Mismatch Tolerance Research Articles

Negative DNA supercoiling induces genome-wide Cas9 off-target activity

CRISPR-Cas9 is a powerful gene-editing technology; however, off-target activity remains an important consideration for therapeutic applications. We have previously shown that force-stretching DNA induces off-target activity and hypothesized that distortions of the DNA topology invivo, such as negative DNA supercoiling, could reduce Cas9 specificity. Using single-molecule optical-tweezers, we demonstrate that negative supercoiling λ-DNA induces sequence-specific Cas9 off-target binding at multiple sites, even at low forces. Using an adapted CIRCLE-seq approach, we detect over 10,000 negative-supercoiling-induced Cas9 off-target double-strand breaks genome-wide caused by increased mismatch tolerance. We further demonstrate invivo that directed local DNA distortion increases off-target activity in cells and that induced off-target events can be detected during Cas9 genome editing. These data demonstrate that Cas9 off-target activity is regulated by DNA topology invitro and invivo, suggesting that cellular processes, such as transcription and replication, could induce off-target activity at previously overlooked sites.

Prediction of base editor off-targets by deep learning

Due to the tolerance of mismatches between gRNA and targeting sequence, base editors frequently induce unwanted Cas9-dependent off-target mutations. Here, to develop models to predict such off-targets, we design gRNA-off- target pairs for adenine base editors (ABEs) and cytosine base editors (CBEs) and stably integrate them into the human cells. After five days of editing, we obtain valid efficiency datasets of 54,663 and 55,727 off-targets for ABEs and CBEs, respectively. We use the datasets to train deep learning models, resulting in ABEdeepoff and CBEdeepoff, which can predict off-target sites. We use these tools to predict off-targets for a panel of endogenous loci and achieve Spearman correlation values varying from 0.710 to 0.859. Finally, we develop an integrated tool that is freely accessible via an online web server http://www.deephf.com/#/bedeep/bedeepoff. These tools could facilitate minimizing the off-target effects of base editing.

Ultra-sensitive and specificity quantitative detection of miRNA using a combined CRISPR/Cas13a and DNA-PAINT

MicroRNAs (miRNAs) are considered to be important biomarkers for the diagnosis of numerous diseases and basic biomedical research. Therefore, a novel rapid diagnostic technique with high sensitivity and specificity is essential for miRNAs detection in clinical trials. Herein, we proposed a sensing platform based on CRISPR/Cas13a and DNA point accumulation in nanoscale topology (DNA-PAINT) to enable an ultra-sensitive analysis of miRNA at the single-molecule level. In this platform, mismatch bases are artificially introduced into the crRNA spacer region, which reduces the mismatch tolerance of the Cas13a/crRNA complex, thus providing high specificity for miRNA detection. Furthermore, signal amplification can be achieved by the dynamic repetitive process of binding and unbinding between fluorescent probes and capture probes in DNA-PAINT imaging, with a detection limit (LOD) as low as 1.12 fM. Convincingly, the practical application capability of the platform for miRNA quantification in complex biological samples is validated. More importantly, given the flexible design of the crRNA spacer region sequence, the strategy could be easily applied to the rapid detection of other miRNAs with excellent reliability, sensitivity and specificity, which has great potential for early diagnosis of miRNA-related diseases.

Study on optical coupling characteristics of a high-radial-tolerance thick-lens beam expanding fiber collimator

In this paper, a high-radial-tolerance fiber collimator (HRTFC) consisting of beam expanding fiber (BEF) and thick-lens (T-lens) is designed. The HRTFC uses BEF to reduce coupling loss, while T-lens are used to increase the outgoing spot to enhance the tolerance of radial mismatch. Firstly, the Gaussian beam ABCD optical transmission matrix is used to analyze transmission properties of light in the T-lens. Then, a beam transmission coupling model is constructed to explore the coupling losses of radial, angular, and axial mismatches under different spots. Finally, a static experimental platform is built to verify the influence of spot size on the coupling mismatch, and a dynamic experimental platform is built to monitor the temperature without contact. The experimental results show that the coupling loss decreases from 1.808 dB to 0.368 dB in the HRTFC, with the fiber mode field diameter (MFD) increasing from 10μm to 28μm. Increasing the spot of the HRTFC to a certain extent can reduce the tolerance of angular mismatch but can significantly increase the tolerance of radial mismatch.

Highly mismatch-tolerant homology testing by RecA could explain how homology length affects recombination.

In E. coli, double strand breaks (DSBs) are resected and loaded with RecA protein. The genome is then rapidly searched for a sequence that is homologous to the DNA flanking the DSB. Mismatches in homologous partners are rare, suggesting that RecA should rapidly reject mismatched recombination products; however, this is not the case. Decades of work have shown that long lasting recombination products can include many mismatches. In this work, we show that in vitro RecA forms readily observable recombination products when 16% of the bases in the product are mismatched. We also consider various theoretical models of mismatch-tolerant homology testing. The models test homology by comparing the sequences of Ltest bases in two single-stranded DNAs (ssDNA) from the same genome. If the two sequences pass the homology test, the pairing between the two ssDNA becomes permanent. Stringency is the fraction of permanent pairings that join ssDNA from the same positions in the genome. We applied the models to both randomly generated genomes and bacterial genomes. For both randomly generated genomes and bacterial genomes, the models show that if no mismatches are accepted stringency is ∼ 99% when Ltest = 14 bp. For randomly generated genomes, stringency decreases with increasing mismatch tolerance, and stringency improves with increasing Ltest. In contrast, in bacterial genomes when Ltest ∼ 75 bp, stringency is ∼ 99% for both mismatch-intolerant and mismatch-tolerant homology testing. Furthermore, increasing Ltest does not improve stringency because most incorrect pairings join different copies of repeats. In sum, for bacterial genomes highly mismatch tolerant homology testing of 75 bp provides the same stringency as homology testing that rejects all mismatches and testing more than ∼75 base pairs is not useful. Interestingly, in vivo commitment to recombination typically requires homology testing of ∼ 75 bp, consistent with highly mismatch intolerant testing.

A programmable pAgo nuclease with RNA target-cleavage specificity from the mesophilic bacterium Verrucomicrobia.

Argonaute (Ago) proteins are conserved programmable nucleases present in eukaryotes and prokaryotes and provide defense against mobile genetic elements. Almost all characterized pAgos prefer to cleave DNA targets. Here, we describe a novel pAgo from Verrucomicrobia bacterium (VbAgo) that can specifically cleave RNA targets rather than DNA targets at 37°C and function as a multiple-turnover enzyme showing prominent catalytic capacity. VbAgo utilizes DNA guides (gDNAs) to cleave RNA targets at the canonical cleavage site. Meanwhile, the cleavage activity is remarkably strengthened at low concentrations of NaCl. In addition, VbAgo presents a weak tolerance for mismatches between gDNAs and RNA targets, and single-nucleotide mismatches at positions 11‒12 and dinucleotide mismatches at positions 3‒15 dramatically reduce target cleavage. Moreover, VbAgo can efficiently cleave highly structured RNA targets at 37°C. These properties of VbAgo broaden our understanding of Ago proteins and expand the pAgo-based RNA manipulation toolbox.

Application of Familial Y-STR Haplotype Mismatch Tolerance in Genealogy Inference.

To provide a guideline for genealogy inference and family lineage investigation through a study of the mismatch tolerance distribution of Y-STR loci in Chinese Han male lineage. Three Han lineages with clear genetic relationships were selected. YFiler Platinum PCR amplification Kit was used to obtain the typing data of 35 Y-STR loci in male samples. The variation of Y-STR haplotypes in generation inheritance and the mismatch tolerance at 1-7 kinship levels were statistically analyzed. Mutations in Y-STR were family-specific with different mutation loci and numbers of mutation in different lineages. Among all the mutations, 66.03% were observed on rapidly and fast mutating loci. At 1-7 kinship levels, the number of mismatch tolerance ranged from 0 to 5 on all 35 Y-STR loci, with a maximum step size of 6. On medium and slow mutant loci, the number of mismatch tolerance ranged from 0 to 2, with a maximum step size of 3; on rapidly and fast mutant loci, the number of mismatch tolerance ranged from 0 to 3, with a maximum step size of 6. Combined use of SNP genealogy inference and Y-STR lineage investigation, both 0 and multiple mismatch tolerance need to be considered. Family lineage with 0-3 mismatch tolerance on all 35 Y-STR loci and 0-1 mismatch tolerance on medium and slow loci can be prioritized for screening. When the number of mismatch tolerance is eligible, family lineages with long steps should be carefully excluded. Meanwhile, adding fast mutant loci should also be handled with caution.

C-band dual-wavelength synchronized mode-locked erbium-doped fiber laser using a common absorber

We demonstrate a C-band dual-wavelength synchronized mode-locked erbium-doped fiber laser based on a common single-wall carbon nanotube saturable absorber and two loop resonators. The synchronous mode-locked pulses laser is with a repetition rate of 16.28 MHz and durations of 1.95 ps and 1.21 ps at 1533 nm and 1562 nm, respectively. The tolerance of a cavity-length mismatch is about 830μm. The proposed dual-wavelength laser system exhibits a flexibly controlled performance and a long-term stability, which offers great potential for a number of applications, such as difference-frequency terahertz-wave generation and field-enhancement Raman microscopy.

CRISPR-Cas effector specificity and cleavage site determine phage escape outcomes.

CRISPR-mediated interference relies on complementarity between a guiding CRISPR RNA (crRNA) and target nucleic acids to provide defense against bacteriophage. Phages escape CRISPR-based immunity mainly through mutations in the protospacer adjacent motif (PAM) and seed regions. However, previous specificity studies of Cas effectors, including the class 2 endonuclease Cas12a, have revealed a high degree of tolerance of single mismatches. The effect of this mismatch tolerance has not been extensively studied in the context of phage defense. Here, we tested defense against lambda phage provided by Cas12a-crRNAs containing preexisting mismatches against the genomic targets in phage DNA. We find that most preexisting crRNA mismatches lead to phage escape, regardless of whether the mismatches ablate Cas12a cleavage in vitro. We used high-throughput sequencing to examine the target regions of phage genomes following CRISPR challenge. Mismatches at all locations in the target accelerated emergence of mutant phage, including mismatches that greatly slowed cleavage in vitro. Unexpectedly, our results reveal that a preexisting mismatch in the PAM-distal region results in selection of mutations in the PAM-distal region of the target. In vitro cleavage and phage competition assays show that dual PAM-distal mismatches are significantly more deleterious than combinations of seed and PAM-distal mismatches, resulting in this selection. However, similar experiments with Cas9 did not result in emergence of PAM-distal mismatches, suggesting that cut-site location and subsequent DNA repair may influence the location of escape mutations within target regions. Expression of multiple mismatched crRNAs prevented new mutations from arising in multiple targeted locations, allowing Cas12a mismatch tolerance to provide stronger and longer-term protection. These results demonstrate that Cas effector mismatch tolerance, existing target mismatches, and cleavage site strongly influence phage evolution.

Hybrid Multitask Learning Reveals Sequence Features Driving Specificity in the CRISPR/Cas9 System

CRISPR/Cas9 technology is capable of precisely editing genomes and is at the heart of various scientific and medical advances in recent times. The advances in biomedical research are hindered because of the inadvertent burden on the genome when genome editors are employed-the off-target effects. Although experimental screens to detect off-targets have allowed understanding the activity of Cas9, that knowledge remains incomplete as the rules do not extrapolate well to new target sequences. Off-target prediction tools developed recently have increasingly relied on machine learning and deep learning techniques to reliably understand the complete threat of likely off-targets because the rules that drive Cas9 activity are not fully understood. In this study, we present a count-based as well as deep-learning-based approach to derive sequence features that are important in deciding on Cas9 activity at a sequence. There are two major challenges in off-target determination-the identification of a likely site of Cas9 activity and the prediction of the extent of Cas9 activity at that site. The hybrid multitask CNN-biLSTM model developed, named CRISP-RCNN, simultaneously predicts off-targets and the extent of activity on off-targets. Employing methods of integrated gradients and weighting kernels for feature importance approximation, analysis of nucleotide and position preference, and mismatch tolerance have been performed.

Nonvolatile magnetoelectric coupling in two-dimensional van der Waals sandwich heterostructure CuInP2S6/MnCl3/CuInP2S6.

Electrical control of magnetism is of great interest for low-energy-consumption spintronic applications. Due to the recent experimental breakthrough in two-dimensional materials, with the absence of hanging bonds on the surface and strong tolerance for lattice mismatch, heterogeneous integration of different two-dimensional materials provides a new opportunity for coupling between different physical properties. Here, we report the realization of nonvolatile magnetoelectric coupling in vdW sandwich heterostructure CuInP2S6/MnCl3/CuInP2S6. Using first-principles calculations, we reveal that when interfacing with ferroelectric CuInP2S6, the Dirac half-metallic state of monolayer MnCl3 will be destroyed. Moreover, depending on the electrically polarized direction of CuInP2S6, MnCl3 can be a half-metal or a ferromagnetic semiconductor. We unveil that the obtained ferromagnetic semiconductor in MnCl3 can be attributed to the different gain and loss of electrons on the two adjacent Mn atoms due to the sublattice symmetry broken by interlayer coupling. The effects of interfacial magnetoelectric coupling on magnetic anisotropy and ferromagnetic Curie temperature of MnCl3 are also investigated, and a multiferroic memory based on this model is designed. Our work not only provides a promising way to design nonvolatile electrical control of magnetism but also renders monolayer MnCl3 an appealing platform for developing low-dimensional memory devices.

Experimental investigation of improved tolerance for concentration mismatch in potassium isotope analysis on a hexapole collision cell MC-ICP-MS (Nu Sapphire)

Effect of K concentration mismatch up to 30% on δ41K value from RF Ref. of 1.05 V.

Formation mechanism of coronal composite secondary phase in titanium-zirconium-molybdenum alloy

The coronal composite secondary phase has a high mismatch tolerance with the molybdenum matrix. TiO 2 particles located within the coronal structure that provide a semi-coherent interface with a mismatch degree of 8.70%, and ZrO 2 particles located outside the coronal structure that provide an incoherent interface with a mismatch degree as high as 41.61%. The ZrO 2 particles of the coronal composite secondary phase are partially attached to the TiO 2 in a coronal structure, which endows the ZrO 2 with the molybdenum matrix's higher interfacial bonding force. Titanium sulfate and zirconium nitrate are realized the mixing of Ti/Zr, TiO 2 /ZrO 2 molecules at atomic levels. Compared with Ti, Zr is more easily combined with oxygen, which leads to Zr in Ti/Zr solid solution being attracted by oxygen at high temperature to form ZrO 2 . The small addition of zirconium element lacks nucleation particles to form of ZrO 2 , so ZrO 2 can only adhere to more TiO 2 particles and nucleate, forming the second phase of the coronal composite structure. This study can guide the design of high-performance secondary phase strengthening alloys. • The coronal composite secondary phase and the Mo matrix have a high tolerance of mismatch degree. • ZrO 2 particles are attached to TiO 2 , which endows ZrO 2 with a higher interfacial bonding force with Mo. • The secondary phase particles have a fine particle size, uniform distribution, and strong adhesion. • Mixing at the molecular or atomic level, Zr is oxyphilic, enabling the formation of the coronal composite secondary phase.

Validating and optimizing mismatch tolerance of Doppler backscattering measurements with the beam model (invited).

We use the beam model of Doppler backscattering (DBS), which was previously derived from beam tracing and the reciprocity theorem, to shed light on mismatch attenuation. This attenuation of the backscattered signal occurs when the wavevector of the probe beam's electric field is not in the plane perpendicular to the magnetic field. Correcting for this effect is important for determining the amplitude of the actual density fluctuations. Previous preliminary comparisons between the model and Mega-Ampere Spherical Tokamak (MAST) plasmas were promising. In this work, we quantitatively account for this effect on DIII-D, a conventional tokamak. We compare the predicted and measured mismatch attenuation in various DIII-D, MAST, and MAST-U plasmas, showing that the beam model is applicable in a wide variety of situations. Finally, we performed a preliminary parameter sweep and found that the mismatch tolerance can be improved by optimizing the probe beam's width and curvature at launch. This is potentially a design consideration for new DBS systems.

Condensation droplet sieve

Large droplets emerging during dropwise condensation impair surface properties such as anti-fogging/frosting ability and heat transfer efficiency. How to spontaneously detach massive randomly distributed droplets with controlled sizes has remained a challenge. Herein, we present a solution called condensation droplet sieve, through fabricating microscale thin-walled lattice structures coated with a superhydrophobic layer. Growing droplets were observed to jump off this surface once becoming slightly larger than the lattices. The maximum radius and residual volume of droplets were strictly confined to 16 μm and 3.2 nl/mm2 respectively. We reveal that this droplet radius cut off is attributed to the large tolerance of coalescence mismatch for jumping and effective isolation of droplets between neighboring lattices. Our work brings forth a strategy for the design and fabrication of high-performance anti-dew materials.

Repair of mismatched templates during Rad51-dependent Break-Induced Replication.

Using budding yeast, we have studied Rad51-dependent break-induced replication (BIR), where the invading 3' end of a site-specific double-strand break (DSB) and a donor template share 108 bp of homology that can be easily altered. BIR still occurs about 10% as often when every 6th base is mismatched as with a perfectly matched donor. Here we explore the tolerance of mismatches in more detail, by examining donor templates that each carry 10 mismatches, each with different spatial arrangements. Although 2 of the 6 arrangements we tested were nearly as efficient as the evenly-spaced reference, 4 were significantly less efficient. A donor with all 10 mismatches clustered at the 3' invading end of the DSB was not impaired compared to arrangements where mismatches were clustered at the 5' end. Our data suggest that the efficiency of strand invasion is principally dictated by thermodynamic considerations, i.e., by the total number of base pairs that can be formed; but mismatch position-specific effects are also important. We also addressed an apparent difference between in vitro and in vivo strand exchange assays, where in vitro studies had suggested that at a single contiguous stretch of 8 consecutive bases was needed to be paired for stable strand pairing, while in vivo assays using 108-bp substrates found significant recombination even when every 6th base was mismatched. Now, using substrates of either 90 or 108 nt-the latter being the size of the in vivo templates-we find that in vitro D-loop results are very similar to the in vivo results. However, there are still notable differences between in vivo and in vitro assays that are especially evident with unevenly-distributed mismatches. Mismatches in the donor template are incorporated into the BIR product in a strongly polar fashion up to ~40 nucleotides from the 3' end. Mismatch incorporation depends on the 3'→ 5' proofreading exonuclease activity of DNA polymerase δ, with little contribution from Msh2/Mlh1 mismatch repair proteins, or from Rad1-Rad10 flap nuclease or the Mph1 helicase. Surprisingly, the probability of a mismatch 27 nt from the 3' end being replaced by donor sequence was the same whether the preceding 26 nucleotides were mismatched every 6th base or fully homologous. These data suggest that DNA polymerase δ "chews back" the 3' end of the invading strand without any mismatch-dependent cues from the strand invasion structure. However, there appears to be an alternative way to incorporate a mismatch at the first base at the 3' end of the donor.

CaSilico: A versatile CRISPR package for in silico CRISPR RNA designing for Cas12, Cas13, and Cas14.

The efficiency of the CRISPR-Cas system is highly dependent on well-designed CRISPR RNA (crRNA). To facilitate the use of various types of CRISPR-Cas systems, there is a need for the development of computational tools to design crRNAs which cover different CRISPR-Cas systems with off-target analysis capability. Numerous crRNA design tools have been developed, but nearly all of them are dedicated to design crRNA for genome editing. Hence, we developed a tool matching the needs of both beginners and experts, named CaSilico, which was inspired by the limitations of the current crRNA design tools for designing crRNAs for Cas12, Cas13, and Cas14 CRISPR-Cas systems. This tool considers a comprehensive list of the principal rules that are not yet well described to design crRNA for these types. Using a list of important features such as mismatch tolerance rules, self-complementarity, GC content, frequency of cleaving base around the target site, target accessibility, and PFS (protospacer flanking site) or PAM (protospacer adjacent motif) requirement, CaSilico searches all potential crRNAs in a user-input sequence. Considering these features help users to rank all crRNAs for a sequence and make an informed decision about whether a crRNA is suited for an experiment or not. Our tool is sufficiently flexible to tune some key parameters governing the design of crRNA and identification of off-targets, which can lead to an increase in the chances of successful CRISPR-Cas experiments. CaSilico outperforms previous crRNA design tools in the following aspects: 1) supporting any reference genome/gene/transcriptome for which an FASTA file is available; 2) designing crRNAs that simultaneously target multiple sequences through conserved region detection among a set of sequences; 3) considering new CRISPR-Cas subtypes; and 4) reporting a list of different features for each candidate crRNA, which can help the user to select the best one. Given these capabilities, CaSilico addresses end-user concerns arising from the use of sophisticated bioinformatics algorithms and has a wide range of potential research applications in different areas, especially in the design of crRNA for pathogen diagnosis. CaSilico was successfully applied to design crRNAs for different genes in the SARS-CoV-2 genome, as some of the crRNAs have been experimentally tested in the previous studies.

Efficient Single-Nucleotide Microbial Genome Editing Achieved Using CRISPR/Cpf1 with Maximally 3'-End-Truncated crRNAs.

Mismatch tolerance, a cause of the off-target effect, impedes accurate genome editing with the CRISPR/Cas system. Herein, we observed that oligonucleotide-directed single-base substitutions could be rarely introduced in the microbial genome using CRISPR/Cpf1-mediated negative selection. Because crRNAs have the ability to recognize and discriminate among specific target DNA sequences, we systematically compared the effects of modified crRNAs with 3′-end nucleotide truncations and a single mismatch on the genomic cleavage activity of FnCpf1 inEscherichia coli. Five nucleotides could be maximally truncated at the crRNA 3′-end for the efficient cleavage of the DNA targets of galK and xylB in the cells. However, target cleavage in the genome was inefficient when a single mismatch was simultaneously introduced in the maximally 3′-end-truncated crRNA. Based on these results, we assumed that the maximally truncated crRNA-Cpf1 complex can distinguish between single-base-edited and unedited targets in vivo. Compared to other crRNAs with shorter truncations, maximally 3′-end-truncated crRNAs showed highly efficient single-base substitutions (>80%) in the DNA targets of galK and xylB. Furthermore, the editing efficiency for the 24 bases in both galK and xylB showed success rates of 79 and 50%, respectively. We successfully introduced single-nucleotide indels in galK and xylB with editing efficiencies of 79 and 62%, respectively. Collectively, the maximally truncated crRNA-Cpf1 complex could perform efficient base and nucleotide editing regardless of the target base location or mutation type; this system is a simple and efficient tool for microbial genome editing, including indel correction, at the single-nucleotide resolution.

Mismatch discrimination and sequence bias during end-joining by DNA ligases.

DNA ligases, critical enzymes for in vivo genome maintenance and modern molecular biology, catalyze the joining of adjacent 3′-OH and 5′-phosphorylated ends in DNA. To determine whether DNA annealing equilibria or properties intrinsic to the DNA ligase enzyme impact end-joining ligation outcomes, we used a highly multiplexed, sequencing-based assay to profile mismatch discrimination and sequence bias for several ligases capable of efficient end-joining. Our data reveal a spectrum of fidelity and bias, influenced by both the strength of overhang annealing as well as sequence preferences and mismatch tolerances that vary both in degree and kind between ligases. For example, while T7 DNA ligase shows a strong preference for ligating high GC sequences, other ligases show little GC-dependent bias, with human DNA Ligase 3 showing almost none. Similarly, mismatch tolerance varies widely among ligases, and while all ligases tested were most permissive of G:T mismatches, some ligases also tolerated bulkier purine:purine mismatches. These comprehensive fidelity and bias profiles provide insight into the biology of end-joining reactions and highlight the importance of ligase choice in application design.

Structural basis for mismatch surveillance by CRISPR\u2013Cas9

CRISPR–Cas9 as a programmable genome editing tool is hindered by off-target DNA cleavage1–4, and the underlying mechanisms by which Cas9 recognizes mismatches are poorly understood5–7. Although Cas9 variants with greater discrimination against mismatches have been designed8–10, these suffer from substantially reduced rates of on-target DNA cleavage5,11. Here we used kinetics-guided cryo-electron microscopy to determine the structure of Cas9 at different stages of mismatch cleavage. We observed a distinct, linear conformation of the guide RNA–DNA duplex formed in the presence of mismatches, which prevents Cas9 activation. Although the canonical kinked guide RNA–DNA duplex conformation facilitates DNA cleavage, we observe that substrates that contain mismatches distal to the protospacer adjacent motif are stabilized by reorganization of a loop in the RuvC domain. Mutagenesis of mismatch-stabilizing residues reduces off-target DNA cleavage but maintains rapid on-target DNA cleavage. By targeting regions that are exclusively involved in mismatch tolerance, we provide a proof of concept for the design of next-generation high-fidelity Cas9 variants.