Simultaneous precise editing of multiple genes in human cells

Riesenberg, Stephan; Chintalapati, Manjusha; Macak, Dominik; Kanis, Philipp; Maričić, Tomislav; Pääbo, Svante

doi:10.1093/nar/gkz669

Cited by 96 publications

(108 citation statements)

References 43 publications

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“…All methods are very effective compared with traditional methods of gene targeting in zygotes. Perhaps future avenues to even more efficient gene targeting lie in the application of small molecule activators for HDR [189][190][191]. 1 Conditional: A critical exon was flanked by loxP sites, so as to produce a Cre-dependent knockout allele.…”

Section: Resultsmentioning

confidence: 99%

Principles of Genetic Engineering

Lanigan

Kopera

Saunders

2020

Genes

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Genetic engineering is the use of molecular biology technology to modify DNA sequence(s) in genomes, using a variety of approaches. For example, homologous recombination can be used to target specific sequences in mouse embryonic stem (ES) cell genomes or other cultured cells, but it is cumbersome, poorly efficient, and relies on drug positive/negative selection in cell culture for success. Other routinely applied methods include random integration of DNA after direct transfection (microinjection), transposon-mediated DNA insertion, or DNA insertion mediated by viral vectors for the production of transgenic mice and rats. Random integration of DNA occurs more frequently than homologous recombination, but has numerous drawbacks, despite its efficiency. The most elegant and effective method is technology based on guided endonucleases, because these can target specific DNA sequences. Since the advent of clustered regularly interspaced short palindromic repeats or CRISPR/Cas9 technology, endonuclease-mediated gene targeting has become the most widely applied method to engineer genomes, supplanting the use of zinc finger nucleases, transcription activator-like effector nucleases, and meganucleases. Future improvements in CRISPR/Cas9 gene editing may be achieved by increasing the efficiency of homology-directed repair. Here, we describe principles of genetic engineering and detail: (1) how common elements of current technologies include the need for a chromosome break to occur, (2) the use of specific and sensitive genotyping assays to detect altered genomes, and (3) delivery modalities that impact characterization of gene modifications. In summary, while some principles of genetic engineering remain steadfast, others change as technologies are ever-evolving and continue to revolutionize research in many fields.

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Section: Resultsmentioning

confidence: 99%

Principles of Genetic Engineering

Lanigan

Kopera

Saunders

2020

Genes

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“…We hypothesised that successful CRISPR-del requires paired DSBs to co-occur before NHEJ has time to act, and thus may be enhanced by pharmacological inhibition of DNA-PK. This is initially counter-intuitive, as DNA-PK is a necessary step in the NHEJ pathway upon which CRISPR-del relies, and its inhibition is widely used to promote HDR 37,40,44,45 . However, rather than permanently blocking NHEJ, our protocol slows the kinetics of NHEJ for a defined period while DSBs are taking place.…”

Section: Discussionmentioning

confidence: 99%

“…We tested DNA-PK, a DNA end-binding factor at the first step of NHEJ pathway, for which a number of small-molecule inhibitors are available 54 . We began by treating HeLa cells with the inhibitor M3814 (IC50=3nM) 45,55,56 at two concentrations (300 nM and 900 nM).…”

Section: Temporary Inhibition Of Dna-pk During Dsb Formation Increasementioning

confidence: 99%

“…Here, editing events are rare, and HR is the rate-limitingstep 38,39 . The two principal strategies to boost efficiency are: (1) direct stimulation of homology directed repair (HDR) 37,40-42 ; (2) suppression of the competing NHEJ pathway at early stages through inhibition of Ku70/80 complex 37,40,43 or DNA-dependent protein kinase (DNA-PK) 37,40,44,45 , or at late phases, via Ligase IV (LigIV) inhibition 37,40,46,47 . To date, however, there are no reported methods for pharmacological enhancement of CRISPR-del.…”

Section: Introductionmentioning

confidence: 99%

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Enhancing CRISPR deletion via pharmacological delay of DNA-PK

Bosch

Medová

Esposito

et al. 2020

Preprint

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CRISPR-Cas9 deletion (CRISPR-del) is the leading approach for eliminating DNA from mammalian cells and underpins a variety of genome-editing applications. Target DNA, defined by a pair of double strand breaks (DSBs), is removed during non-homologous end-joining (NHEJ). However, the low efficiency of CRISPR-del results in laborious experiments and false negative results. Using an endogenous reporter system, we demonstrate that temporary inhibition of DNA-dependent protein kinase (DNA-PK) -an early step in NHEJ -yields up to 17-fold increase in DNA deletion. This is observed across diverse cell lines, gene delivery methods, commercial inhibitors and guide RNAs, including those that otherwise display negligible activity. Importantly, the method is compatible with pooled functional screens employing lentivirally-delivered guide RNAs. Thus, delaying the kinetics of NHEJ relative to DSB formation is a simple and effective means of enhancing CRISPR-deletion.

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“…A number of approaches have been devised to shift the balance between competing NHEJ and HDR pathways toward the latter. These include the use of chemical and biochemical compounds to interfere with the DNA repair machineryeither by blocking the NHEJ pathway, with DNA-dependent protein kinase inhibitors like M3814 [41] , or by stimulating the HDR pathway, with mRNA from HR effectors like RAD51 [42] or with stimulating molecules like RS-1 [43] . As pharmacological inhibitors can be cytotoxic and yield variable efficiency, HDR can also be enhanced by coupling Cas9 expression to cell cycle effectors at the late S to G2 phase [44,45] or by tethering the DNA template to the gRNA/Cas9 RNP complex, juxtaposing the template DNA to the DSB repair site [46][47][48] .…”

Section: Optimizing Hdr Outcomesmentioning

confidence: 99%

Embryo-mediated genome editing for accelerated genetic improvement of livestock

McLean¹,

Oback²,

Laible³

2020

Front. Agr. Sci. Eng.

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Selecting beneficial DNA variants is the main goal of animal breeding. However, this process is inherently inefficient because each animal only carries a fraction of all desirable variants. Genome editing technology with its ability to directly introduce beneficial sequence variants offers new opportunities to modernize animal breeding by overcoming this biological limitation and accelerating genetic gains. To realize rapid genetic gain, precise edits need to be introduced into genomicallyselected embryos, which minimizes the genetic lag. However, embryo-mediated precision editing by homology-directed repair (HDR) mechanisms is currently an inefficient process that often produces mosaic embryos and greatly limits the numbers of available edited embryos. This review provides a summary of genome editing in bovine embryos and proposes an embryo-mediated accelerated breeding scheme that overcomes the present efficiency limitations of HDR editing in bovine embryos. It integrates embryo-based genomic selection with precise multi-editing and uses embryonic cloning with elite edited blastomeres or embryonic pluripotent stem cells to resolve mosaicism, enable multiplex editing and multiply rare elite genotypes. Such a breeding strategy would enable a more targeted, accelerated approach for livestock improvement that allows stacking of beneficial variants, even including novel traits from outside the breeding population, in the most recent elite genetic background, essentially within a single generation.

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Simultaneous precise editing of multiple genes in human cells

Cited by 96 publications

References 43 publications

Principles of Genetic Engineering

Principles of Genetic Engineering

Enhancing CRISPR deletion via pharmacological delay of DNA-PK

Embryo-mediated genome editing for accelerated genetic improvement of livestock

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