and Cu(II) coordination) interactions. Relative to natural laccases, the amyloid-like F-Cu(II) architecture exhibited comparable substrate affinity (Michaelis constant, Km = 0.75 mM) and higher catalytic efficiency (kcat/Km = 773.33 × 10-3 g-1 min-1L). Moreover, it exhibited remarkable tolerances in pH (4 ~ 10), temperature (room temperature ~ 200 ℃), organic solvent, and long-term storage (> 15 days). These stabilities were superior among the reported nature and artificial laccases, presenting a more promising candidate in various chemo- or bio-applications. In addition, F-Cu(II) fibrils could catalyze the oxidation of dopamine (DA) to a brown product, in which a new absorption band at 470 nm was observed. Based on this, a simple colorimetric assay for the detection of DA could be performed. We reported a novel amyloid-like phenylalanine-Cu fibrils, in which F-Cu+ complex can mimick the T1 site of natural laccase to oxidize the substrates. Then electrons transferred to F-Cu2+ complex via N-H···O=C hydrogen binding pathway. Finally, the dioxygen was transformed to water though radical reaction.

Synthesis of water-soluble, ultrabright Cu nanoclusters with core-shell structure via facile reduction approach for determination of 4-nitrophenol.

In this work, we reported a facile reduction approach for fabrication of water-soluble and ultrabright Cu nanoclusters with core-shell structure. A certain amount of reducing agent as NaBH4was introduced into the polyethyleneimine-stabilized Cu nanoclusters (CuNCs@PEI) system, which exhibited 4-fold fluorescence enhancement along with a blue shift of the emission peak. The variations of morphology, valence states and functional groups demonstrated that a Cu shell was formed surround CuNCs (defined as CuNCs-Cu@PEI), attributable to metal complex (PEI-Cu+and PEI-Cu2+) reduction. The effect of core-shell morphology on luminous and electron relaxation mechanism of CuNCs-Cu@PEI was investigated via temperature-dependent steady and time-resolved fluorescence measurements. The CuNCs-Cu@PEI with a high fluorescence quantum yields of 22.59% were able to homogeneously disperse in aqueous phase, indicating their potential applications in biological labeling, sensing and invivoimaging. Finally, the CuNCs-Cu@PEI was employed as a fluorescence probe to determine 4-nitrophenol, of which the detection limit was much lower than initial CuNCs@PEI.

Nitrogen-terminated silicon nanoparticles obtained via chemical etching and passivation are specific fluorescent probes for creatinine.

A method is described here to prepare water-dispersible nitrogen-functionalized silicon nanoparticles (N-SiNPs). It consists of two steps, viz. etching of the oxidized shell of SiNPs and nitrogen-passivation of the exposed silicon. The resulting N-SiNPs have an average diameter of 2.6±0.7 nm and show blue fluorescence (with excitation/emission peaks at 340/420 nm). The fluorescence quantum yield is 23% and the decay time is in the nanosecond regime. Compared to etching methods using a plasma or hydrofluoric acid, the process described here (etching and passivation) is mild, continuous, fast, and air-compatible. The N-SiNPs modified with chlorotetracycline are shown to be a viable fluorescent probe for creatinine. Fluorescence drops in the 0 to 20 µM creatinine concentration range, and the limit of detection is 0.14 µM.

Catechol oxidase nanozyme based colorimetric sensors array for highly selective distinction among multiple catecholamines.

In order to effectively monitor multiple catecholamine (CA) neurotransmitters with extreme similar structures, a rapid, sensitive and selective detection strategy has become an urgent problem to be solved. In this paper, a novel colorimetric sensors array based on CuNCs protected by various ligands such as tannic acid, ascorbic acid and polymethylacrylic acid (CuNCs@TA, CuNCs@AA and CuNCs@PMAA) was constructed. All of these CuNCs could mimic catechol oxidase to selective catalyze catechol-type analogues (such as CAs) to corresponding quinones along with color changes. Furthermore, experiments and theory calculations demonstrated that Cr6+-modification on the surface of CuNCs facilitated the steady-state kinetics of enzymatic activity. Based on these CuNCs as sensing probes, this sensors array can quickly detect different CAs (such as epinephrine (EP), including dopamine (DA), norepinephrine (NE) and l-dopa) with similar structures. When those analogues were added to the CuNC-based colorimetric array sensors, different absorbance changes were produced at 485 nm. Linear discriminant analysis (LDA) showed that the tri-probe colorimetric array sensors could recognize and distinguish these analogues, and corresponding binary and ternary mixtures could be well categorized. The value of Factor 1 of an array with varied CA concentrations had a good linear correlation, and the detection limit (LOD) was as low as 10-8∼10-9 mol/L. Four CA analogues in real samples were identified by CuNCs-based colorimetric array sensors. This work provides a fast and convenient experimental basis for monitoring the complex structure CAs neurotransmitters.

Cohesin maintains replication timing to suppress DNA damage on cancer genes.

Cohesin loss-of-function mutations are frequently observed in tumors, but the mechanism underlying its role in tumorigenesis is unclear. Here, we found that depletion of RAD21, a core subunit of cohesin, leads to massive genome-wide DNA breaks and 147 translocation hotspot genes, co-mutated with cohesin in multiple cancers. Increased DNA damages are independent of RAD21-loss-induced transcription alteration and loop anchor disruption. However, damage-induced chromosomal translocations coincide with the asymmetrically distributed Okazaki fragments of DNA replication, suggesting that RAD21 depletion causes replication stresses evidenced by the slower replication speed and increased stalled forks. Mechanistically, approximately 30% of the human genome exhibits an earlier replication timing after RAD21 depletion, caused by the early initiation of >900 extra dormant origins. Correspondingly, most translocation hotspot genes lie in timing-altered regions. Therefore, we conclude that cohesin dysfunction causes replication stresses induced by excessive DNA replication initiation, resulting in gross DNA damages that may promote tumorigenesis.

Human 8-cell embryos enable efficient induction of disease-preventive mutations without off-target effect by cytosine base editor.

Approximately 140 million people worldwide are homozygous carriers of APOE4 (ε4), a strong genetic risk factor for late onset familial and sporadic Alzheimer's disease (AD), 91% of whom will develop AD at earlier age than heterozygous carriers and noncarriers. Susceptibility to AD could be reduced by targeted editing of APOE4, but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene therapies. Here, we first screened eight cytosine base editor variants at four injection stages (from 1- to 8-cell stage), and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate (up to 100%) with the lowest bystander effects. In particular, 80% of AD-susceptible ε4 allele copies were converted to the AD-neutral ε3 allele in human ε4-carrying embryos. Stringent control measures combined with targeted deep sequencing, whole genome sequencing, and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem cells. Furthermore, base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst stage. Finally, we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial hypercholesterolemia. Our study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos, a potential approach for reducing human susceptibility to AD or other genetic diseases.

NH2-MIL-101(Fe) nanozyme-based dual-modality sensor for determination of alendronate sodium and study of two-dimensional correlation spectroscopy.

We developed a dual-modality sensing platform for ratiometric fluorescence and colorimetric determination of alendronate sodium (ALDS). This platform was performed by using a NH2- MIL-101(Fe) as a peroxidase mimic. Since preferential complexing between Fe3+ (active site for peroxidase) and ALDS, the production of 2,3-diaminophenazine (DAP, oxidized product of OPD) has been inhibited in the presence of H2O2. As a result, the ratiometric fluorescence value of F556/F456 and absorbance at 450 nm exhibited significant changes, which could be used as the dual-modality sensing platform. In addition, Two-dimensional correlation spectroscopy (2D-COS) analysis on Fourier-transform infrared (FTIR), ultraviolet visible and ratiometric fluorescence spectra were applied to investigate the binding features. Synchronous and asynchronous maps of these spectra confirmed our above hypothesis, in which Fe3+-ALDS complex was the critical factor that regulated dual-modality signals. To our knowledge, the 2D-COS method was applied to study the catalytic and sensing mechanism of nanozyme as NH2- MIL-101(Fe) for the first time. This technique was helpful to understand interaction of substrates on nanozyme and develop more sensitive sensors for assaying.

PEM-seq comprehensively quantifies DNA repair outcomes during gene-editing and DSB repair.

The repair products of double-stranded DNA breaks (DSBs) are crucial for investigating the mechanism underlying DNA damage repair as well as evaluating the safety and efficiency of gene-editing; however, a comprehensively quantitative assay remains to be established. Here, we describe the step-by-step instructions of the primer extension-mediated sequencing (PEM-seq), followed by the framework of data processing and statistical analysis. PEM-seq presents a full spectrum of repair outcomes for both genome-editing-induced and endogenous DSBs in mouse and human cells. For complete details on the use and execution of this profile, please refer to Gan et al. (2021), Yin et al. (2019), Liu et al. (2021a), and Zhang et al. (2021).

Comprehensive assessment of miniature CRISPR-Cas12f nucleases for gene disruption.

Because of their small size, the recently developed CRISPR-Cas12f nucleases can be effectively packaged into adeno-associated viruses for gene therapy. However, a systematic evaluation of the editing outcomes of CRISPR-Cas12f is lacking. In this study, we apply a high-throughput sequencing method to comprehensively assess the editing efficiency, specificity, and safety of four Cas12f proteins in parallel with that of Cas9 and two Cas12a proteins at multiple genomic sites. Cas12f nucleases achieve robust cleavage at most of the tested sites and mainly produce deletional fragments. In contrast, Cas9 and Cas12a show relatively higher editing efficiency at the vast majority of the tested sites. However, the off-target hotspots identified in the Cas9- and Cas12a-edited cells are negligibly detected in the Cas12f-edited cells. Moreover, compared to Cas9 and Cas12a nucleases, Cas12f nucleases reduce the levels of chromosomal translocations, large deletions, and integrated vectors by 2- to 3-fold. Therefore, our findings confirm the editing capacity of Cas12f and reveal the ability of this nuclease family to preserve genome integrity during genome editing.

Safeguarding genome integrity during gene-editing therapy in a mouse model of age-related macular degeneration.

Ensuring genome safety during gene editing is crucial for clinical translation of the high-efficient CRISPR-Cas9 toolbox. Therefore, the undesired events including chromosomal translocations, vector integrations, and large deletions arising during therapeutic gene editing remain to be adequately addressed or tackled in vivo. Here, we apply CRISPR-Cas9TX in comparison to CRISPR-Cas9 to target Vegfa for the treatment of age-related macular degeneration (AMD) disease in a mouse model. AAV delivery of both CRISPR-Cas9 and CRISPR-Cas9TX can efficiently inhibit laser-induced neovascularization. Importantly, Cas9TX almost eliminates chromosomal translocations that occur at a frequency of approximately 1% in Cas9-edited mouse retinal cells. Strikingly, the widely observed AAV integration at the target Vegfa site is also greatly reduced from nearly 50% of edited events to the background level during Cas9TX editing. Our findings reveal that chromosomal structural variations routinely occur during in vivo genome editing and highlight Cas9TX as a superior form of Cas9 for in vivo gene disruption.

Cas9 exo-endonuclease eliminates chromosomal translocations during genome editing.

The mechanism underlying unwanted structural variations induced by CRISPR-Cas9 remains poorly understood, and no effective strategy is available to inhibit the generation of these byproducts. Here we find that the generation of a high level of translocations is dependent on repeated cleavage at the Cas9-targeting sites. Therefore, we employ a strategy in which Cas9 is fused with optimized TREX2 to generate Cas9TX, a Cas9 exo-endonuclease, which prevents perfect DNA repair and thereby avoids repeated cleavage. In comparison with CRISPR-Cas9, CRISPR-Cas9TX greatly suppressed translocation levels and enhanced the editing efficiency of single-site editing. The number of large deletions associated with Cas9TX was also reduced to very low level. The application of CRISPR-Cas9TX for multiplex gene editing in chimeric antigen receptor T cells nearly eliminated deleterious chromosomal translocations. We report the mechanism underlying translocations induced by Cas9, and propose a general strategy for reducing chromosomal abnormalities induced by CRISPR-RNA-guided endonucleases.

Improved HTGTS for CRISPR/Cas9 off-target detection.

Precise genome editing is essential for scientific research and clinical application. At present, linear amplification-mediated high-throughput genome-wide translocation sequencing (LAM-HTGTS) is one of most effective methods to evaluate the off-target activity of CRISPR-Cas9, which is based on chromosomal translocation and employs a "bait" DNA double-stranded break (DSB) to capture genome-wide "prey" DNA DSBs. Here, we described an improved HTGTS (iHTGTS) method, in which size-selection beads were used to enhance reaction efficiency and a new primer system was designed to be compatible with Illumina Hiseq sequencing. Compared with LAM-HTGTS, iHTGTS is lower cost and has much higher sensitivity for off-target detection in HEK293T, K562, U2OS and HCT116 cell lines. So we believe that iHTGTS is a powerful method for comprehensively assessing Cas9 off-target effect.

Erratum: Correction Notice: Improved HTGTS for CRISPR/Cas9 Off-target Detection.

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Optimizing genome editing strategy by primer-extension-mediated sequencing.

Efficient and precise genome editing is essential for clinical applications and generating animal models, which requires engineered nucleases with high editing ability while low off-target activity. Here we present a high-throughput sequencing method, primer-extension-mediated sequencing (PEM-seq), to comprehensively assess both editing ability and specificity of engineered nucleases. We showed CRISPR/Cas9-generated breaks could lead to chromosomal translocations and large deletions by PEM-seq. We also found that Cas9 nickase possessed lower off-target activity while with some loss of target cleavage ability. However, high-fidelity Cas9 variants, including both eCas9 and the new FeCas9, could significantly reduce the Cas9 off-target activity with no obvious editing retardation. Moreover, we found AcrIIA4 inhibitor could greatly reduce the activities of Cas9, but off-target loci were not so effectively suppressed as the on-target sites. Therefore, PEM-seq fully evaluating engineered nucleases could help choose better genome editing strategy at given loci than other methods detecting only off-target activity.

11-Mercaptoundecanoic acid capped gold nanoclusters as a fluorescent probe for specific detection of folic acid via a ratiometric fluorescence strategy.

A novel ratiometric fluorescence strategy is developed for specific detection of folic acid (FA) by using 11-mercaptoundecanoic acid protected gold nanoclusters (AuNCs@MUA). In this design, the fluorescence color of the probe can be switched among red, pink, violet and blue by varying the concentration of FA. AuNCs@MUA possesses strong fluorescence peaking at 612 nm (R-signal) and FA exhibits blue emissive auto-fluorescence at 446 nm (B-signal), showing a large emission shift of ∼170 nm. When AuNCs@MUA approaches FA through electrostatic binding, the R-signal decreases while the B-signal increases with titration of FA. Based on the above phenomenon, a radiometric analysis platform is constructed for FA target detection, with a wide linear response range from 0 to 20 µM, and an excellent detection limit of 26 nM. This new ratiometric strategy exhibits low background, and wide signal changes in a low concentration range, which presents obvious advantages over most previous FA detections based on single-responsive fluorescence methods. Furthermore, the proposed method is successfully applied to determine FA in human serum samples.

Polyethyleneimine protected silver nanoclusters luminescence probe for sensitive detection of cobalt (II) in living cells.

A simple luminescence sensor, based on polyethyleneimine protected silver nanoclusters (AgNCs@PEI) is successfully fabricated via one-pot reduction method. The obtained AgNCs@PEI are characterized by high-resolution transmission electron microscopy (HR-TEM), Dynamic light scattering (DLS), transient and steady-state fluorescence, and UV-vis spectroscopy. The NCs show large Stocks-shift (∼130nm), high tolerability to extreme pH and high ionic strengths, and excellent photo-stability under UV irradiation, laying the basement for the practical applications. In addition, the sensor is used to detect the Co2+ basing on the luminescence quenching, which is interfered by pH conditions (from pH4.0 to pH7.4). As a luminescence probe for Co2+ ions, the detection limit of AgNCs@PEI is as low as 0.25nM, which is much lower than that of many other reports. Additionally, the AgNCs@PEI possess the advantages of good selectivity, fast response and abroad linear detection. A linear response range in 0.5nM-50µM is achieved for Co2+ when using 20µM AgNCs@PEI in BR buffer solution (neutral condition pH7.4). Incubation time of AgNCs@PEI toward Co2+ is only 2min and it can distinguish Co2+ from other 13 metal ions. Furthermore, the probe (AgNCs@PEI) is applied to sensing and imaging of HeLa cells, showing low cytotoxicity and good sensitivity.