Simplified pipelines for genetic engineering of mammalian embryos by CRISPR-Cas9 electroporation†

Miao, De-Qiang; Giassetti, Mariana Ianello; Ciccarelli, Michela; Lopez-Biladeau, Blanca; Oatley, Jon M.

doi:10.1093/biolre/ioz075

Cited by 35 publications

(36 citation statements)

References 28 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…In vitro produced embryos, as were used in this study, have reduced development potential relative to in vivo produced embryos 20 . Electroporation offers a promising approach to deliver editing reagents to zygotes without the physical damage of the microinjection technique 24,25 which may increase viability, although to date efficiencies of HDR editing in electroporated bovine embryos are low 26 .…”

Section: Discussionmentioning

confidence: 99%

Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos

Owen

Hennig

McNabb

et al. 2020

Preprint

View full text Add to dashboard Cite

11Introducing useful traits into livestock breeding programs through gene knock-ins has 12 proven challenging. Typically, targeted insertions have been performed in cell lines, followed by 13 somatic cell nuclear transfer cloning, which can be inefficient. An alternative is to introduce 14 genome editing reagents and a homologous recombination (HR) donor template into embryos to 15 trigger homology-directed repair (HDR). However, the HR pathway is primarily restricted to 16 actively dividing cells (S/G2-phase) and its efficiency is low in zygotes, especially for the 17 introduction of large DNA sequences. The homology-mediated end joining (HMEJ)-based 18 strategy harnesses HDR by direct injection of embryos, and has been shown to have an improved 19 knock-in efficiency in non-dividing cells. The knock-in efficiency for a 1.8kb gene was contrasted 20 when combining a gRNA/Cas9 ribonucleoprotein complex with either a traditional HR donor 21 template, or a HMEJ template in bovine zygotes. The HMEJ template resulted in a significantly 22 higher rate of gene knock-in as compared to the HR template (37.0% and 13.8%; P < 0.05). 23Additionally, more than a third of the knock-in embryos (36.9%) were non-mosaic. This approach 24 will facilitate the one-step introduction of gene constructs at a specific location of the bovine 25 genome and contribute to the next generation of elite cattle. 26 27Many attempts have been made to increase the rate of homologous recombination (HR) or 47 decrease the rate of non-homologous end joining (NHEJ) for gene insertion when using the 48 CRISPR/Cas9 system via CPI of zygotes 11 . However, these approaches have been unsuccessful in 49 bovine embryos as HR is primarily restricted to actively dividing cells 12 . Alternative homology 50 directed repair (HDR) approaches have been utilized for KI using a donor template via the 51 homology-mediated end joining (HMEJ) method 13 . This method has been shown to be active in 52 gametes and early stage 1-cell embryos, in which proteins necessary for pushing DNA repair 53 machinery towards the end-joining pathways are at their highest concentration 11 . While NHEJ 54 utilizes the Ku proteins and a ligase to mend the double-strand break (DSB) by blunt end ligation, 55 the HMEJ approach utilizes proteins for resection of the 5' ends and annealing of homologous 56 regions between the DSB and donor template similar to that in the microhomology-mediated end 57 joining (MMEJ) pathway 14 . 58In this study, we employed the HMEJ method for the precise insertion of the sex-59 determining region Y (SRY) gene into a region 10kb downstream of the zinc finger, X-linked (ZFX) 60 gene on the X chromosome of bovine embryos by injecting either in vitro matured oocytes prior 61 to in vitro fertilization, or presumptive zygotes 6hpi. We used two donor templates to compare KI 62 efficiency using the HMEJ and HR approaches, and show an increased rate of gene insertion at 63 the target location when using the HMEJ donor template. 64 RESULTS 65 Guide-RNA design and testing 66...

show abstract

Section: Discussionmentioning

confidence: 99%

Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos

Owen

Hennig

McNabb

et al. 2020

Preprint

View full text Add to dashboard Cite

show abstract

“…Cytoplasmic injection (CPI) has been the go-to technique for delivering genome editing components directly into livestock zygotes. Electroporation has only recently begun to show its potential for this purpose with effective introduction of indel mutations (through NHEJ) into zygotes of pigs and cattle (Hirata et al, 2019;Miao et al, 2019;Tanihara et al, 2018Tanihara et al, , 2016. Unlike CPI, where a needle is used to deliver genome editing reagents into zygotes individually, electroporation allows the manipulation of zygotes en masse, reducing the time and expertise required.…”

Section: Nuclease and Repair Template Deliverymentioning

confidence: 99%

Genome editing approaches to augment livestock breeding programs

Bishop

Eenennaam

2020

Journal of Experimental Biology

View full text Add to dashboard Cite

The prospect of genome editing offers a number of promising opportunities for livestock breeders. Firstly, these tools can be used in functional genomics to elucidate gene function, and identify causal variants underlying monogenic traits. Secondly, they can be used to precisely introduce useful genetic variation into structured livestock breeding programs. Such variation may include repair of genetic defects, the inactivation of undesired genes, and the moving of useful alleles and haplotypes between breeds in the absence of linkage drag. Editing could also be used to accelerate the rate of genetic progress by enabling the replacement of the germ cell lineage of commercial breeding animals with cells derived from genetically elite lines. In the future, editing may also provide a useful complement to evolving approaches to decrease the length of the generation interval through in vitro generation of gametes. For editing to be adopted, it will need to seamlessly integrate with livestock breeding schemes. This will likely involve introducing edits into multiple elite animals to avoid genetic bottlenecks. It will also require editing of different breeds and lines to maintain genetic diversity, and enable structured crossbreeding. This requirement is at odds with the process-based trigger and event-based regulatory approach that has been proposed for the products of genome editing by several countries. In the absence of regulatory harmony, researchers in some countries will have the ability to use genome editing in food animals, while others will not, resulting in disparate access to these tools, and ultimately the potential for global trade disruptions.

show abstract

“…Delivering the editing tools as plasmid DNA is convenient but carries the risk of unwanted insertions of plasmid vectors as has been observed in cattle that were edited for the Celtic polled mutation [35] . Unwanted insertion can be avoided by delivering the editors in a different form and several studies have successfully demonstrated on-target mutagenesis in zygotes with preformed gRNA/Cas9 ribonucleoprotein (RNP) complexes [30][31][32][33] . Since designing gRNAs for Cas9 is far simpler than TALENs or ZFNs, delivering preformed gRNA/Cas9 RNP complexes is likely to be the preferred option for future accelerated breeding programs.…”

Section: Editing Zygotesmentioning

confidence: 99%

“…This approach has been reported to deliver gRNA/Cas9 RNPs, Cas9 mRNA and gRNAs into rodent embryos, achieving high biallelic editing efficiencies [36,37] . This was followed by the validation of electroporation as a suitable method to edit bovine embryos [32,33] . In addition, the feasibility was demonstrated to HDR-edit bovine zygotes by electroporating gRNA/Cas9 RNPs and an exogenous repair template [30] .…”

Section: Editing Zygotesmentioning

confidence: 99%

Embryo-mediated genome editing for accelerated genetic improvement of livestock

McLean¹,

Oback²,

Laible³

2020

Front. Agr. Sci. Eng.

View full text Add to dashboard Cite

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.

show abstract

Simplified pipelines for genetic engineering of mammalian embryos by CRISPR-Cas9 electroporation†

Cited by 35 publications

References 28 publications

Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos

Harnessing endogenous repair mechanisms for targeted gene knock-in of bovine embryos

Genome editing approaches to augment livestock breeding programs

Embryo-mediated genome editing for accelerated genetic improvement of livestock

Contact Info

Product

Resources

About