‘Cold shock’ increases the frequency of homology directed repair gene editing in induced pluripotent stem cells

Guo, Qi; Mintier, Gabe; Ma-Edmonds, Manling; Storton, Donna; Wang, X; Xiao, Xia; Kienzle, Bernadette; Zhao, David; Feder, John N.

doi:10.1038/s41598-018-20358-5

Cited by 61 publications

(61 citation statements)

References 24 publications

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“…The The distribution of efficiencies is shown in Figure 1, clearly demonstrating the 3-fold lower efficiency for HDR (median = 0.199) compared to NHEJ (median = 0.606) when choosing targets traditionally. These values are comparable to previous work (21)(22)(23).…”

Section: A Dataset Of Genome-wide Hdr Efficienciessupporting

confidence: 92%

Unlocking HDR-mediated Nucleotide Editing by identifying high-efficiency target sites using machine learning

O’Brien

Wilson

Burgio

et al. 2018

Preprint

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Editing individual nucleotides is a crucial component for validating genomic disease association. It currently is hampered by CRISPR-Cas-mediated "base editing" being limited to certain nucleotide changes, and only achievable within a small window around CRISPR-Cas target sites. The more versatile alternative, HDR (homology directed repair), has a 4fold lower efficiency with known optimization factors being largely immutable in experiments.Here, we investigated the variable efficiency-governing factors on a novel mouse dataset using machine learning. We found the sequence composition of the repair template (ssODN) to be a governing factor, where different regions of the ssODN have variable influence, which reflects the underlying biophysical mechanism. Our model improves HDR efficiency by 83% compared to traditionally chosen targets. Using our findings, we develop CUNE (Computational Universal Nucleotide Editor), which enables users to identify and design the optimal targeting strategy using traditional base editing or --for-the-first-time --HDRmediated nucleotide changes. CUNE can be run via the web at: https://gt-scan.net/cune

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Section: A Dataset Of Genome-wide Hdr Efficienciessupporting

confidence: 92%

Unlocking HDR-mediated Nucleotide Editing by identifying high-efficiency target sites using machine learning

O’Brien

Wilson

Burgio

et al. 2018

Preprint

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“…Our results corroborate these findings and reveal that cut-to-insertion-site distance is a critical factor for successful knock-in. Lower knock-in efficiency achieved with the distal-cutting gRNAs could be attributed to ‘partial HDR’ events ( Guo et al, 2018 ), which are not detectable in our ICC end-point assays. Although beyond the scope of our present study, whole-genome sequencing of the CRISPR-edited bulk cells can shed light on the extent of ‘partial HDR’ events in epitope knock-in experiments.…”

Section: Discussionmentioning

confidence: 83%

An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein

Dewari

Southgate

Mccarten

et al. 2018

eLife

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CRISPR/Cas9 can be used for precise genetic knock-in of epitope tags into endogenous genes, simplifying experimental analysis of protein function. However, Cas9-assisted epitope tagging in primary mammalian cell cultures is often inefficient and reliant on plasmid-based selection strategies. Here, we demonstrate improved knock-in efficiencies of diverse tags (V5, 3XFLAG, Myc, HA) using co-delivery of Cas9 protein pre-complexed with two-part synthetic modified RNAs (annealed crRNA:tracrRNA) and single-stranded oligodeoxynucleotide (ssODN) repair templates. Knock-in efficiencies of ~5–30%, were achieved without selection in embryonic stem (ES) cells, neural stem (NS) cells, and brain-tumor-derived stem cells. Biallelic-tagged clonal lines were readily derived and used to define Olig2 chromatin-bound interacting partners. Using our novel web-based design tool, we established a 96-well format pipeline that enabled V5-tagging of 60 different transcription factors. This efficient, selection-free and scalable epitope tagging pipeline enables systematic surveys of protein expression levels, subcellular localization, and interactors across diverse mammalian stem cells.

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“…For example, if the eggs are kept at 6 °C, it takes about three months until hatching, in stark contrast to two days for zebrafish. Interestingly, it has been reported that cold shock-treatment increases the frequency of HDR gene editing in induced pluripotent stem cells 51 .…”

Section: Discussionmentioning

confidence: 99%

Indel locations are determined by template polarity in highly efficient in vivo CRISPR/Cas9-mediated HDR in Atlantic salmon

Straume

Kjærner-Semb

Skaftnesmo

et al. 2020

Sci Rep

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Precise gene editing such as CRISPR/Cas9-mediated homology directed repair (HDR) can increase our understanding of gene function and improve traits of importance for aquaculture. This fine-tuned technology has not been developed for farmed fish including Atlantic salmon. We performed knock-in (KI) of a FLAG element in the slc45a2 gene in salmon using sense (S), anti-sense (AS) and doublestranded (ds) oligodeoxynucleotide (ODN) templates with short (24/48/84 bp) homology arms. We show in vivo ODN integration in almost all the gene edited animals, and demonstrate perfect HDR rates up to 27% in individual F0 embryos, much higher than reported previously in any fish. HDR efficiency was dependent on template concentration, but not homology arm length. Analysis of imperfect HDR variants suggest that repair occurs by synthesis-dependent strand annealing (SDSA), as we show for the first time in any species that indel location is dependent on template polarity. Correct ODN polarity can be used to avoid 5′-indels interrupting the reading frame of an inserted sequence and be of importance for HDR template design in general. Aquaculture continues to grow faster than any other major food production sector and is quickly becoming the main source of seafood in human diets. In this context, Norway is the largest producer of farmed Atlantic salmon (Salmo salar) worldwide. In later years, the production of salmon in Norway has ceased to grow due to sustainability challenges linked to open sea-cage rearing. Genetic introgression of farmed salmon into wild stocks and the marine parasite, salmon louse, are recognized as the two major concerns 1. The high prevalence of salmon lice in most Norwegian fjords, due to open sea-cage farming, cause high lethality in wild salmonids and is hindering expansion of sea-cage farming. The consequences of genetic introgression caused by escapees remain uncertain, but existing knowledge indicates that it may lead to changes in life-history traits, with potential ecological impacts 2-5. Sequencing of the salmon genome 6 has permitted more detailed studies on the link between genes and key traits, and we and others have shown that single nucleotide polymorphisms (SNPs) to a certain degree can explain the time of maturity 1 and disease resistance 7,8. In this context, New Breeding Technologies (NBTs) by gene editing may offer a solution to some of the problems in salmon farming, with a possible production of salmon displaying traits such as disease resistance and sterility 9-12. We have previously demonstrated the feasibility of double allelic KO in F0 salmon using CRISPR/Cas9, by targeting genes essential for pigmentation 9 , elongation of polyunsaturated fatty acids 13 and reproduction 10. At the same time, CRISPR/Cas9 KO-mutations targeting various phenotypes have been shown by others in several farmed fish species such as tilapia 14-21 , sea bream 22 , sterlet 23 , channel catfish 24,25 , southern catfish 26 , common carp 27 , sturgeon 28 and rainbow trout 29. CRISPR/Cas9 KOs are produced by a Cas9-...

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‘Cold shock’ increases the frequency of homology directed repair gene editing in induced pluripotent stem cells

Cited by 61 publications

References 24 publications

Unlocking HDR-mediated Nucleotide Editing by identifying high-efficiency target sites using machine learning

Unlocking HDR-mediated Nucleotide Editing by identifying high-efficiency target sites using machine learning

An efficient and scalable pipeline for epitope tagging in mammalian stem cells using Cas9 ribonucleoprotein

Indel locations are determined by template polarity in highly efficient in vivo CRISPR/Cas9-mediated HDR in Atlantic salmon

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