Uchida, N. | Search bioRxiv

bioRxiv2018

Dynamic gait transition in the Scolopendromorpha scolopocryptops rubiginosus L. Koch centipede

Crawling using locomotory waves is a common method of locomotion for limbless and many-legged invertebrates. It is generally believed that the direction of locomotory waves is fixed for a given species. However, by recording and performing detailed analyses of the gait patterns of a Scolopendromorpha scolopocryptops rubiginosus L. Koch centipede in various conditions, we found that it dynamically generated its gait to allow for locomotory waves that varied in direction. By introducing the wave-index order parameter to characterise locomotory waves, we showed that gait patterns were associated with control of stride rather than rotation frequency.

animal behavior and cognition↗

bioRxiv2018

Selection-free, high frequency genome editing by homologous recombination of human pluripotent stem cells using Cas9 RNP and AAV6

Combination of genome editing and human pluripotent stem cells (hPSCs) offers a platform for in vitro disease modeling, drug discovery and personalized stem cell therapeutics. However, incorporation of large modifications using CRISPR/Cas9-based genome editing in hPSCs typically requires the use of selection markers due to low editing efficiencies. Here we report a novel editing technology in hPSCs using Cas9 protein complexed with chemically modified single guide RNA (sgRNA) and recombinant AAV6 (rAAV6) vectors for donor delivery without marker selection. With these components, we demonstrate targeted integration of a 2.2 kb DNA expression cassette in hPSCs at frequencies up to 94% and 67% at the HBB and MYD88 loci, respectively. We used this protocol to correct the homozygous sickle cell disease (SCD) mutation in an iPSC line derived from a SCD patient with a frequency of 63%. This Cas9/AAV6 system allows for both the integration of large gene cassettes and the creation of single nucleotide changes in hPSCs at high frequencies, eliminating the need for multiple editing steps and marker selection, thus increasing the potential of editing human pluripotent cells for both research and translational applications.

bioengineering↗