Abstract
The CRISPR/Cas9 gene editing system has enhanced the development of genetically engineered animals for use in xenotransplantation. Potential limitations to the CRISPR/Cas9 system impacting the development of genetically engineered cells and animals include the creation of off-target mutations. We sought to develop a method to reduce the likelihood of off-target mutation while maintaining a high efficiency rate of desired genetic mutations for the GGTA1 gene. Extension of sgRNA length, responsible for recognition of the target DNA sequence for Cas9 cleavage, resulted in improved specificity for the GGTA1 gene and less off-target DNA cleavage. Three PAM sites were selected within exon 1 of the porcine GGTA1 gene and ten sgRNA of variable lengths were designed across these three sites. The sgRNA was tested against synthetic double stranded DNA templates replicating both the native GGTA1 DNA template and the two most likely off-target binding sites in the porcine genome. Cleavage ability for native and off-target DNA was determined by in vitro cleavage assays. Resulting cleavage products were analyzed to determine the cleavage efficiency of the Cas9/sgRNA complex. Extension of sgRNA length did not have a statistical impact on the specificity of the Cas9/sgRNA complex for PAM1 and PAM2 sites. At the PAM3 site, however, an observed increase in specificity for native versus off-target templates was seen with increased sgRNA length. In addition, distance between PAM site and the start codon had a significant impact on cleavage efficiency and target specificity, regardless of sgRNA length. Although the in vitro assays showed off-target cleavage, Sanger sequencing revealed that no off-target mutations were found in GGTA1 knockout cell lines or piglet. These results demonstrate an optimized method for improvement of the CRIPSR/Cas9 gene editing system by reducing the likelihood of damaging off-target mutations in GGTA1 knocked out cells destined for xenotransplant donor production.
Highlights
Xenotransplantation could provide a much-needed source of donor organs for use in the treatment of a wide array of human diseases
Guides were tested against the porcine GGTA1 gene that was asymmetrically PCR amplified at the targeted protospacer adjacent motif (PAM) sites
No significant differences were seen between the three guides at the PAM2 cut site; a cleavage efficiency of 63.47% was seen for 20 bp, 76.12% for 30 bp, and 68.98% for 40 bp (Fig 3B, Table 3)
Summary
Xenotransplantation could provide a much-needed source of donor organs for use in the treatment of a wide array of human diseases. Pigs are considered appropriate candidates for xenotransplantation to humans due to similarities in organ size and metabolism, multiple human genetic mutations produce phenotypes known to cause immune responses leading to xenograft rejection[1]. The most commonly targeted porcine genes include α-1,3-galactosyltransferase (GGTA1), cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH), and β-1,4N-acetylgalactosaminyltransferase (β4GalNT2) [1]. Recent advances in precision gene editing have allowed for the successful development of genetically engineered (GE) porcine donors targeting these genes for greater compatibility with the human immune system in order to reduce the likelihood of xenograft rejection[1]
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
