Aim
Distinguishing common HLA null allele is required for both solid organ and hematopoietic stem cell transplantation. A fast, easy and precise tool to complement current low and high resolution typing strategies for null allele assignment would be beneficial. ASHI standards requires detection of at least HLA-A*24:09N, -B*51:11N, -C*04:09N, -DRB4*01:03N, -DRB5*01:08N. These null alleles are a result of different molecular modifications including; nucleotide substitution, homopolymer extension, homopolymer shortening or indels.
Methods
We developed a CRISPR-Cas based assay to detect null alleles in a gDNA sample. The method combines an isothermal LAMP reaction with CRISPR-Cas recognition of a specific target sequence and a fluorescent reporter assay in a singly tube. After adding DNA (5ng) to the assay tube, the reaction is incubated at 65°C for 50 minutes and fluorescent signal is monitored every minute.
Results
CRISPR-Cas Assays for HLA-A*24:09N, -B*51:11N, -C*04:09N, -DRB4*01:03N, -DRB5*01:08N were developed and tested using gBlocks DNA, cell lines and clinical samples. All samples with a specific null allele were tested positive between 20 and 40 minutes after addition of 5ng of sample DNA. We tested the assay using 58 samples from the HLA diverse GeT-RM panel (Coriell institute) and none of these samples without any null allele tested positive. Additionally, we tested a limit of detection for the HLA-A*24:09N assay. We successfully tested a DNA input range from 50 to only 0.05ng per assay.
Conclusions
The new CRISPR-Cas assays we developed here are highly sensitive and specific for HLA-A*24:09N, -B*51:11N, -C*04:09N, -DRB4*01:03N, -DRB5*01:08N.