Browse
Submit Data
Databases
API
Help

Dataset Information

17 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Ligation-assisted homologous recombination enables precise genome editing by deploying both MMEJ and HDR.

ABSTRACT: CRISPR/Cas12a is a single effector nuclease that, like CRISPR/Cas9, has been harnessed for genome editing based on its ability to generate targeted DNA double strand breaks (DSBs). Unlike the blunt-ended DSB generated by Cas9, Cas12a generates sticky-ended DSB that could potentially aid precise genome editing, but this unique feature has thus far been underutilized. In the current study, we found that a short double-stranded DNA (dsDNA) repair template containing a sticky end that matched one of the Cas12a-generated DSB ends and a homologous arm sharing homology with the genomic region adjacent to the other end of the DSB enabled precise repair of the DSB and introduced a desired nucleotide substitution. We termed this strategy 'Ligation-Assisted Homologous Recombination' (LAHR). Compared to the single-stranded oligo deoxyribonucleotide (ssODN)-mediated homology directed repair (HDR), LAHR yields relatively high editing efficiency as demonstrated for both a reporter gene and endogenous genes. We found that both HDR and microhomology-mediated end joining (MMEJ) mechanisms are involved in the LAHR process. Our LAHR genome editing strategy, extends the repertoire of genome editing technologies and provides a broader understanding of the type and role of DNA repair mechanisms involved in genome editing.

SUBMITTER: Zhao Z

PROVIDER: S-EPMC9226534 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Similar Datasets

Precise genome editing by homologous recombination.

Project description:Simple and efficient methods are presented for creating precise modifications of the zebrafish genome. Edited alleles are generated by homologous recombination between the host genome and double-stranded DNA (dsDNA) donor molecules, stimulated by the induction of double-strand breaks at targeted loci in the host genome. Because several kilobase-long tracts of sequence can be exchanged, multiple genome modifications can be generated simultaneously at a single locus. Methods are described for creating: (1) alleles with simple sequence changes or in-frame additions, (2) knockin/knockout alleles that express a reporter protein from an endogenous locus, and (3) conditional alleles in which exons are flanked by recombinogenic loxP sites. Significantly, our approach to genome editing allows the incorporation of a linked reporter gene into the donor sequences so that successfully edited alleles can be identified by virtue of expression of the reporter. Factors affecting the efficiency of genome editing are discussed, including the finding that dsDNA products of I-SceI meganuclease enzyme digestion are particularly effective as donor molecules for gene-editing events. Reagents and procedures are described for accomplishing efficient genome editing in the zebrafish.

| S-EPMC5127288 | biostudies-literature

Deploying MMEJ using MENdel in precision gene editing applications for gene therapy and functional genomics.

Project description:Gene-editing experiments commonly elicit the error-prone non-homologous end joining for DNA double-strand break (DSB) repair. Microhomology-mediated end joining (MMEJ) can generate more predictable outcomes for functional genomic and somatic therapeutic applications. We compared three DSB repair prediction algorithms - MENTHU, inDelphi, and Lindel - in identifying MMEJ-repaired, homogeneous genotypes (PreMAs) in an independent dataset of 5,885 distinct Cas9-mediated mouse embryonic stem cell DSB repair events. MENTHU correctly identified 46% of all PreMAs available, a ∼2- and ∼60-fold sensitivity increase compared to inDelphi and Lindel, respectively. In contrast, only Lindel correctly predicted predominant single-base insertions. We report the new algorithm MENdel, a combination of MENTHU and Lindel, that achieves the most predictive coverage of homogeneous out-of-frame mutations in this large dataset. We then estimated the frequency of Cas9-targetable homogeneous frameshift-inducing DSBs in vertebrate coding regions for gene discovery using MENdel. 47 out of 54 genes (87%) contained at least one early frameshift-inducing DSB and 49 out of 54 (91%) did so when also considering Cas12a-mediated deletions. We suggest that the use of MENdel helps researchers use MMEJ at scale for reverse genetics screenings and with sufficient intra-gene density rates to be viable for nearly all loss-of-function based gene editing therapeutic applications.

| S-EPMC7797032 | biostudies-literature

Dynamics and competition of CRISPR-Cas9 ribonucleoproteins and AAV donor-mediated NHEJ, MMEJ and HDR editing.

Project description:Investigations of CRISPR gene knockout editing profiles have contributed to enhanced precision of editing outcomes. However, for homology-directed repair (HDR) in particular, the editing dynamics and patterns in clinically relevant cells, such as human iPSCs and primary T cells, are poorly understood. Here, we explore the editing dynamics and DNA repair profiles after the delivery of Cas9-guide RNA ribonucleoprotein (RNP) with or without the adeno-associated virus serotype 6 (AAV6) as HDR donors in four cell types. We show that editing profiles have distinct differences among cell lines. We also reveal the kinetics of HDR mediated by the AAV6 donor template. Quantification of T50 (time to reach half of the maximum editing frequency) indicates that short indels (especially +A/T) occur faster than longer (>2 bp) deletions, while the kinetics of HDR falls between NHEJ (non-homologous end-joining) and MMEJ (microhomology-mediated end-joining). As such, AAV6-mediated HDR effectively outcompetes the longer MMEJ-mediated deletions but not NHEJ-mediated indels. Notably, a combination of small molecular compounds M3814 and Trichostatin A (TSA), which potently inhibits predominant NHEJ repairs, leads to a 3-fold increase in HDR efficiency.

| S-EPMC7826255 | biostudies-literature

Enhancement of Precise Gene Editing by the Association of Cas9 With Homologous Recombination Factors.

Project description:The CRISPR-Cas9 system is used for genome editing in mammalian cells by introducing double-strand breaks (DSBs) which are predominantly repaired via non-homologous end joining (NHEJ) or to lesser extent by homology-directed repair (HDR). To enhance HDR for improving the introduction of precise genetic modifications, we tested fusion proteins of Cas9 nuclease with HDR effectors to enforce their localization at DSBs. Using a traffic-light DSB repair reporter (TLR) system for the quantitative detection of HDR and NHEJ events in human HEK cells we found that Cas9 fusions with CtIP, Rad52, and Mre11, but not Rad51C promote HDR up to twofold in human cells and significantly reduce NHEJ events. We further compared, as an alternative to the direct fusion with Cas9, two components configurations that associate CtIP fusion proteins with a Cas9-SunTag fusion or with guide RNA that includes MS2 binding loops. We found that the Cas9-CtIP fusion and the MS2-CtIP system, but not the SunTag approach increase the ratio of HDR/NHEJ 4.5-6-fold. Optimal results are obtained by the combined use of Cas9-CtIP and MS2-CtIP, shifting the HDR/NHEJ ratio by a factor of 14.9. Thus, our findings provide a simple and effective tool to promote precise gene modifications in mammalian cells.

| S-EPMC6503098 | biostudies-literature

Targeted recombination between homologous chromosomes for precise breeding in tomato.

Project description:Homologous recombination (HR) between parental chromosomes occurs stochastically. Here, we report on targeted recombination between homologous chromosomes upon somatic induction of DNA double-strand breaks (DSBs) via CRISPR-Cas9. We demonstrate this via a visual and molecular assay whereby DSB induction between two alleles carrying different mutations in the PHYTOENE SYNTHASE (PSY1) gene results in yellow fruits with wild type red sectors forming via HR-mediated DSB repair. We also show that in heterozygote plants containing one psy1 allele immune and one sensitive to CRISPR, repair of the broken allele using the unbroken allele sequence template is a common outcome. In another assay, we show evidence of a somatically induced DSB in a cross between a psy1 edible tomato mutant and wild type Solanum pimpinellifolium, targeting only the S. pimpinellifolium allele. This enables characterization of germinally transmitted targeted somatic HR events, demonstrating that somatically induced DSBs can be exploited for precise breeding of crops.

| S-EPMC5458649 | biostudies-literature

Homologous Recombination-Independent Large Gene Cassette Knock-in in CHO Cells Using TALEN and MMEJ-Directed Donor Plasmids.

Project description:Gene knock-in techniques have rapidly evolved in recent years, along with the development and maturation of genome editing technology using programmable nucleases. We recently reported a novel strategy for microhomology-mediated end-joining-dependent integration of donor DNA by using TALEN or CRISPR/Cas9 and optimized targeting vectors, named PITCh (Precise Integration into Target Chromosome) vectors. Here we describe TALEN and PITCh vector-mediated integration of long gene cassettes, including a single-chain Fv-Fc (scFv-Fc) gene, in Chinese hamster ovary (CHO) cells, with comparison of targeting and cloning efficiency among several donor design and culture conditions. We achieved 9.6-kb whole plasmid integration and 7.6-kb backbone-free integration into a defined genomic locus in CHO cells. Furthermore, we confirmed the reasonable productivity of recombinant scFv-Fc protein of the knock-in cells. Using our protocol, the knock-in cell clones could be obtained by a single transfection and a single limiting dilution using a 96-well plate, without constructing targeting vectors containing long homology arms. Thus, the study described herein provides a highly practical strategy for gene knock-in of large DNA in CHO cells, which accelerates high-throughput generation of cell lines stably producing any desired biopharmaceuticals, including huge antibody proteins.

| S-EPMC4632728 | biostudies-literature

Improved Double-Nicking Strategies for COL7A1-Editing by Homologous Recombination.

Project description:Current gene-editing approaches for treatment of recessive dystrophic epidermolysis bullosa (RDEB), an inherited, severe form of blistering skin disease, suffer from low efficiencies and safety concerns that complicate implementation in clinical settings. We present a strategy for efficient and precise repair of RDEB-associated mutations in the COL7A1 gene. We compared the efficacy of double-strand breaks (induced by CRISPR/Cas9), single nicks, or double nicks (induced by Cas9n) in mediating repair of a COL7A1 splice-site mutation in exon 3 by homologous recombination (HR). We accomplished remarkably high HR frequencies of 89% with double nicking while at the same time keeping unwanted repair outcomes, such as non-homologous end joining (NHEJ), at a minimum (11%). We also investigated the effects of subtle differences in repair template design on HR rates and found that strategic template-nicking can enhance COL7A1-editing efficiency. In RDEB patient keratinocytes, application of double-nicking led to restoration and subsequent secretion of type VII collagen at high efficiency. Comprehensive analysis of 25 putative off-target sites revealed no off-target activity for double-nicking, while usage of Cas9 resulted in 54% modified alleles at one site. Taken together, our work provides a framework for efficient, precise, and safe repair of COL7A1, which lies at the heart of a future curative therapy of RDEB.

| S-EPMC6838546 | biostudies-literature

Precise Editing at DNA Replication Forks Enables Multiplex Genome Engineering in Eukaryotes.

Project description:We describe a multiplex genome engineering technology in Saccharomyces cerevisiae based on annealing synthetic oligonucleotides at the lagging strand of DNA replication. The mechanism is independent of Rad51-directed homologous recombination and avoids the creation of double-strand DNA breaks, enabling precise chromosome modifications at single base-pair resolution with an efficiency of >40%, without unintended mutagenic changes at the targeted genetic loci. We observed the simultaneous incorporation of up to 12 oligonucleotides with as many as 60 targeted mutations in one transformation. Iterative transformations of a complex pool of oligonucleotides rapidly produced large combinatorial genomic diversity >105. This method was used to diversify a heterologous ?-carotene biosynthetic pathway that produced genetic variants with precise mutations in promoters, genes, and terminators, leading to altered carotenoid levels. Our approach of engineering the conserved processes of DNA replication, repair, and recombination could be automated and establishes a general strategy for multiplex combinatorial genome engineering in eukaryotes.

| S-EPMC5995112 | biostudies-literature

Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination.

Project description:Direct cloning represents the most efficient strategy to access the vast number of uncharacterized natural product biosynthetic gene clusters (BGCs) for the discovery of novel bioactive compounds. However, due to their large size, repetitive nature, or high GC-content, large-scale cloning of these BGCs remains an overwhelming challenge. Here, we report a scalable direct cloning method named Cas12a-assisted precise targeted cloning using in vivo Cre-lox recombination (CAPTURE) which consists of Cas12a digestion, a DNA assembly approach termed T4 polymerase exo + fill-in DNA assembly, and Cre-lox in vivo DNA circularization. We apply this method to clone 47 BGCs ranging from 10 to 113 kb from both Actinomycetes and Bacilli with ~100% efficiency. Heterologous expression of cloned BGCs leads to the discovery of 15 previously uncharacterized natural products including six cyclic head-to-tail heterodimers with a unique 5/6/6/6/5 pentacyclic carbon skeleton, designated as bipentaromycins A-F. Four of the bipentaromycins show strong antimicrobial activity to both Gram-positive and Gram-negative bacteria such as methicillin-resistant Staphylococcus aureus, vancomycinresistant Enterococcus faecium, and bioweapon Bacillus anthracis. Due to its robustness and efficiency, our direct cloning method coupled with heterologous expression provides an effective strategy for large-scale discovery of novel natural products.

| S-EPMC7896053 | biostudies-literature

Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination.

Project description:Cas9 is an RNA-guided double-stranded DNA nuclease that participates in clustered regularly interspaced short palindromic repeats (CRISPR)-mediated adaptive immunity in prokaryotes. CRISPR-Cas9 has recently been used to generate insertion and deletion mutations in Caenorhabditis elegans, but not to create tailored changes (knock-ins). We show that the CRISPR-CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome. The targeted double-strand breaks generated by CRISPR are substrates for transgene-instructed gene conversion. This allows customized changes in the C. elegans genome by homologous recombination: sequences contained in the repair template (the transgene) are copied by gene conversion into the genome. The possibility to edit the C. elegans genome at selected locations will facilitate the systematic study of gene function in this widely used model organism.

| S-EPMC3814388 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data