Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9

Prykhozhij, Sergey V.; Fuller, Charlotte; Steele, Shelby L.; Veinotte, Chansey J.; Razaghi, Babak; Robitaille, Johane; McMaster, Christopher R.; Shlien, Adam; Malkin, David; Berman, Jason N.

doi:10.1093/nar/gky674

Cited by 54 publications

(45 citation statements)

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“…[37][38][39][40]42,44,46 . Only three studies in zebrafish have used NGS, showing lower perfect repair rates of 1-4% 41 , 1.7-3.5% 45 and <1% 43 .…”

Section: Discussionmentioning

confidence: 99%

“…With the exception of very low efficiency KI of a gene encoding red fluorescent protein in rohu carp 34 , such advanced and fine-tuned genome editing has not been developed for farmed fish, and it may be useful to learn from protocols already established in model fish species. While only a few studies have reported HDR in medaka 35,36 , several KI-strategies have been reported in zebrafish using either donor plasmids [37][38][39] or single-stranded oligodeoxynucleotides (ssODNs) [40][41][42][43][44][45] , or both 46 . Knowledge from other fish studies are somewhat inconclusive when it comes to deciding the strategy for applying HDR in salmon, as there is a lack of consensus regarding the impact of different repair templates, homology arm length and strand complementarity in the above-mentioned studies.…”

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confidence: 99%

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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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“…[37][38][39][40]42,44,46 . Only three studies in zebrafish have used NGS, showing lower perfect repair rates of 1-4% 41 , 1.7-3.5% 45 and <1% 43 .…”

Section: Discussionmentioning

confidence: 99%

mentioning

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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“…However, the incorporation of restriction enzyme site efficiency was still only~5% (8 out of 186 fish) [2], and subsequently just a single founder (1/46) was identified for the smaller template and three founders (3/77) for the longer template, both using ssODN to introduce point mutations [13]. Anti-sense asymmetric oligo design was also found to be possible in zebrafish achieving around 2% efficiency of correct HDR knock-in as assessed by high-throughput sequencing analysis [14].…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas9-mediated precise genome modification by a long ssDNA template in zebrafish

Bai

Liu

et al. 2020

BMC Genomics

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Background: Gene targeting by homology-directed repair (HDR) can precisely edit the genome and is a versatile tool for biomedical research. However, the efficiency of HDR-based modification is still low in many model organisms including zebrafish. Recently, long single-stranded DNA (lssDNA) molecules have been developed as efficient alternative donor templates to mediate HDR for the generation of conditional mouse alleles. Here we report a method, zLOST (zebrafish long single-stranded DNA template), which utilises HDR with a long singlestranded DNA template to produce more efficient and precise mutations in zebrafish. Results: The efficiency of knock-ins was assessed by phenotypic rescue at the tyrosinase (tyr) locus and confirmed by sequencing. zLOST was found to be a successful optimised rescue strategy: using zLOST containing a tyr repair site, we restored pigmentation in at least one melanocyte in close to 98% of albino tyr 25del/25del embryos, although more than half of the larvae had only a small number of pigmented cells. Sequence analysis showed that there was precise HDR dependent repair of the tyr locus in these rescued pigmented embryos. Furthermore, quantification of zLOST knock-in efficiency at the rps14, nop56 and th loci by next generation sequencing demonstrated that zLOST showed a clear improvement. We utilised the HDR efficiency of zLOST to precisely model specific human disease mutations in zebrafish with ease. Finally, we determined that this method can achieve a germline transmission rate of up to 31.8%. Conclusions: In summary, these results show that zLOST is a useful method of zebrafish genome editing, particularly for generating desired mutations by targeted DNA knock-in through HDR.

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“…While the mutagenesis method employed by us is likely feasible for the majority of MOs targeting 5' UTRs, there will inevitably be a subset where PAM sites will be absent or located closer to the middle or the 3' of the target site. In such cases, oligonucleotide-mediated repair of double strand breaks can be used to engineer desired mutations (Bedell et al, 2012;Burg et al, 2018;Burg et al, 2016;Dong et al, 2014;Gagnon et al, 2014;Gibb et al, 2018;Hruscha et al, 2013;Prykhozhij et al, 2018).…”

Section: Suppression Of the B-catenin Morphant Phenotype By Maternal mentioning

confidence: 99%

Deletion of MOrpholino Binding Sites (DeMOBS) to Assess Specificity of Morphant Phenotypes

Cunningham

Bellipanni

Habas

et al. 2020

Preprint

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Two complimentary approaches are widely used to study gene function in zebrafish:induction of genetic mutations, usually using targeted nucleases such as CRISPR/Cas9, and suppression of gene expression, typically using Morpholino oligomers. Neither method is perfect. Morpholinos (MOs) sometimes produce offtarget or toxicity-related effects that can be mistaken for true phenotypes.Conversely, genetic mutants can be subject to compensation, or may fail to yield a null phenotype due to leakiness. When discrepancy between mutant and morpholino-induced (morphant) phenotypes is observed, experimental validation of such phenotypes becomes very labor intensive. We have developed a simple genetic method to differentiate between genuine morphant phenotypes and those produced due to off-target effects. We speculated that indels within 5' untranslated regions would be unlikely to have a significant negative effect on gene expression.Mutations induced within a MO target site would result in a Morpholino-refractive allele thus suppressing true MO phenotypes whilst non-specific phenotypes would remain. We tested this hypothesis on one gene with an exclusively zygotic function, tbx5a, and one gene with strong maternal effect, ctnnb2. We found that indels within the Morpholino binding site are indeed able to suppress both zygotic and maternal morphant phenotypes. We also observed that the ability of such indels to suppress Morpholino phenotypes does depend on the size and the location of the deletion.Nonetheless, mutating the morpholino binding sites in both maternal and zygotic genes can ascertain the specificity of morphant phenotypes.

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Optimized knock-in of point mutations in zebrafish using CRISPR/Cas9

Cited by 54 publications

References 1 publication

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

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

CRISPR/Cas9-mediated precise genome modification by a long ssDNA template in zebrafish

Deletion of MOrpholino Binding Sites (DeMOBS) to Assess Specificity of Morphant Phenotypes

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