Parallelized engineering of mutational models using piggyBac transposon delivery of CRISPR libraries

Novel gene and variant discoveries have reached unprecedented scale with the emergence of exome and genome sequencing studies across a spectrum of human disease initiatives. Highly parallelized functional characterization of these variants is now paramount to deciphering disease mechanisms, and approaches that facilitate editing of induced pluripotent stem cells (iPSCs) to derive otherwise inaccessible tissues of interest (e.g., brain) have become critical in genomics research. Here, we sought to facilitate scalable editing of multiple genes and variants by developing a genome engineering approach that incorporates libraries of CRISPR/Cas9 guide RNAs (gRNAs) into a piggyBac (PB) transposon system. To test the efficiency of inducing small indels, targeted deletions, and large reciprocal copy number variants (CNVs), we simultaneously delivered to human iPSCs both Cas9 and a library including 59 single gRNAs targeting segmental duplications, 70 paired gRNAs flanking particular genic regions, and three single gRNAs targeting the coding sequence of an individual gene, MAGEL2. After editing, we isolated single cells, expanded resultant colonies, and genotyped their gRNA contents and mutational outcomes. We observed that 97.7% of gRNA constructs were integrated into at least one colony, with 85.6% of colonies containing three or fewer PB integrations. This PB editing method generated 354 cell lines with 57.8% of sequenced gRNA cleavage sites modified in at least one line, 14.4% of these lines altered at multiple targets, and single-copy indel mutagenesis predominating. Among the edits generated were eight targeted genomic deletions, including pathogenic microdeletions at chromosome 15q11-q13 (∼5.3 Mbp), chromosome 16p11.2 (∼740 kbp), and chromosome 17q11.2 (∼1.4 Mbp). These data highlight PB editing as a powerful platform for gene inactivation and testify to its strong potential for oligogenic modeling. The ability to rapidly establish high-quality mutational models at scale will facilitate the development of near-isogenic cellular collections and catalyze comparative functional genomic studies, better positioning us to investigate the roles of hundreds of genes and mutations in development and disease.

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Systematic functional characterization of antisense eRNA of protocadherin α composite enhancer

Genes & Development ◽

10.1101/gad.348621.121 ◽

2021 ◽

Vol 35 (19-20) ◽

pp. 1383-1394

Author(s):

Yuxiao Zhou ◽

Siyuan Xu ◽

Mo Zhang ◽

Qiang Wu

Keyword(s):

Gene Expression ◽

Molecular Mechanisms ◽

Single Cells ◽

Functional Characterization ◽

Three Dimensional ◽

Elongation Factor ◽

Locked Nucleic Acid ◽

Loop Formation ◽

Long Distance ◽

Enhancer Region

Enhancers generate bidirectional noncoding enhancer RNAs (eRNAs) that may regulate gene expression. At present, the eRNA function remains enigmatic. Here, we report a 5′ capped antisense eRNA PEARL (Pcdh eRNA associated with R-loop formation) that is transcribed from the protocadherin (Pcdh) α HS5-1 enhancer region. Through loss- and gain-of-function experiments with CRISPR/Cas9 DNA fragment editing, CRISPRi, and CRISPRa, as well as locked nucleic acid strategies, in conjunction with ChIRP, MeDIP, DRIP, QHR-4C, and HiChIP experiments, we found that PEARL regulates Pcdhα gene expression by forming local RNA–DNA duplexes (R-loops) in situ within the HS5-1 enhancer region to promote long-distance chromatin interactions between distal enhancers and target promoters. In particular, increased levels of eRNA PEARL via perturbing transcription elongation factor SPT6 lead to strengthened local three-dimensional chromatin organization within the Pcdh superTAD. These findings have important implications regarding molecular mechanisms by which the HS5-1 enhancer regulates stochastic Pcdhα promoter choice in single cells in the brain.

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Isolation of Neurospora crassa A mating type mutants by repeat induced point (RIP) mutation.

Genetics ◽

10.1093/genetics/132.1.125 ◽

1992 ◽

Vol 132 (1) ◽

pp. 125-133 ◽

Cited By ~ 1

Author(s):

N L Glass ◽

L Lee

Keyword(s):

Neurospora Crassa ◽

Mating Type ◽

Single Copy ◽

Open Reading Frame ◽

Sexual Cycle ◽

Reading Frame ◽

Heterokaryon Incompatibility ◽

Functional Regions ◽

A Genome ◽

Ascospore Formation

Abstract In the filamentous fungus, Neurospora crassa, mating type is regulated by a single locus with alternate alleles, termed A and a. The mating type alleles control entry into the sexual cycle, but during vegetative growth they function to elicit heterokaryon incompatibility, such that fusion of A and a hypha results in death of cells along the fusion point. Previous studies have shown that the A allele consists of 5301 bp and has no similarity to the a allele; it is found as a single copy and only within the A genome. The a allele is 3235 bp in length and it, too, is found as a single copy within the a genome. Within the A sequence, a single open reading frame (ORF) of 288 amino acids (mt A-1) is thought to confer fertility and heterokaryon incompatibility. In this study, we have used repeat induced point (RIP) mutation to identify functional regions of the A idiomorph. RIP mutations in mt A-1 resulted in the isolation of sterile, heterokaryon-compatible mutants, while RIP mutations generated in a region outside of mt A-1 resulted in the isolation of mutants capable of mating, but deficient in ascospore formation.

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A multiplex guide RNA expression system and its efficacy for plant genome engineering

10.21203/rs.2.21279/v1 ◽

2020 ◽

Author(s):

Youngbin Oh ◽

Hyeonjin Kim ◽

Bora Lee ◽

Sang-Gyu Kim

Keyword(s):

Genome Engineering ◽

Binary Vector ◽

Expression System ◽

Plant Genome ◽

Nicotiana Attenuata ◽

Specific Sequence ◽

Editing Efficiency ◽

Guide Rna ◽

Assembly Method ◽

A Genome

Abstract BackgroundThe Streptococcus pyogenes CRISPR system is composed of a Cas9 endonuclease (SpCas9) and a single-stranded guide RNA (gRNA) harboring a target-specific sequence. Theoretically, SpCas9 proteins could cleave as many targeted loci as gRNAs bind in a genome.ResultsWe introduce a PCR-free multiple gRNA cloning system for editing plant genomes. This method consists of two steps: (1) cloning annealed products of two oligonucleotides harboring target-binding sequence between tRNA and gRNA scaffold sequences in a pGRNA vector; and (2) assembling tRNA-gRNA units from several pGRNA vectors with a plant binary vector containing a SpCas9 expression cassette using the Golden Gate assembly method. We validated the editing efficiency and patterns of the multiplex gRNA expression system in wild tobacco (Nicotiana attenuata) protoplasts and in transformed plants by performing targeted deep sequencing. Two proximal cleavages by SpCas9-gRNA largely increased the editing efficiency and induced large deletions between two cleavage sites.ConclusionsThis multiplex gRNA expression system enables high-throughput production of a single binary vector and increases the efficiency of plant genome editing.

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16p12.1 deletion orthologs are expressed in motile neural crest cells and are important for regulating craniofacial development in Xenopus laevis

10.1101/2020.12.11.421347 ◽

2020 ◽

Author(s):

Micaela Lasser ◽

Jessica Bolduc ◽

Luke Murphy ◽

Caroline O'Brien ◽

Sangmook Lee ◽

...

Keyword(s):

Xenopus Laevis ◽

Neural Crest ◽

Expression Patterns ◽

Copy Number Variants ◽

Neural Crest Cell ◽

Underlying Mechanism ◽

Craniofacial Development ◽

Pharyngeal Arches ◽

Individual Gene ◽

Vertebrate Model

Copy number variants (CNVs) associated with neurodevelopmental disorders are characterized by extensive phenotypic heterogeneity. In particular, one CNV was identified in a subset of children clinically diagnosed with intellectual disabilities (ID) that results in a hemizygous deletion of multiple genes at chromosome 16p12.1. In addition to ID, individuals with this deletion display a variety of symptoms including microcephaly, seizures, cardiac defects, and growth retardation. Moreover, patients also manifest severe craniofacial abnormalities, such as micrognathia, cartilage malformation of the ears and nose, and facial asymmetries; however, the function of the genes within the 16p12.1 region have not been studied in the context of vertebrate craniofacial development. The craniofacial tissues affected in patients with this deletion all derive from the same embryonic precursor, the cranial neural crest, leading to the hypothesis that one or more of the 16p12.1 genes may be involved in regulating neural crest cell (NCC)-related processes. To examine this, we characterized the developmental role of the 16p12.1-affected gene orthologs, polr3e, mosmo, uqcrc2, and cdr2, during craniofacial morphogenesis in the vertebrate model system, Xenopus laevis. While the currently-known cellular functions of these genes are diverse, we find that they share similar expression patterns along the neural tube, pharyngeal arches, and later craniofacial structures. As these genes show co-expression in the pharyngeal arches where NCCs reside, we sought to elucidate the effect of individual gene depletion on craniofacial development and NCC migration. We find that reduction of several 16p12.1 genes significantly disrupts craniofacial and cartilage formation, pharyngeal arch migration, as well as NCC specification and motility. Thus, we have determined that some of these genes play an essential role during vertebrate craniofacial patterning by regulating specific processes during NCC development, which may be an underlying mechanism contributing to the craniofacial defects associated with the 16p12.1 deletion.

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Isolation and Sequencing of a Single Copy of an Introgressed Chromosome From a Complex Genome for Gene and SNP Identification

10.21203/rs.3.rs-629403/v1 ◽

2021 ◽

Author(s):

Cushla J Metcalfe ◽

Jingchuan Li ◽

Bangyou Zheng ◽

Jiri Stiller ◽

Adam Healey ◽

...

Keyword(s):

Flow Cytometry ◽

New Technologies ◽

Single Copy ◽

Snp Markers ◽

Crop Species ◽

Single Chromosome ◽

Flow Karyotype ◽

Large Complex ◽

A Genome ◽

Complex Genome

Abstract The large complex genomes of many crops constrain the use of new technologies for genome-assisted selection and genetic improvement. One method to simplify a genome is to break it into individual chromosomes by flow cytometry, however, in many crop species most chromosomes cannot be isolated individually. Flow sorting of a single copy of a chromosome has been developed in wheat and here we demonstrate its use to identify markers of interest in an Erianthus/Sacchurum hybrid. Erianthus/Saccharum hybrids are of interest because Erianthus is known to be highly resistant to soil borne diseases which cause extensive sugarcane yield losses in Australia. Sugarcane (Saccharum) cultivars are autopolyploids with a highly complex genome and over 100 chromosomes. Flow cytometry for sugarcane, as in most crops, does not resolve individual chromosomes to a karyotype peak for sorting. To isolate a single chromosome, we used genomic in situ hybridisation (GISH) to identify the flow karyotype region containing the Erianthus chromosomes, flow sorted single chromosomes from this region, PCR screened for the Erianthus chromosomes and sequenced them. One Erianthus chromosome amplified and sequenced well, and from this data we could identify 57 resistant type genes and SNPs in nearly half of these genes. We developed KASP SNP assays and demonstrated that the identified SNP markers segregated as expected in a small introgression population. The pipeline we developed here to flow sort and sequence single chromosomes could be used in any crop with a large complex genome to rapidly discover and develop markers to important loci.

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Building a genome engineering toolbox in nonmodel prokaryotic microbes

Biotechnology and Bioengineering ◽

10.1002/bit.26727 ◽

2018 ◽

Vol 115 (9) ◽

pp. 2120-2138 ◽

Cited By ~ 16

Author(s):

Emily Freed ◽

Jacob Fenster ◽

Sharon L. Smolinski ◽

Julie Walker ◽

Calvin A. Henard ◽

...

Keyword(s):

Genome Engineering ◽

A Genome

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Genomic deletions in Rhodococcus based on transformation of linear heterologous DNA

Microbiology ◽

10.1099/mic.0.001028 ◽

2021 ◽

Author(s):

Divya Singhi ◽

Shabnam Parwin ◽

Preeti Srivastava

Keyword(s):

Rapid Method ◽

Genome Engineering ◽

Cost Effective ◽

Cell Disruption ◽

Content Type ◽

First Report ◽

Genomic Deletions ◽

Linear Dna ◽

Link Type ◽

Successful Expression

Several genome engineering methods have been developed for Rhodococcus . However, they suffer from limitations such as extensive cloning, multiple steps, successful expression of heterologous genes via plasmid etc. Here, we report a rapid method for performing genomic deletions/disruptions in Rhodococcus spp. using heterologous linear DNA. The method is cost effective and less labour intensive. The applicability of the method was demonstrated by successful disruption of rodA and orphan parA. None of the disrupted genes were found to be essential for the viability of the cell. Disruption of orphan parA and rodA resulted in elongated cells and short rods, respectively. This is the first report demonstrating disruption of rodA and orphan parA genes by electroporation of heterologous linear DNA in Rhodococcus spp.

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Genomic Syndromes in Schizophrenia

Neurobiology of Mental Illness ◽

10.1093/med/9780199934959.003.0019 ◽

2013 ◽

pp. 247-255

Author(s):

George Kirov ◽

Michael C. O’Donovan ◽

Michael J. Owen

Keyword(s):

Intellectual Disability ◽

Developmental Delay ◽

Neurodevelopmental Disorders ◽

Copy Number ◽

De Novo ◽

Copy Number Variants ◽

Individual Risk ◽

Genomic Deletions ◽

Pathogenic Cnvs ◽

Cognition And Learning

Several submicroscopic genomic deletions and duplications known as copy number variants (CNVs) have been reported to increase susceptibility to schizophrenia. Those for which the evidence is particularly strong include deletions at chromosomal segments 1q21.1, 3q29, 15q11.2, 15q13.3, 17q12 and 22q11.2, duplications at 15q11.2-q13.1, 16p13.1, and 16p11.2, and deletions atthe gene NRXN1. The effect of each on individual risk is relatively large, but it does not appear that any of them is alone sufficient to cause disorder in carriers. These CNVs often arise as new mutations(de novo). Analyses of genes enriched among schizophrenia implicated CNVs highlight the involvement in the disorder of post-synaptic processes relevant to glutamatergicsignalling, cognition and learning. CNVs that contribute to schizophrenia risk also contribute to other neurodevelopmental disorders, including intellectual disability, developmental delay and autism. As a result of selection, all known pathogenic CNVs are rare, and none makes a sizeable contribution to overall population risk of schizophrenia, although the study of these mutations is nevertheless providing important insights into the origins of the disorder.

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CRISPR/Cas9: a historical and chemical biology perspective of targeted genome engineering

Chemical Society Reviews ◽

10.1039/c6cs00197a ◽

2016 ◽

Vol 45 (24) ◽

pp. 6666-6684 ◽

Cited By ~ 16

Author(s):

Amrita Singh ◽

Debojyoti Chakraborty ◽

Souvik Maiti

Keyword(s):

Genome Editing ◽

Chemical Biology ◽

Genome Engineering ◽

A Genome

The development and adaptation of CRISPR–Cas9 as a genome editing tool and chemical biology approaches for modulating its activity.

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Genome-Wide Identification and Analysis of VQ Motif-containing Gene Family in Brassica napus and Functional Characterization of BnMKS1 in Response to Leptosphaeria maculans

Phytopathology ◽

10.1094/phyto-04-20-0134-r ◽

2020 ◽

Cited By ~ 1

Author(s):

Zhongwei Zou ◽

Fei Liu ◽

Shuanglong Huang ◽

DILANTHA GERARD FERNANDO

Keyword(s):

Brassica Napus ◽

Gene Family ◽

Functional Characterization ◽

Leptosphaeria Maculans ◽

Defense Responses ◽

Marker Genes ◽

Blackleg Disease ◽

Genome Wide ◽

A Genome

Proteins containing Valine-glutamine (VQ) motifs play important roles in plant growth and development, as well as in defense responses to both abiotic and biotic stresses. Blackleg disease, which is caused by Leptosphaeria maculans, is the most important disease in canola (Brassica napus L.) worldwide. H; however, the identification of B. napus VQs and their functions in response to blackleg disease have not yet been reported. In this study, we conducted a genome genome-wide identification and characterization of the VQ gene family in B. napus, including chromosome location, phylogenetic relations, gene structure, motif domain, synteny analysis, and cis-elements categorization of their promoter regions. To understand B. napus VQ gene function in response to blackleg disease, we overexpressed BnVQ7 (BnaA01g36880D, also known as the mitogen-activated protein kinase4 substrate1 (MKS1) gene) in a blackleg-susceptible canola variety Westar. Overexpression The overexpression of BnMKS1 in canola did not improve its resistance to blackleg disease at the seedling stage. H; however, transgenic canola plants overexpressing BnMKS1 displayed an enhanced resistance to L. maculans infection at the adult plant stage. Expression levels of downstream and defense marker genes in cotyledons increased significantly at the necrotrophic stage of L. maculans infection in the overexpression line of BnMKS1, suggesting that the SA salicylic acid (SA)- and jasmonic acid (JA )-mediated signaling pathways were both involved in the defense responses. Together, these results suggest that BnMKS1 might play an important role in the defense against L. maculans.

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