Highly efficient stem cell engineering via Cas9 protein transfection

  • Staff Scientist, Thermo Fisher Scientific
      Xiquan Liang graduated from City College of New York in 1999. He joined Thermo Fisher Scientific as a staff scientist in 2004. Since then he has led development of product development leadership in protein analysis, including High Molecular Weight MS standard, SILAC for protein identification and quantification, and Dynabeads-TiO2 for phosphopeptide enrichment. He is now engaged in product development in Synthetic Biology, including Bluegrass, GeneArt seamless DNA assembly tools, site-directed mutagenesis , and CRISPR-mediated mammalian cell engineering in the Life Sciences Solutions Group of Thermo Fisher Scientific in Carlsbad, CA. He is focused on developing tools for the entire Synthetic Biology workflow, specifically DNA cloning, Gene Synthesis and Assembly, and Molecular and Cell Engineering.


    CRISPR-Cas9 systems provide a platform for high efficiency genome editing that are enabling innovative applications of mammalian cell engineering.  The delivery of Cas9 plasmid DNA or mRNA involves in transcription and/or translation.  On the other hand, the direct delivery of Cas9 protein/gRNA ribonucleoprotein complexes (Cas9 RNPs) approves to be more effective.  In this endeavor, we have developed robust methods to purify and delivery Cas9 RNPs into a variety of mammalian cells through liposome-mediated transfection or electroporation. Using these methods, we report nuclease-mediated indel rates of up to 94% in Jurkat T cells and 87% in induced pluripotent stem cells (iPSC) for a single target. When we used this approach for multigene targeting in Jurkat cells we found that two-locus and three-locus indels were achieved in approximately 93% and 65% of the resulting isolated cell lines, respectively. Further, we found that the off-target cleavage rate is reduced using Cas9 protein when compared to plasmid DNA transfection.  Recently, we enhanced CRISPR/Cas9-mediated precise genome editing by improved design and delivery of gRNA, Cas9 nuclease, and donor DNA.  Under optimal conditions, we achieved precise genome editing rates of up to 24% in induced pluripotent stem cells (iPSCs) and 40% in Cas9-expressing iPSCs for a single nucleotide substitution at multiple genomic loci.   One of the keys to high HDR efficiency was placement of the cleavage site in close proximity to the intended site of editing.  Secondarily, asymmetric PAM and non-PAM single stranded (ss) DNA donors were also found to enhance HDR efficiency.   Taken together, we provide simple and highly efficient approaches for modulation of the mammalian genome and for generation of knock-in and knock-out cell lines.

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