Two alternative approaches gave more promising results: the use of Cas9 nickase
(Cas9n) or fCas9.
Efficient genome modification by CRISPR-Cas9 nickase
with minimal off-target effects.
The TadA A106V/D108N mutant was fused to the N-terminus of the Cas9 (D10A mutant with nickase
deactivated the catalytic domain of the nuclease creating a nickase
Though, increasing specificity by titrating Cas9-sgRNA concentration also reduces on-target frequencies ; (b) wild-type cas9 is mutated to D10 mutant nickase
and paired with two sgRNAs to induce nick in the opposite strands of two nearby target sites.
This strategy known as nickase
Cas9 can be achieved by inactivation of one of the two nuclease domains of the Cas9, resulting in the cleavage of only one DNA strand.
Zhang et al., "Efficient genome modification by CRISPR-Cas9 nickase
with minimal off-target effects," Nature Methods, vol.
Single nucleotide mutation in two domains (RuvC, HNH) of Cas9 transforms it into Cas9 nickase
This cassette was integrated into the target site by homologous recombination using the clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins 9 (CRISPR/Cas9) nickase
Cas9 specificity was increase 100-1500 fold by the use of a pair of Cas9 nickase
variants, which were directed to target on opposite strands up to 100 bp apart.