AGBE: a dual deaminase-mediated base editor by fusing CGBE with ABE for creating a saturated mutant population with multiple editing patterns

Establishing saturated mutagenesis in a specific gene through gene editing is an efficient approach for identifying the relationships between mutations and the corresponding phenotypes. CRISPR/Cas9-based sgRNA library screening often creates indel mutations with multiple nucleotides. Single base editors and dual deaminase-mediated base editors can achieve only one and two types of base substitutions, respectively. A new glycosylase base editor (CGBE) system, in which the uracil glycosylase inhibitor (UGI) is replaced with uracil-DNA glycosylase (UNG), was recently reported to efficiently induce multiple base conversions, including C-to-G, C-to-T and C-to-A. In this study, we fused a CGBE with ABE to develop a new type of dual deaminase-mediated base editing system, the AGBE system, that can simultaneously introduce 4 types of base conversions (C-to-G, C-to-T, C-to-A and A-to-G) as well as indels with a single sgRNA in mammalian cells. AGBEs can be used to establish saturated mutant populations for verification of the functions and consequences of multiple gene mutation patterns, including single-nucleotide variants (SNVs) and indels, through high-throughput screening.     

Multiplex knockout of trichome-regulating MYB duplicates in hybrid poplar using a single gRNA

As the focus for CRISPR/Cas-edited plants moves from proof-of-concept to real-world applications, precise gene manipulation will increasingly require concurrent multiplex editing for polygenic traits. A common approach for editing across multiple sites is to design one guide RNA (gRNA) per target; however, this complicates construct assembly and increases the possibility of off-target mutations. In this study, we utilized one gRNA to target MYB186, a known positive trichome regulator, as well as its paralogs MYB138 and MYB38 at a consensus site for mutagenesis in hybrid poplar (Populus tremula × P. alba INRA 717-1B4). Unexpected duplications of MYB186 and MYB138 resulted in eight alleles for the three targeted genes in the hybrid poplar. Deep sequencing and polymerase chain reaction analyses confirmed editing across all eight targets in nearly all of the resultant glabrous mutants, ranging from small indels to large genomic dropouts, with no off-target activity detected at four potential sites. This highlights the effectiveness of a single gRNA targeting conserved exonic regions for multiplex editing. Additionally, cuticular wax and whole-leaf analyses showed a complete absence of triterpenes in the trichomeless mutants, hinting at a previously undescribed role for the nonglandular trichomes of poplar.

Targeting conserved sequences with a single gRNA allows efficient mutagenesis of a multigene family and the recovery of trichomeless and triterpene-free poplar mutants.     

Evolutionary dynamics of wheat mitochondrial gene structure with special remarks on the origin and effects of RNA editing in cereals

We investigated the evolutionary dynamics of wheat mitochondrial genes with respect to their structural differentiation during organellar evolution, and to mutations that occurred during cereal evolution. First, we compared the nucleotide sequences of three wheat mitochondrial genes to those of wheat chloroplast, alpha-proteobacterium and cyanobacterium orthologs. As a result, we were able to (1) differentiate the conserved and variable segments of the orthologs, (2) reveal the functional importance of the conserved segments, and (3) provide a corroborative support for the alpha-proteobacterial and cyanobacterial origins of those mitochondrial and chloroplast genes, respectively. Second, we compared the nucleotide sequences of wheat mitochondrial genes to those of rice and maize to determine the types and frequencies of base changes and indels occurred in cereal evolution. Our analyses showed that both the evolutionary speed, in terms of number of base substitutions per site, and the transition/transversion ratio of the cereal mitochondrial genes were less than two-fifths of those of the chloroplast genes. Eight mitochondrial gene groups differed in their evolutionary variability, RNA and Complex I (nad) genes being most stable whereas Complex V (atp) and ribosomal protein genes most variable. C-to-T transition was the most frequent type of base change; C-to-G and G-to-C transversions occurred at lower rates than all other changes. The excess of C-to-T transitions was attributed to C-to-U RNA editing that developed in early stage of vascular plant evolution. On the contrary, the editing of C residues at cereal T-to-C transition sites developed mostly during cereal divergence. Most indels were associated with short direct repeats, suggesting intra- and intermolecular recombination as an important mechanism for their origin. Most of the repeats associated with indels were di- or trinucleotides, although no preference was noticed for their sequences. The maize mt genome was characterized by a high incidence of indels, comparing to the wheat and rice mt genomes.     

利用rBE3和rBE4系统进行水稻基因单碱基编辑的特异性和遗传稳定性分析

对基于rBE3(Rice base editor)和rBE4碱基编辑系统创制获得Os SERK1(D428N)和pi-ta(S918F)等基因的单碱基编辑突变体材料进行编辑特异性和遗传稳定性分析,旨在全面了解和更好地利用该碱基编辑系统。首先对Os SERK1(D428N)和pi-ta(S918F)sg RNA的潜在脱靶位点进行预测,并对T0代材料中的各脱靶位点进行PCR扩增和Sanger测序检测;同时对该两个基因的突变体材料自交获得的T1代植株的靶位点序列和外源T-DNA分离进行检测。结果显示各T0代材料均未检测到潜在脱靶位点发生单碱基编辑;此外,Os SERK1(D428N)和Os08g07774含有相同的sg RNA位点,且两个位点均能发生单碱基编辑;rBE3或rBE4系统介导产生的碱基编辑可稳定遗传至T1代,并在T1代可获得无外源T-DNA的纯合突变体。上述结果表明由rBE3或rBE4介导的碱基编辑具有较高的特异性,可进行多位点编辑,引入的碱基替换可稳定遗传至后代。     

Discovering single nucleotide variants and indels from bulk and single-cell ATAC-seq

Genetic variants and de novo mutations in regulatory regions of the genome are typically discovered by whole-genome sequencing (WGS), however WGS is expensive and most WGS reads come from non-regulatory regions. The Assay for Transposase-Accessible Chromatin (ATAC-seq) generates reads from regulatory sequences and could potentially be used as a low-cost ‘capture’ method for regulatory variant discovery, but its use for this purpose has not been systematically evaluated. Here we apply seven variant callers to bulk and single-cell ATAC-seq data and evaluate their ability to identify single nucleotide variants (SNVs) and insertions/deletions (indels). In addition, we develop an ensemble classifier, VarCA, which combines features from individual variant callers to predict variants. The Genome Analysis Toolkit (GATK) is the best-performing individual caller with precision/recall on a bulk ATAC test dataset of 0.92/0.97 for SNVs and 0.87/0.82 for indels within ATAC-seq peak regions with at least 10 reads. On bulk ATAC-seq reads, VarCA achieves superior performance with precision/recall of 0.99/0.95 for SNVs and 0.93/0.80 for indels. On single-cell ATAC-seq reads, VarCA attains precision/recall of 0.98/0.94 for SNVs and 0.82/0.82 for indels. In summary, ATAC-seq reads can be used to accurately discover non-coding regulatory variants in the absence of whole-genome sequencing data and our ensemble method, VarCA, has the best overall performance.     

Scalable and Versatile Genome Editing Using Linear DNAs with Microhomology to Cas9 Sites in Caenorhabditis elegans

Homology-directed repair (HDR) of double-strand DNA breaks is a promising method for genome editing, but is thought to be less efficient than error-prone nonhomologous end joining in most cell types. We have investigated HDR of double-strand breaks induced by CRISPR-associated protein 9 (Cas9) in Caenorhabditis elegans. We find that HDR is very robust in the C. elegans germline. Linear repair templates with short (∼30–60 bases) homology arms support the integration of base and gene-sized edits with high efficiency, bypassing the need for selection. Based on these findings, we developed a systematic method to mutate, tag, or delete any gene in the C. elegans genome without the use of co-integrated markers or long homology arms. We generated 23 unique edits at 11 genes, including premature stops, whole-gene deletions, and protein fusions to antigenic peptides and GFP. Whole-genome sequencing of five edited strains revealed the presence of passenger variants, but no mutations at predicted off-target sites. The method is scalable for multi-gene editing projects and could be applied to other animals with an accessible germline.     

DAJIN enables multiplex genotyping to simultaneously validate intended and unintended target genome editing outcomes

Genome editing can introduce designed mutations into a target genomic site. Recent research has revealed that it can also induce various unintended events such as structural variations, small indels, and substitutions at, and in some cases, away from the target site. These rearrangements may result in confounding phenotypes in biomedical research samples and cause a concern in clinical or agricultural applications. However, current genotyping methods do not allow a comprehensive analysis of diverse mutations for phasing and mosaic variant detection. Here, we developed a genotyping method with an on-target site analysis software named Determine Allele mutations and Judge Intended genotype by Nanopore sequencer (DAJIN) that can automatically identify and classify both intended and unintended diverse mutations, including point mutations, deletions, inversions, and cis double knock-in at single-nucleotide resolution. Our approach with DAJIN can handle approximately 100 samples under different editing conditions in a single run. With its high versatility, scalability, and convenience, DAJIN-assisted multiplex genotyping may become a new standard for validating genome editing outcomes.

Genome editing can introduce designed mutations into a target genomic site, but also into unintended off-target sites. DAJIN, a novel nanopore sequencing data analysis tool, identifies and quantifies allele numbers and their mutation patterns, reporting consensus sequences and visualizing mutations in alleles at single-nucleotide resolution.     

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.     

Efficient Cas9 multiplex editing using unspaced sgRNA arrays engineering in a Potato virus X vector

Systems based on the clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR-associated proteins (Cas) have revolutionized genome editing in many organisms, including plants. Most CRISPR-Cas strategies in plants rely on genetic transformation using Agrobacterium tumefaciens to supply the gene editing reagents, such as Cas nucleases or the synthetic guide RNA (sgRNA). While Cas nucleases are constant elements in editing approaches, sgRNAs are target-specific and a screening process is usually required to identify those most effective. Plant virus-derived vectors are an alternative for the fast and efficient delivery of sgRNAs into adult plants, due to the virus capacity for genome amplification and systemic movement, a strategy known as virus-induced genome editing. We engineered Potato virus X (PVX) to build a vector that easily expresses multiple sgRNAs in adult solanaceous plants. Using the PVX-based vector, Nicotiana benthamiana genes were efficiently targeted, producing nearly 80% indels in a transformed line that constitutively expresses Streptococcus pyogenes Cas9. Interestingly, results showed that the PVX vector allows expression of arrays of unspaced sgRNAs, achieving highly efficient multiplex editing in a few days in adult plant tissues. Moreover, virus-free edited progeny can be obtained from plants regenerated from infected tissues or infected plant seeds, which exhibit a high rate of heritable biallelic mutations. In conclusion, this new PVX vector allows easy, fast and efficient expression of sgRNA arrays for multiplex CRISPR-Cas genome editing and will be a useful tool for functional gene analysis and precision breeding across diverse plant species, particularly in Solanaceae crops.     

Functional correction of CFTR mutations in human airway epithelial cells using adenine base editors

Mutations in the CFTR gene that lead to premature stop codons or splicing defects cause cystic fibrosis (CF) and are not amenable to treatment by small-molecule modulators. Here, we investigate the use of adenine base editor (ABE) ribonucleoproteins (RNPs) that convert A•T to G•C base pairs as a therapeutic strategy for three CF-causing mutations. Using ABE RNPs, we corrected in human airway epithelial cells premature stop codon mutations (R553X and W1282X) and a splice-site mutation (3849 + 10 kb C > T). Following ABE delivery, DNA sequencing revealed correction of these pathogenic mutations at efficiencies that reached 38–82% with minimal bystander edits or indels. This range of editing was sufficient to attain functional correction of CFTR-dependent anion channel activity in primary epithelial cells from CF patients and in a CF patient-derived cell line. These results demonstrate the utility of base editor RNPs to repair CFTR mutations that are not currently treatable with approved therapeutics.     

Delivery of CRISPR/Cas Components into Higher Plant Cells for Genome Editing

Russian Journal of Plant Physiology - CRISPR/Cas genome editing of plants is realized in three basic variants, including knockout mutations as indels, insertion of alien DNA fragments, and base...     

GCAT|Panel,a comprehensive structural variant haplotype map of the Iberian population from high-coverage whole-genome sequencing

The combined analysis of haplotype panels with phenotype clinical cohorts is a common approach to explore the genetic architecture of human diseases. However, genetic studies are mainly based on single nucleotide variants (SNVs) and small insertions and deletions (indels). Here, we contribute to fill this gap by generating a dense haplotype map focused on the identification, characterization, and phasing of structural variants (SVs). By integrating multiple variant identification methods and Logistic Regression Models (LRMs), we present a catalogue of 35 431 441 variants, including 89 178 SVs (≥50 bp), 30 325 064 SNVs and 5 017 199 indels, across 785 Illumina high coverage (30x) whole-genomes from the Iberian GCAT Cohort, containing a median of 3.52M SNVs, 606 336 indels and 6393 SVs per individual. The haplotype panel is able to impute up to 14 360 728 SNVs/indels and 23 179 SVs, showing a 2.7-fold increase for SVs compared with available genetic variation panels. The value of this panel for SVs analysis is shown through an imputed rare Alu element located in a new locus associated with Mononeuritis of lower limb, a rare neuromuscular disease. This study represents the first deep characterization of genetic variation within the Iberian population and the first operational haplotype panel to systematically include the SVs into genome-wide genetic studies.     

Performance comparison of whole-genome sequencing platforms

Whole-genome sequencing is becoming commonplace, but the accuracy and completeness of variant calling by the most widely used platforms from Illumina and Complete Genomics have not been reported. Here we sequenced the genome of an individual with both technologies to a high average coverage of ～76×, and compared their performance with respect to sequence coverage and calling of single-nucleotide variants (SNVs), insertions and deletions (indels). Although 88.1% of the ～3.7 million unique SNVs were concordant between platforms, there were tens of thousands of platform-specific calls located in genes and other genomic regions. In contrast, 26.5% of indels were concordant between platforms. Target enrichment validated 92.7% of the concordant SNVs, whereas validation by genotyping array revealed a sensitivity of 99.3%. The validation experiments also suggested that >60% of the platform-specific variants were indeed present in the genome. Our results have important implications for understanding the accuracy and completeness of the genome sequencing platforms.     

Quality control and integration of genotypes from two calling pipelines for whole genome sequence data in the Alzheimer's disease sequencing project

The Alzheimer's Disease Sequencing Project (ADSP) performed whole genome sequencing (WGS) of 584 subjects from 111 multiplex families at three sequencing centers. Genotype calling of single nucleotide variants (SNVs) and insertion-deletion variants (indels) was performed centrally using GATK-HaplotypeCaller and Atlas V2. The ADSP Quality Control (QC) Working Group applied QC protocols to project-level variant call format files (VCFs) from each pipeline, and developed and implemented a novel protocol, termed “consensus calling,” to combine genotype calls from both pipelines into a single high-quality set. QC was applied to autosomal bi-allelic SNVs and indels, and included pipeline-recommended QC filters, variant-level QC, and sample-level QC. Low-quality variants or genotypes were excluded, and sample outliers were noted. Quality was assessed by examining Mendelian inconsistencies (MIs) among 67 parent-offspring pairs, and MIs were used to establish additional genotype-specific filters for GATK calls. After QC, 578 subjects remained. Pipeline-specific QC excluded ~12.0% of GATK and 14.5% of Atlas SNVs. Between pipelines, ~91% of SNV genotypes across all QCed variants were concordant; 4.23% and 4.56% of genotypes were exclusive to Atlas or GATK, respectively; the remaining ~0.01% of discordant genotypes were excluded. For indels, variant-level QC excluded ~36.8% of GATK and 35.3% of Atlas indels. Between pipelines, ~55.6% of indel genotypes were concordant; while 10.3% and 28.3% were exclusive to Atlas or GATK, respectively; and ~0.29% of discordant genotypes were. The final WGS consensus dataset contains 27,896,774 SNVs and 3,133,926 indels and is publicly available.     

Efficient genome editing and gene knockout in Setaria viridis with CRISPR/Cas9 directed gene editing by the non-homologous end-joining pathway

The CRISPR/Cas9 system has been used for genome editing in several organisms, including higher plants. This system induces site-specific mutations in the genome based on the nucleotide sequence of engineered guide RNAs. The complex genomes of C4 grasses makes genome editing a challenge in key grass crops like maize (Zea mays), sorghum (Sorghum bicolor), Brachiaria spp., switchgrass (Panicum virgatum), and sugarcane (Saccharum spp.). Setaria viridis is a diploid C4 grass widely used as a model for these C4 crop plants. Here, an optimized CRISPR/Cas9 binary vector that exploits the non-homologous end joining (NHEJ) system was used to knockout a green fluorescent protein (gfp) transgene in S. viridis accession A10.1. Transformation of embryogenic callus by A. tumefaciens generated ten glufosinate-ammonium resistant transgenic events. In the T0 generation, 60% of the events were biallelic mutants in the gfp transgene with no detectable accumulation of GFP protein and without insertions or deletions in predicted off-target sites. The gfp mutations generated by CRISPR/Cas9 were stable and displayed Mendelian segregation in the T1 generation. Altogether, the system described here is a highly efficient genome editing system for S. viridis, an important model plant for functional genomics studies in C4 grasses. Also, this system is a potential tool for improvement of agronomic traits in C4 crop plants with complex genomes.     

Efficient and error-free fluorescent gene tagging in human organoids without double-strand DNA cleavage

CRISPR-associated nucleases are powerful tools for precise genome editing of model systems, including human organoids. Current methods describing fluorescent gene tagging in organoids rely on the generation of DNA double-strand breaks (DSBs) to stimulate homology-directed repair (HDR) or non-homologous end joining (NHEJ)-mediated integration of the desired knock-in. A major downside associated with DSB-mediated genome editing is the required clonal selection and expansion of candidate organoids to verify the genomic integrity of the targeted locus and to confirm the absence of off-target indels. By contrast, concurrent nicking of the genomic locus and targeting vector, known as in-trans paired nicking (ITPN), stimulates efficient HDR-mediated genome editing to generate large knock-ins without introducing DSBs. Here, we show that ITPN allows for fast, highly efficient, and indel-free fluorescent gene tagging in human normal and cancer organoids. Highlighting the ease and efficiency of ITPN, we generate triple fluorescent knock-in organoids where 3 genomic loci were simultaneously modified in a single round of targeting. In addition, we generated model systems with allele-specific readouts by differentially modifying maternal and paternal alleles in one step. ITPN using our palette of targeting vectors, publicly available from Addgene, is ideally suited for generating error-free heterozygous knock-ins in human organoids.

A major downside of double-strand break-mediated genome editing is the need to verify the genomic integrity of the targeted locus and confirm the absence of off-target indels. This study shows that in-trans paired nicking is a mutation-free CRISPR strategy to introduce precise knock-ins into human organoids; its genomic fidelity allows all knock-in cells to be pooled, accelerating the establishment of new organoid models.