Abstract
<p class="MsoBodyTextIndent" style="text-align:justify; text-indent:14.15pt; margin-top:8px"><span style="font-size:12pt"><span style="line-height:200%"><span style="font-family:"Times New Roman",serif"><a name="_Hlk63785465">Calvarial bone healing is challenging, especially for individuals with osteoporosis because stem cells from osteoporotic patients are highly prone to adipogenic differentiation.</a> <a name="_Hlk63785654">Based on previous findings that chondrogenic induction of adipose-derived stem cells (ASC) can augment calvarial bone healing, </a>we hypothesized that <a name="_Hlk63785706">activating chondroinductive Sox Trio genes (<i>Sox5</i>, <i>Sox6</i>, <i>Sox9</i>) and repressing adipoinductive genes (<i>C/ebp-</i></a><i><span style="font-family:Symbol">a</span></i>, <i>Ppar-</i><i><span style="font-family:Symbol">g</span></i>) in osteoporotic ASC can reprogram cell differentiation and improve calvarial bone healing after implantation. However, simultaneous gene activation and repression in ASC is difficult. To tackle this problem, <a name="_Hlk63785765">we built a CRISPR-BiD system</a> for Bi-Directional gene regulation. Specifically, <a name="_Hlk63785881">we built a CRISPR-AceTran system that exploited </a>both histone acetylation and transcription activation for synergistic Sox Trio activation. <a name="_Hlk63785978">We also developed a CRISPR interference (CRISPRi) system that exploited </a><span style="line-height:200%">DNA methylation </span>for repression of adipoinductive<i> </i>genes. <a name="_Hlk63786173">We combined CRISPR-AceTran and CRISPRi to form the CRISPR-BiD system </a><a name="_Hlk78622515">which harnessed three mechanisms (</a>transcription activation, histone acetylation and DNA methylation). <a name="_Hlk63786198">A</a>fter delivery into osteoporotic rat ASC, CRISPR-BiD significantly enhanced chondrogenesis and <i>in vitro</i> cartilage formation. Implantation of the engineered osteoporotic ASC into critical-sized calvarial bone defects significantly improved bone healing in osteoporotic rats. These results implicated the potential of CRISPR-BiD system for bi-directional regulation of cell fate and regenerative medicine.</span></span></span></p>