Dataset Information

One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes.

ABSTRACT:

Background

The homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. The homology-mediated end joining (HMEJ)-based strategy has been shown to increase knock-in efficiency relative to HR, non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) strategies in non-dividing cells.

Results

By introducing gRNA/Cas9 ribonucleoprotein complex and a HMEJ-based donor template with 1?kb homology arms flanked by the H11 safe harbor locus gRNA target site, knock-in rates of 40% of a 5.1?kb bovine sex-determining region Y (SRY)-green fluorescent protein (GFP) template were achieved in Bos taurus zygotes. Embryos that developed to the blastocyst stage were screened for GFP, and nine were transferred to recipient cows resulting in a live phenotypically normal bull calf. Genomic analyses revealed no wildtype sequence at the H11 target site, but rather a 26?bp insertion allele, and a complex 38?kb knock-in allele with seven copies of the SRY-GFP template and a single copy of the donor plasmid backbone. An additional minor 18?kb allele was detected that looks to be a derivative of the 38?kb allele resulting from the deletion of an inverted repeat of four copies of the SRY-GFP template.

Conclusion

The allelic heterogeneity in this biallelic knock-in calf appears to have resulted from a combination of homology directed repair, homology independent targeted insertion by blunt-end ligation, NHEJ, and rearrangement following editing of the gRNA target site in the donor template. This study illustrates the potential to produce targeted gene knock-in animals by direct cytoplasmic injection of bovine embryos with gRNA/Cas9, although further optimization is required to ensure a precise single-copy gene integration event.

SUBMITTER: Owen JR

PROVIDER: S-EPMC7881600 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes.

Owen Joseph R JR Hennig Sadie L SL McNabb Bret R BR Mansour Tamer A TA Smith Justin M JM Lin Jason C JC Young Amy E AE Trott Josephine F JF Murray James D JD Delany Mary E ME Ross Pablo J PJ Van Eenennaam Alison L AL

BMC genomics 20210212 1

<h4>Background</h4>The homologous recombination (HR) pathway is largely inactive in early embryos prior to the first cell division, making it difficult to achieve targeted gene knock-ins. The homology-mediated end joining (HMEJ)-based strategy has been shown to increase knock-in efficiency relative to HR, non-homologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) strategies in non-dividing cells.<h4>Results</h4>By introducing gRNA/Cas9 ribonucleoprotein complex and a HMEJ-b ...[more]

PMID: 33581720

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Project description:NANOS3 is expressed in migrating primordial germ cells (PGCs) to protect them from apoptosis, and it is known to be a critical factor for germline development of both sexes in several organisms. However, to date, live NANOS3 knockout (KO) cattle have not been reported, and the specific role of NANOS3 in male cattle, or bulls, remains unexplored. This study generated NANOS3 KO cattle via cytoplasmic microinjection of the CRISPR/Cas9 system in vitro produced bovine zygotes and evaluated the effect of NANOS3 elimination on bovine germline development, from fetal development through reproductive age. The co-injection of two selected guide RNA (gRNA)/Cas9 ribonucleoprotein complexes (i.e., dual gRNA approach) at 6 h post fertilization achieved a high NANOS3 KO rate in developing embryos. Subsequent embryo transfers resulted in a 31% (n = 8/26) pregnancy rate. A 75% (n = 6/8) total KO rate (i.e., 100% of alleles present contained complete loss-of-function mutations) was achieved with the dual gRNA editing approach. In NANOS3 KO fetal testes, PGCs were found to be completely eliminated by 41-day of fetal age. Importantly, despite the absence of germ cells, seminiferous tubule development was not impaired in NANOS3 KO bovine testes during fetal, perinatal, and adult stages. Moreover, a live, NANOS3 KO, germline-ablated bull was produced and at sexual maturity he exhibited normal libido, an anatomically normal reproductive tract, and intact somatic gonadal development and structure. Additionally, a live, NANOS3 KO, germline-ablated heifer was produced. However, it was evident that the absence of germ cells in NANOS3 KO cattle compromised the normalcy of ovarian development to a greater extent than it did testes development. The meat composition of NANOS3 KO cattle was unremarkable. Overall, this study demonstrated that the absence of NANOS3 in cattle leads to the specific deficiency of both male and female germ cells, suggesting the potential of NANOS3 KO cattle to act as hosts for donor-derived exogenous germ cell production in both sexes. These findings contribute to the understanding of NANOS3 function in cattle and have valuable implications for the development of novel breeding technologies using germline complementation in NANOS3 KO germline-ablated hosts.

Dataset Information

One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes.

Background

Results

Conclusion

Publications

One-step generation of a targeted knock-in calf using the CRISPR-Cas9 system in bovine zygotes.

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