Apr 15, 2016 · Here, we describe a Cas9(D10A)-based screening approach that combines an All-in-One Cas9(D10A) nickase vector with fluorescence-activated cell sorting enrichment followed by high-throughput genotypic and phenotypic clonal screening strategies to generate isogenic knockouts and knock-ins highly efficiently, with minimal off-target effects.

Mar 30, 2023 · To increase their editing efficiency, dCas9 is replaced with nCas9 (D10A). Unlike dCas9, nCas9 (D10A) nicking of the target strand stimulates cellular repair mechanisms, which leads to increased...

Apr 15, 2016 · Here, we describe a Cas9 D10A -based screening approach that combines an All-in-One Cas9 D10A nickase vector with fluorescence-activated cell sorting enrichment followed by high-throughput...

In this study, we showed: (i) the in vivo cleavage efficiency of the HNH domain of Cas9 in mammalian cells is higher than that of the RuvC domain, (ii) paired Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption.

D10A nickase outperformed both WT Cas9 sites tested by a wide margin, exhibiting >20% repair efficiency. Clearly, a nickase strategy can expand the targetable region, which is especially useful if there are no available guides close to the desired mutation site.

Mar 20, 2025 · Explore how the Cas9 D10A nickase enables precise single-strand DNA editing, its structural features, and its role in experimental applications.

A mutant form known as Cas9 D10A, which lacks just the RuvC-like nuclease domain activity, only nicks the DNA strand complementary to its crRNA, and is characterized as a Cas9 nickase (Cas9n).

Jul 6, 2018 · In this study, we showed: (i) the in vivo cleavage efficiency of the HNH domain of Cas9 in mammalian cells is higher than that of the RuvC domain, (ii) paired Cas9 nickases are sometimes more efficient than individual nucleases for gene disruption.

Oct 31, 2017 · The CRISPR-Cas9 D10A nickase-based genome editing in Lactobacillus casei, an important food industrial microorganism, was demonstrated in this study. This genetic tool allows efficient single-gene deletion and insertion to be accomplished by one-step transformation, and the cycle time is reduced to 9 days.

Nov 11, 2017 · Overview of CRISPR-Cas9 D10A nickase-assisted recombineering in L. casei. Step 1 is the delivery of pLCNICK into wild-type cells. sgRNA-guided Cas9 D10A (nickase) generates a nick in chromosome and triggers the repair process. In step 2, pLCNICK provides the homologous arms (repair donors), which are utilized in homology-dependent repair (step ...