Mitigating xenogeneic barriers to chimerism through Cas13-induced host attenuation

Area of Science:

• Developmental Biology
• Stem Cell Biology
• Biotechnology

Background:

• Interspecies chimeras are crucial for studying mammalian development and organ generation.
• Xenogeneic barriers like developmental timing and cell competition limit human pluripotent stem cell (hPSC) contribution to non-human embryos.

Purpose of the Study:

• To develop a method to overcome xenogeneic barriers and enhance donor cell chimerism.
• To investigate the use of Cas13 to modulate host cell proliferation and improve interspecies developmental potential.

Main Methods:

• Engineered mouse epiblast stem cells (mEpiSCs) with inducible Cas13 (iCas13).
• Utilized Cas13's collateral RNA cleavage to selectively attenuate host cell proliferation.
• Performed co-culture and in vivo chimera experiments with wild-type and engineered cells, including hPSCs.

Main Results:

• Cas13 activation in mEpiSCs reduced proliferation and bystander transcripts while maintaining pluripotency.
• Selective host cell attenuation enhanced the competitiveness and survival of donor cells (mEpiSCs and hPSCs) in vitro.
• Blastocysts with iCas13 demonstrated increased donor cell integration in chimeric embryos and adult tissues.

Conclusions:

• Cas13-mediated host cell attenuation is a viable strategy to boost donor cell chimerism.
• This approach effectively mitigates xenogeneic barriers in interspecies developmental models.
• The findings offer a generalizable method for advancing interspecies organogenesis and developmental research.