Designer Platelets: Crispr/Cas-Mediated Conversion of Human Platelet Alloantigen Allotypes
Authors: Zhang Nanyan , Zhi Huiying , Rao Sridhar , Newman Peter J. 
Published: October 2019

Abstract
Human platelet alloantigens (HPAs) reside on functionally important platelet membrane glycoproteins, and are caused by single nucleotide polymorphisms in the genes that encode them. Antibodies that form against HPAs are responsible for several clinically important alloimmune bleeding disorders, including fetal and neonatal alloimmune thrombocytopenia, posttransfusion purpura, and multitransfusion platelet refractoriness. The HPA-1a/HPA-1b alloantigen system, also known as the PlA1/PlA2 polymorphism, is the most frequently implicated HPA among Caucasians, and a single C29523T nucleotide substitution, resulting in a Leu33Pro amino acid polymorphism within the PSI domain of the integrin β3 subunit (platelet glycoprotein IIIa) was shown 25 years ago to be responsible for generating the HPA-1a/HPA-1b alloantigenic epitopes. Like other low-frequency alloantigens, HPA-1b/b platelets are relatively rare in the population, and therefore often difficult to obtain for purposes of transfusion therapy and diagnostic testing. As a first step in producing designer platelets expressing low-frequency human platelet alloantigens, we employed a CRISPR/Cas9 RNA-guided nicking nuclease system to transform megakaryocyte-like cells expressing the Leu33 allele of integrin β3 to the Pro33 form. Two different guide RNAs that target the ITGB3 gene with a 13-base pair offset 53 bases and 0 nucleotides upstream of the C/T polymorphism site were designed and cloned into plasmids that co-express GFP as well as a mutated form of Cas9 that nicks only one strand of DNA (Cas9n). Such a double-nicking strategy has been shown in other systems to increase the specificity of gene targeting while minimizing off-target effects. A 200 bp single-stranded DNA oligonucleotide encompassing the single base C29523T mismatch was also synthesized to be used for homology-directed repair (HDR) of the endogenous ITGB3 gene sequence. The HDR oligo was then transfected, together with the two plasmids encoding the guide RNAs+Cas9n+GFP, into megakaryocyte-like DAMI cells. Twenty-four hours post-transfection, GFP positive cells were sorted by flow cytometry and isolated as single clones. Surveyor endonuclease assays revealed that ~30% of the GFP positive clones had been cleaved at the expected location, indicating efficient double nicking directed by the pair of guide RNAs. Additionally, two out of twenty seven isolated clones had incorporated the HDR repair template, as reported by a diagnostic NciI restriction enzyme site that is specific for the T29523-bearing HPA-1b allele. Sequence analysis further confirmed conversion of C29523 to T in these two clones. Finally, Western blotting using HPA-1b-specific human alloantisera verified that these DAMI cells now expressed the HPA-1b (PlA2) alloantigenic epitope. Taken together, these results establish that the CRISPR/Cas system can be successfully employed to genetically edit this and other clinically-important HPAs in human cells. Application of this technology for the generation of alloantigen-specific human induced pluripotent stem cells holds great potential as a general tool for producing designer platelets for diagnostic and therapeutic use.

Disclosures
No relevant conflicts of interest to declare.