Making ends meet: targeted integration of DNA fragments by genome editing

Targeted insertion of large pieces of DNA is an important goal of genetic engineering. However, this goal has been elusive since classical methods for homology-directed repair are inefficient and often not feasible in many systems. Recent advances are described here that enable site-specific genomic...

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Bibliographic Details
Published in:Chromosoma 2018-12, Vol.127 (4), p.405-420
Main Authors: Yamamoto, Yutaka, Gerbi, Susan A.
Format: Article
Language:English
Subjects:
Animal Genetics and Genomics
Animals
Biochemistry
Biomedical and Life Sciences
Cell Biology
Chromosomes
CRISPR-Cas Systems
Deoxyribonucleic acid
Developmental Biology
DNA
DNA Breaks, Double-Stranded
DNA End-Joining Repair
DNA Repair
Efficiency
Eukaryotic Microbiology
Gene Editing - methods
Genetic engineering
Genome
Genome editing
Genomes
Homology
Human Genetics
Humans
Insertion
Life Sciences
Plasmids - genetics
Recombination, Genetic
Review
RNA, Guide, CRISPR-Cas Systems
Transcription Activator-Like Effector Nucleases - genetics
Zinc Finger Nucleases - genetics
Zinc Finger Nucleases - metabolism
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Summary:Targeted insertion of large pieces of DNA is an important goal of genetic engineering. However, this goal has been elusive since classical methods for homology-directed repair are inefficient and often not feasible in many systems. Recent advances are described here that enable site-specific genomic insertion of relatively large DNA with much improved efficiency. Using the preferred repair pathway in the cell of nonhomologous end-joining, DNA of up to several kb could be introduced with remarkably good precision by the methods of HITI and ObLiGaRe with an efficiency up to 30–40%. Recent advances utilizing homology-directed repair (methods of PITCh; short homology arms including ssODN; 2H2OP) have significantly increased the efficiency for DNA insertion, often to 40–50% or even more depending on the method and length of DNA. The remaining challenges of integration precision and off-target site insertions are summarized. Overall, current advances provide major steps forward for site-specific insertion of large DNA into genomes from a broad range of cells and organisms.
ISSN:0009-5915
1432-0886
DOI:10.1007/s00412-018-0677-6