MENU

Highly efficient stem cell engineering via Cas9 protein transfection

Speaker

Abstract

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.


Show Resources
You May Also Like
SEP 14, 2021 7:00 AM PDT
C.E. CREDITS
SEP 14, 2021 7:00 AM PDT
Date: September 14, 2021 Time: 7am PDT, 10am EDT, 4pm CEST A conventional thermal cycler has long been a commodity product in the lab and end-point PCR techniques can be completed almost wit...
NOV 09, 2021 11:00 AM PST
C.E. CREDITS
NOV 09, 2021 11:00 AM PST
Date: November 09, 2021 Time: 11:00am (PDT), 02:00pm (EDT) Clinical translation of human pluripotent stem cells (hPSCs) requires advanced strategies that ensure safe and robust long-term gro...
NOV 17, 2021 8:00 AM PST
C.E. CREDITS
NOV 17, 2021 8:00 AM PST
Date: November 17, 2021 Time: 8:00am (PDT), 11:00am (EDT) From waste disposal to promising biomarkers and therapeutic agents, exosomes and other extracellular vesicles are valuable in resear...
SEP 28, 2021 9:00 AM PDT
C.E. CREDITS
SEP 28, 2021 9:00 AM PDT
Date: September 28, 2021 Time: 9:00am (PDT), 12:00pm (EDT) Standardization and reproducibility are prerequisites for peripheral blood mononuclear cell (PBMC) generation from donor or patient...
OCT 21, 2021 9:00 AM PDT
C.E. CREDITS
OCT 21, 2021 9:00 AM PDT
Date: October 21, 2021 Time: 9:00am (PDT), 12:00pm (EDT) Industry guidelines for host cell protein impurity analysis emphasize the importance of process-specific and product-specific assessm...
NOV 04, 2021 8:00 AM PDT
NOV 04, 2021 8:00 AM PDT
Date: November 4, 2021 Time: 8:00am (PDT), 11:00am (EDT) The promise of tissue clearing techniques in combination with 3D-immunofluorescence (IF) microscopy is revolutionary. Complex biologi...

Highly efficient stem cell engineering via Cas9 protein transfection



Show Resources
Loading Comments...
Show Resources