CRISPR/Cas9 microinjection in oocytes disables pancreas development in sheep

Abstract

One of the ultimate goals of regenerative medicine is the generation of patient-specific organs from pluripotent stem cells (PSCs). Sheep are potential hosts for growing human organs through the technique of blastocyst complementation. We report here the creation of pancreatogenesis-disabled sheep by oocyte microinjection of CRISPR/Cas9 targeting PDX1, a critical gene for pancreas development. We compared the efficiency of target mutations after microinjecting the CRISPR/Cas9 system in metaphase II (MII) oocytes and zygote stage embryos. MII oocyte microinjection reduced lysis, improved blastocyst rate, increased the number of targeted bi-allelic mutations, and resulted in similar degree of mosaicism when compared to zygote microinjection. While the use of a single sgRNA was efficient at inducing mutated fetuses, the lack of complete gene inactivation resulted in animals with an intact pancreas. When using a dual sgRNA system, we achieved complete PDX1 disruption. This PDX1−/− fetus lacked a pancreas and provides the basis for the production of gene-edited sheep as a host for interspecies organ generation. In the future, combining gene editing with CRISPR/Cas9 and PSCs complementation could result in a powerful approach for human organ generation.

Evaluation of Mosaicism and Off Target Mutations in CRISPR-Mediated Genome Edited Bovine Embryos

“…Many of these studies characterized mosaicism by sequencing the PCR amplicon of the genomic regions flanking the gRNA target sequence and then decomposing the resulting chromatogram data with the TIDE bioinformatics package 9 . Although this approach is cost-effective and rapid, next generation sequencing of the PCR products allows for a more accurate characterization of the different alleles that are present in a mosaic individual, and their relative abundance 10 .…”

“…Guides were selected with no less than three mismatches in the guide sequence for off-target sites using the UMD3.1.1 bovine reference genome 24 , and at least one mismatch in the seed region (8-11bp upstream of the PAM sequence). Oligonucleotides were ordered from Eurofins USA (Louisville, KY) for the top four guides for construction of the gRNA and were used for in vitro transcription using the AmpliScribe T7-Flash Transcription kit (Lucigen, Palo Alto, CA) and purified using the MEGAclear Transcription Clean-Up kit (Thermo Fisher, Chicago, IL) as described by Vilarino et al 10 . Cleavage efficiency was tested using an in vitro cleavage assay by combining 60ng of PCR amplified product, 100ng of gRNA, 150ng of Cas9 protein (PNA Bio, Inc., Newbury Park, CA), 1μL of 10X BSA, 1μL of NEB Buffer 3.1 and water bringing the total volume to 10μL in a 0.2μL tube and incubating at 37°C for 1 hour.…”

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

“…Knock-in of donor plasmids was attempted using laser-assisted cytoplasmic injection 37 of in vitro matured oocytes after 18 hours of maturation and in vitro fertilized embryos 6 hours post insemination (hpi) with 6pL of a solution containing 67ng/μL of in vitro transcribed gRNA, 167ng/μL of Cas9 protein (PNA Bio, Inc., Newbury Park, CA) and 133ng/μL of donor plasmid. Injected MII oocytes were subsequently co-cultured with cumulus-oocyte-complexes (COCs) and in vitro fertilized following procedures previously described for sheep 22 . Embryos were scored for developmental stage reached at day 7-8.…”

Directions and applications of CRISPR technology in livestock research

“…A strategy to increase the percentage of KO out of edited embryos may be the use of multiple guides for the same gene (Wang et al, 2015b;Chuang et al, 2016;Wang et al, 2016a;Wang et al, 2016b;Vilarino et al, 2017). Multiple guides lead to multiple DSB that may result in either the deletion of a large fragment within them, which may include the start codon, or in the alteration of the ORF at different points.…”

“…This is phenomenon is called mosaicism, as it results in mosaic individuals composed by more than one cell population. Mosaicism was initially overlooked, as it is not a common problem in the generation of murine KO models (Bermejo-Alvarez et al, 2015), but most of the publications that have performed allele screening following CRISPR direct injection in zygotes have observed mosaicism in different species such as pigs (Hai et al, 2014;Sato et al, 2015;Wang et al, 2015c;Chuang et al, 2016;Kang et al, 2016;Petersen et al, 2016;Yu et al, 2016;Zhou et al, 2016;Burkard et al, 2017;Park et al, 2017;Whitworth et al, 2017), goats ), sheep (Crispo et al, 2015Wang et al, 2016c;Vilarino et al, 2017;Zhang et al, 2017), cattle (Bevacqua et al, 2016 and rabbits (Yan et al, 2014;Honda et al, 2015;Guo et al, 2016;Lv et al, 2016;Song et al, 2016a;Song et al, 2016b;Sui et al, 2016;Yang et al, 2016;.…”