28 Generation of Immunoglobulin Heavy Constant Mu (IGHM) Knockout Goats Using CRISPR/Cas9 and Somatic Cell Nuclear Transfer

Abstract

Towards the goals of inactivating endogenous goat immunoglobulin (Ig) genes and producing fully human polyclonal antibodies in transchromosomal goats carrying a human artificial chromosome comprising the entire human Ig gene repertoire, we report here the successful generation of IgM heavy chain knockout (IGHM^−/−) goats using CRISPR/Cas9 and somatic cell nuclear transfer (SCNT) techniques. Two single-guide RNAs (sgRNAs) were designed specific for exon 1 of the goat IGHM constant region (GenBank: EU182621.1). The gene-targeting vectors were constructed by using pX330 plasmid (Plasmid No. 42230, Addgene, Cambridge, MA, USA) and transfected into 2 × 10⁵ goat fetal fibroblast cells. Gene-targeting efficiency of each targeting vector was determined by PCR-restriction fragment length polymorphism (RFLP) assay 3 days post-transfection. Our results showed that one of the sgRNAs, 5′-GAAAGGCGCTTGAGGAATGC-3′, was efficient in directing Cas9 to generate targeted cleavages in exon 1 of IGHM constant region, with a mutation efficiency of 20%. We established single cell-derived fetal fibroblast colonies by limiting dilution of the cells transfected with the targeting vector. Colony screening with the PCR-RFLP assay confirmed that we achieved targeted gene disruption in exon 1 in 11/49 (22.4%) of the colonies (7 colonies with biallelic and 4 with monoallelic gene disruption). Sanger sequencing analysis of genomic DNA isolated from cell colonies with biallelic mutations showed that typical nucleotide deletions and insertions (indels), caused by repairing double-strand DNA breaks during the error-prone non-homologous end joining (NHEJ) process, were generated at the targeting site of exon 1. One of the colonies harboring a 1-nucleotide (nt) deletion was used as nuclear donors for SCNT. A total of 102 1-cell-stage cloned embryos were generated and surgically transferred into 6 synchronized recipients. Three of the recipients (3/6, 50.0%) were confirmed pregnant by ultrasonography on Day 40 to 45 of gestation. Two pregnancies were sacrificed for IGHM^−/− fetal fibroblast isolation and one pregnancy was allowed to go to term, which led to the birth of a live and seemingly healthy kid. This goat was reared conventionally and appeared healthy until 5 weeks of age when it was killed because of commensal virus infection. The PCR-RFLP assay and sequence analysis showed that this cloned goat carried a biallelic 1-nt deletion in exon 1 of IGHM, which was identical to the donor colony it was originated from. No lymphoid follicles were observed in lymph nodes and spleen by histology, and immunohistochemistry for B cells (CD20) and T cells (CD3) demonstrated a lack of B cells in lymph nodes and spleen but the presence of T cells, confirming that IGHM has been successfully knocked out.