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Genome Editing in Mammalian Cell Lines using CRISPR-Cas
07:56

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Published on: April 11, 2019

22.3K

Optimized design parameters for CRISPR Cas9 and Cas12a homology-directed repair

Mollie S Schubert1, Bernice Thommandru1, Jessica Woodley1

  • 1Integrated DNA Technologies, Inc., 1710 Commercial Park, Coralville, IA, 52241, USA.

Scientific Reports
|October 1, 2021
PubMed

Insights

Optimized CRISPR-Cas gene editing methods enhance homology-directed repair (HDR) efficiency using single-stranded oligodeoxynucleotide (ssODN) donors. This breakthrough facilitates precise genetic modifications in mammalian cells, overcoming limitations of traditional repair pathways.

Area of Science:

  • Molecular Biology
  • Genetics
  • Biotechnology

Background:

  • CRISPR-Cas systems introduce targeted double-stranded DNA breaks (DSBs) for gene editing.
  • Cellular repair pathways, non-homologous end joining (NHEJ) and homology-directed repair (HDR), resolve DSBs.
  • HDR enables precise genetic modifications using a DNA template, but is often less efficient than NHEJ.

Purpose of the Study:

  • To optimize homology-directed repair (HDR) efficiency using single-stranded oligodeoxynucleotide (ssODN) donor templates with CRISPR-Cas systems.
  • To investigate key design factors influencing HDR efficiency, including guide RNA selection and donor template modifications.
  • To develop a user-friendly tool for designing HDR donor templates.

Main Methods:

  • Utilized S.p. Cas9, S.p. Cas9 D10A nickase, and A.s. Cas12a CRISPR-Cas systems delivered as ribonucleoprotein (RNP) complexes.
  • Investigated guide RNA selection, donor strand preference, and blocking mutations within ssODN donor templates.
  • Developed and implemented a novel online tool for HDR donor template design.

Main Results:

  • Achieved highly efficient homology-directed repair (HDR) across multiple CRISPR-Cas systems.
  • Identified critical design parameters for maximizing HDR outcomes with ssODN donors.
  • Demonstrated high frequencies of precise genetic repair in various mammalian cell lines.

Conclusions:

  • Optimized design strategies significantly enhance HDR efficiency for precise gene editing.
  • The developed online tool facilitates the design of effective HDR donor templates.
  • These advancements enable robust and precise genetic modifications in mammalian cells using CRISPR-Cas technology.

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