Table 2 Dual overlapping gRNA strategy and outcomes of Gaaem1935C>A mouse generation.

From: CRISPR-mediated generation and characterization of a Gaa homozygous c.1935C>A (p.D645E) Pompe disease knock-in mouse model recapitulating human infantile onset-Pompe disease

Dual gRNA strategy components

Concentration

Founder mice with any Gaa mutation (% Positive)

Founder mice with Gaaem1935C>A mutation (% Positive)

Gaac.1935 crRNAs (gRNA-2, gRNA-3)

3 µM

RNP complex

89.7% (35 of 39 founders)

38.5% (15 of 39 founders)

tracrRNA

3xNLS SpCas9

Donor ssODN

10 ng/µL

  1. Dual overlapping gRNA components and concentrations used in pronuclear microinjection of C57BL/6NJ fertilized zygotes are outlined. Each crRNA was hybridized with tracrRNA at a 1:1 ratio to form gRNA duplexes. Equimolar amounts of gRNAs were then combined with 3xNLS SpCas9 at a 1:1 ratio to form the RNP complex. Numbers of founder mice positive for any Gaa mutation and founder mice with the Gaac.1935 KI mutation are reported as percentages.
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