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b1-tubulin role in platelet function
CRISPR knockout of TUBB1 results in a complete loss of proplatelet formation
To date, the loss of TUBB1 has not been studied in human MK. We generated an iPSC line with a CRISPR mediated bi-allelic loss of function mutation in the N-ter- minus of the coding region of TUBB1 (Figure 3G; Online Supplementary Figure S5). The mutation of the TUBB1 start codon on both alleles results in a complete loss of expres-
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sion (absence of T2 band in Figure 3H) and proplatelet for- mation in vitro (Figure 3I and J, right panel). Unfortunately, our attempts to generate C-terminal truncations through CRISPR in iPSC-MK were unsuccessful as multiple guides targeting the 3’ end of the TUBB1 gene failed to cleave the genomic sequence. Interestingly while TUBB1 KO clones stain positively for polyglutamylated and polyglycylated tubulin, the distribution of these residues is disturbed
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Figure 2. Continued on following page.
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