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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 activity) [52].
deactivated the catalytic domain of the nuclease creating a nickase [75].
Though, increasing specificity by titrating Cas9-sgRNA concentration also reduces on-target frequencies [43]; (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 [5, 9].
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 system [6-8].
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.
The company offers not just plasmids for CRISPR gene editing, but also custom-guide RNA design and validation (to provide customers with confidence that they are working with a suitably active guide); custom donor design and manufacturing; QuickStart cell lines that constitutively produce Cas9 nickase; and more than 550 X-MAN cell lines ready for further modification.