One-Step Homology Mediated CRISPR-Cas Editing in Zygotes for Generating Genome Edited Cattle

Abstract

Selective breeding and genetic modification have been the cornerstone of animal agriculture. However, the current strategy of breeding animals over multiple generations to introgress novel alleles is not practical in addressing global challenges such as climate change, pandemics, and the predicted need to feed a population of 9 billion by 2050. Consequently, genome editing in zygotes to allow for seamless introgression of novel alleles is required, especially in cattle with long generation intervals. We report for the first time the use of CRISPR-Cas genome editors to introduce novel PRNP allelic variants that have been shown to provide resilience towards human prion pandemics. From one round of embryo injections, we have established six pregnancies and birth of seven edited offspring, with two founders showing >90% targeted homology-directed repair modifications. This study lays out the framework for in vitro optimization, unbiased deep-sequencing to identify editing outcomes, and generation of high frequency homology-directed repair-edited calves.

FIG. 1.

Identifying optimal CRISPR-Cas targeting reagents. (A) Schematic outlining the target site of the endogenous bovine PRNP locus. The coding sequence was truncated for convenience (indicated by “//”). The site coding for glycine (GGC) at amino acid position 127 targeted for conversion to valine (GTA) is highlighted in yellow. Successful gene targeting will result in the generation of AccI restriction enzyme site (GTATAC). Two CRISPR spacer sequences targeting the sense strand (F-guide) and antisense strand (R-guide) are shown above the target site. Putative cut-site is shown as a red triangle for each guide. (B) Representative agarose gel electrophoresis image indicating that AccI site introduction into genomic DNA results only from R-guide, and not from F-guide. Targeted genomic region was PCR amplified from embryos (numbered), amplicon fragment gel purified, AccI digested, and digestion fragments resolved on a 2% agarose gel. (C) Results from gene targeting with F- and R-guide from three triplicate experiments. No successful gene targeting was identified with F- guide (n = 14 blastocysts), whereas R-guide resulted in successful targeting with a statistically significant finding (n = 16 blastocysts; *P < 0.05). (D) Results from gene targeting with R-guide and oligos targeting either sense (n = 39 blastocysts) or antisense strand (n = 39 blastocysts). Antisense oligo resulted in better targeting efficiencies from duplicate experiments (*P < 0.05). (E) Schematic outlining Illumina miSeq targeting amplicon sequencing of blastocysts from gene targeting of embryos with various iterations of antisense oligos (sense, antisense, reverse-asymmetric). FastQ files from the miSeq run are trimmed and aligned to reference sequence. Wildtype, unmodified homology-directed repair (HDR) and nonhomologous end-joining (NHEJ) events from representative blastocysts were binned. All embryos were mosaic showing various combination of editing events. (F) The results from collating replicates over several weeks and showing editing events were shown (120 blastocysts total: 43 symmetrical, 24 asymmetrical, and 53 reverse-assymmetrical). Percentage of embryos showing no HDR reads, <10% HDR reads (low HDR frequency), and >10% reads are shown. Asymmetric oligo was least efficient among the three oligos tested. (G) Percentage of embryos showing one-third of gene targeted alleles (>33 % HDR); and (H) greater than half of the modified alleles (>50 % HDR) are shown. Reverse-asymmetric oligos resulted in better targeting efficiencies with greater number of embryos showing high HDR frequency. FWD, forward; REV, reverse.

FIG. 2.

Genotyping of edited calves. (A) Ear and blood biopsies from calves were utilized for DNA isolation and for genotyping. The PCR amplicons were purified, digested with AccI and resolved on an agarose gel to identify successful gene targeting outcomes. (B) High throughput targeted amplicon sequencing on the iSeq platform. FastQ sequence output files were analyzed using the CRISPResso 2.0 platform. (C) % of HDR, NHEJ, unmodified, and other events (insertions, transpositions, other modifications) were binned and shown in the graph. All offspring were edited and are mosaic and have a combination of HDR, NHEJ, and other events. Calves 752 and 774 had low HDR events. Calves 769, 786, and 789 have high HDR frequencies. Calves 769 and 789 have a high frequency of 1 bp and 4 bp out-of-frame mutations.

FIG. 3.

Genotyping of several tissues from a live calf with high HDR frequencies. (A) Biopsies from several tissues in addition to ear and blood, including intestine, spleen, liver, lung, heart, pancreas, kidney, and tail were harvested and DNA isolated for genotyping. The PCR amplicons were purified, digested with AccI and resolved on agarose gel to identify successful gene targeting outcomes. (B) High throughput targeted amplicon sequencing on the iSeq platform. FastQ sequence output files were analyzed using the CRISPResso 2.0 platform. % of HDR, NHEJ, unmodified and other events (insertions, transpositions, other events) were binned and shown in the graph. Results from all tissues identified events within a smaller range, highlighting the homogeneity of editing across all tissues.

FIG. 4.

Genotyping of spermatozoa from a live calf with high HDR frequencies. (A) Genomic DNA from spermatozoa was isolated for genotyping. The PCR amplicons were purified, digested with AccI, and resolved on agarose gel to identify successful gene targeting outcomes. Genomic DNA from euthanized calf with high HDR edits (789 E) and wild type cells were used as controls. We could not recover semen from one of the electro-ejaculated bull calf (827) and was not incorporated in the iSeq run. (B) The % of HDR, NHEJ, unmodified and other events (insertions, transpositions, other events) were binned and shown in the graph.