Optimizing Genome Editing in Bovine Cells: A Comparative Study of Cas9 Variants and CRISPR Delivery Methods

To assist in the establishment of an efficient CRISPR/Cas9 gene editing workflow in bovine cells, we compared the efficiency of four Streptococcus pyogenes Cas9 (SpCas9) nuclease variants (produced in-house or commercially) and two different Cas9/sgRNA ribonucleoprotein (RNP) delivery methods applied to Madin-Darby bovine kidney (MDBK) cells (Bos taurus). We targeted three genes for simple sequence deletion or modification via a single-stranded oligodeoxynucleotide donor template: the testis-determining gene Sry (sex-determining region on Y chromosome), germ cell-specific gene Nanos2 (nanos C2HC-type zinc finger 2) and PRLR (prolactin receptor). RNPs and donor templates were delivered into cells via lipofectamine CRISPRMAX transfection or Neon electroporation. The efficiency of gene editing was determined by target-specific PCR genotyping, real-time PCR assays and next generation sequencing analyses. When targeting Sry, the commercial Alt-R High-Fidelity (HiFi) SpCas9 nuclease exhibited the highest deletion efficiency, followed by the in-house generated Sniper2L, HiFi SpCas9 and wild-type SpCas9. Notably, for PRLR and Nanos2, Sniper2L induced comparable editing outcomes to Alt-R HiFi SpCas9. The two delivery methods, lipofectamine CRISPRMAX transfection and Neon electroporation, demonstrated similar efficiency (60% to 83%) in producing indels in all three target genes. However, Neon electroporation (5.5% to 11%) was superior to CRISPRMAX lipofection (1.5% to 4.8%) at inducing target-specific sequence knock-ins. Strategies to inhibit NHEJ repair and/or enhance HDR may be necessary to improve HDR efficiency in these bovine cells. These findings provide valuable insights for improving gene editing outcomes in bovines and may assist in accelerating the widespread application of genome editing technology in large animals.