scholarly journals A heterodimer of evolved designer-recombinases precisely excises a human genomic DNA locus

2019 ◽  
Vol 48 (1) ◽  
pp. 472-485 ◽  
Author(s):  
Felix Lansing ◽  
Maciej Paszkowski-Rogacz ◽  
Lukas Theo Schmitt ◽  
Paul Martin Schneider ◽  
Teresa Rojo Romanos ◽  
...  
Keyword(s):  
Human Genome ◽  
Genome Editing ◽  
Large Scale ◽  
Genome Engineering ◽  
Genomic Sequence ◽  
Safe Alternative ◽  
Protein Coding ◽  
Dna Binding Specificity ◽  
Human Genomic ◽  
Human Genomic Sequence

Abstract Site-specific recombinases (SSRs) such as the Cre/loxP system are useful genome engineering tools that can be repurposed by altering their DNA-binding specificity. However, SSRs that delete a natural sequence from the human genome have not been reported thus far. Here, we describe the generation of an SSR system that precisely excises a 1.4 kb fragment from the human genome. Through a streamlined process of substrate-linked directed evolution we generated two separate recombinases that, when expressed together, act as a heterodimer to delete a human genomic sequence from chromosome 7. Our data indicates that designer-recombinases can be generated in a manageable timeframe for precision genome editing. A large-scale bioinformatics analysis suggests that around 13% of all human protein-coding genes could be targetable by dual designer-recombinase induced genomic deletion (dDRiGD). We propose that heterospecific designer-recombinases, which work independently of the host DNA repair machinery, represent an efficient and safe alternative to nuclease-based genome editing technologies.

Computational Comparison of Human Genomic Sequence Assemblies for a Region of Chromosome 4

2002 ◽  
Vol 12 (3) ◽  
pp. 424-429 ◽  
Author(s):  
C. A.M. Semple ◽  
S. W. Morris ◽  
D. J. Porteous ◽  
K. L. Evans
Keyword(s):  
Genomic Sequence ◽  
Chromosome 4 ◽  
Human Genomic ◽  
Computational Comparison ◽  
Human Genomic Sequence

scholarly journals Large-scale discovery of recombinases for integrating DNA into the human genome

2021 ◽  
Author(s):  
Matthew G Durrant ◽  
Alison Fanton ◽  
Josh Tycko ◽  
Michaela Hinks ◽  
Sita Chandrasekaran ◽  
...  
Keyword(s):  
Human Genome ◽  
Large Scale ◽  
Genome Engineering ◽  
Human Cells ◽  
Sequencing Data ◽  
Dna Breaks ◽  
Serine Recombinases ◽  
Attachment Sites ◽  
Dna Attachment ◽  
Computational Discovery

Recent microbial genome sequencing efforts have revealed a vast reservoir of mobile genetic elements containing integrases that could be useful genome engineering tools. Large serine recombinases (LSRs), such as Bxb1 and PhiC31, are bacteriophage-encoded integrases that can facilitate the insertion of phage DNA into bacterial genomes. However, only a few LSRs have been previously characterized and they have limited efficiency in human cells. Here, we developed a systematic computational discovery workflow that searches across the bacterial tree of life to expand the diversity of known LSRs and their cognate DNA attachment sites by >100-fold. We validated this approach via experimental characterization of LSRs, leading to three classes of LSRs distinguished from one another by their efficiency and specificity. We identify landing pad LSRs that efficiently integrate into native attachment sites in a human cell context, human genome-targeting LSRs with computationally predictable pseudosites, and multi-targeting LSRs that can unidirectionally integrate cargos with similar efficiency and superior specificity to commonly used transposases. LSRs from each category were functionally characterized in human cells, overall achieving up to 7-fold higher plasmid recombination than Bxb1 and genome insertion efficiencies of 40-70% with cargo sizes over 7 kb. Overall, we establish a paradigm for the large-scale discovery of microbial recombinases directly from sequencing data and the reconstruction of their target sites. This strategy provided a rich resource of over 60 experimentally characterized LSRs that can function in human cells and thousands of additional candidates for large-payload genome editing without double-stranded DNA breaks.

scholarly journals Analysis of EST-Driven Gene Annotation in Human Genomic Sequence

1998 ◽  
Vol 8 (4) ◽  
pp. 362-376 ◽  
Author(s):  
L. Charles Bailey ◽  
David B. Searls ◽  
G. Christian Overton
Keyword(s):  
Genomic Sequence ◽  
Gene Annotation ◽  
Human Genomic ◽  
Human Genomic Sequence

scholarly journals Cloning and Characterization of a Human Genomic Sequence that Alleviates Repeat-Induced Gene Silencing

PLoS ONE ◽  
2016 ◽  
Vol 11 (4) ◽  
pp. e0153338 ◽  
Author(s):  
Miki Fukuma ◽  
Yuto Ganmyo ◽  
Osamu Miura ◽  
Takashi Ohyama ◽  
Noriaki Shimizu
Keyword(s):  
Gene Silencing ◽  
Genomic Sequence ◽  
Human Genomic ◽  
Human Genomic Sequence

scholarly journals Double nicking by RNA-directed Cascade-nCas3 for high-efficiency large-scale genome engineering

Open Biology ◽  
2022 ◽  
Vol 12 (1) ◽  
Author(s):  
Yile Hao ◽  
Qinhua Wang ◽  
Jie Li ◽  
Shihui Yang ◽  
Yanli Zheng ◽  
...  
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Genome Engineering ◽  
High Efficiency ◽  
Helicase Domain ◽  
Type I ◽  
Strand Breaks ◽  
Gene Insertion ◽  
Type V

New CRISPR-based genome editing technologies are developed to continually drive advances in life sciences, which, however, are predominantly derived from systems of Type II CRISPR-Cas9 and Type V CRISPR-Cas12a for eukaryotes. Here we report a novel CRISPR-n(nickase)Cas3 genome editing tool established upon a Type I-F system. We demonstrate that nCas3 variants can be created by alanine-substituting any catalytic residue of the Cas3 helicase domain. While nCas3 overproduction via plasmid shows severe cytotoxicity, an in situ nCas3 introduces targeted double-strand breaks, facilitating genome editing without visible cell killing. By harnessing this CRISPR-nCas3 in situ gene insertion, nucleotide substitution and deletion of genes or genomic DNA stretches can be consistently accomplished with near-100% efficiencies, including simultaneous removal of two large genomic fragments. Our work describes the first establishment of a CRISPR-nCas3-based genome editing technology, thereby offering a simple, yet useful approach to convert the naturally most abundantly occurring Type I systems into advanced genome editing tools to facilitate high-throughput prokaryotic engineering.

scholarly journals Double nicking by RNA-directed Cascade-nCas3 for high-efficiency large-scale genome engineering

2021 ◽  
Author(s):  
Yile Hao ◽  
Qinhua Wang ◽  
Jie Li ◽  
Shihui Yang ◽  
Lixin Ma ◽  
...  
Keyword(s):  
Genome Editing ◽  
Large Scale ◽  
Genome Engineering ◽  
High Efficiency ◽  
Double Strand Breaks ◽  
Helicase Domain ◽  
Type I ◽  
Strand Breaks ◽  
Type V

New CRISPR-based genome editing technologies are developed to continuedly drive advances in life sciences, which, however, are predominantly derived from systems of Type II CRISPR-Cas9 and Type V CRISPR-Cas12a for eukaryotes. Here we report a novel CRISPR-n(nickase)Cas3 genome editing tool established upon an endogenous Type I system of Zymomonas mobilis. We demonstrate that nCas3 variants can be created by alanine-substituting any catalytic residue of the Cas3 helicase domain. While nCas3 overproduction via plasmid shows severe cytotoxicity; an in situ nCas3 introduces targeted double-strand breaks, facilitating genome editing, without visible cell killing. By harnessing this CRISPR-nCas3, deletion of genes or genomic DNA stretches can be consistently accomplished with near-100% efficiencies, including simultaneous removal of two large genomic fragments. Our work describes the first establishment of a CRISPR-nCas3-based genome editing technology, thereby offering a simple, easy, yet useful approach to convert many endogenous Type I systems into advanced genome editing tools. We envision that many CRISPR-nCas3-based toolkits would be soon available for various industrially important non-model bacteria that carry active Type I systems to facilitate high-throughput prokaryotic engineering.

scholarly journals The shrinking human protein coding complement: are there fewer than 20,000 genes?

2014 ◽  
Author(s):  
Iakes Ezkurdia ◽  
David Juan ◽  
Jose Manuel Rodriguez ◽  
Adam Frankish ◽  
Mark Deikhans ◽  
...  
Keyword(s):  
Protein Expression ◽  
Human Genome ◽  
Genome Annotation ◽  
Large Scale ◽  
Cellular Protein ◽  
Human Protein ◽  
Protein Coding ◽  
Detection Rates ◽  
Protein Coding Genes ◽  
Peptide Mass

Determining the full complement of protein-coding genes is a key goal of genome annotation. The most powerful approach for confirming protein coding potential is the detection of cellular protein expression through peptide mass spectrometry experiments. Here we map the peptides detected in 7 large-scale proteomics studies to almost 60% of the protein coding genes in the GENCODE annotation the human genome. We find that conservation across vertebrate species and the age of the gene family are key indicators of whether a peptide will be detected in proteomics experiments. We find peptides for most highly conserved genes and for practically all genes that evolved before bilateria. At the same time there is almost no evidence of protein expression for genes that have appeared since primates, or for genes that do not have any protein-like features or cross-species conservation. We identify 19 non-protein-like features such as weak conservation, no protein features or ambiguous annotations in major databases that are indicators of low peptide detection rates. We use these features to describe a set of 2,001 genes that are potentially non-coding, and show that many of these genes behave more like non-coding genes than protein-coding genes. We detect peptides for just 3% of these genes. We suggest that many of these 2,001 genes do not code for proteins under normal circumstances and that they should not be included in the human protein coding gene catalogue. These potential non-coding genes will be revised as part of the ongoing human genome annotation effort.

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