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View article: A cleavage rule for selection of increased-fidelity SpCas9 variants with high efficiency and no detectable off-targets
A cleavage rule for selection of increased-fidelity SpCas9 variants with high efficiency and no detectable off-targets Open
View article: Position-dependent sequence motif preferences of SpCas9 are largely determined by scaffold-complementary spacer motifs
Position-dependent sequence motif preferences of SpCas9 are largely determined by scaffold-complementary spacer motifs Open
Streptococcus pyogenes Cas9 (SpCas9) nuclease exhibits considerable position-dependent sequence preferences. The reason behind these preferences is not well understood and is difficult to rationalise, since the protein establishes interact…
View article: Functional Characterization of the N-Terminal Disordered Region of the piggyBac Transposase
Functional Characterization of the N-Terminal Disordered Region of the piggyBac Transposase Open
The piggyBac DNA transposon is an active element initially isolated from the cabbage looper moth, but members of this superfamily are also present in most eukaryotic evolutionary lineages. The functionally important regions of the transpos…
View article: Target-matched increased fidelity SpCas9 variants can edit any target without genome-wide off-target effects
Target-matched increased fidelity SpCas9 variants can edit any target without genome-wide off-target effects Open
Streptococcus pyogenes Cas9 (SpCas9) has been employed as a genome engineering tool with a promising potential within therapeutics. However, its off- target effects pose a major limitation for applications requiring high specificity. Appro…
View article: A method for characterizing Cas9 variants via a one-million target sequence library of self-targeting sgRNAs
A method for characterizing Cas9 variants via a one-million target sequence library of self-targeting sgRNAs Open
Detailed target-selectivity information and experiment-based efficacy prediction tools are primarily available for Streptococcus pyogenes Cas9 (SpCas9). One obstacle to develop such tools is the rarity of accurate data. Here, we report a m…
View article: Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases
Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases Open
The widespread use of Cas12a (formerly Cpf1) nucleases for genome engineering is limited by their requirement for a rather long TTTV protospacer adjacent motif (PAM) sequence. Here we have aimed to loosen these PAM constraints and have gen…
View article: Mb- and FnCpf1 nucleases are active in mammalian cells: activities and PAM preferences of four wild-type Cpf1 nucleases and of their altered PAM specificity variants
Mb- and FnCpf1 nucleases are active in mammalian cells: activities and PAM preferences of four wild-type Cpf1 nucleases and of their altered PAM specificity variants Open
Cpf1s, the RNA-guided nucleases of the class II clustered regularly interspaced short palindromic repeats system require a short motive called protospacer adjacent motif (PAM) to be present next to the targeted sequence for their activity.…
View article: Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage
Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage Open
View article: Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage
Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage Open
Background The propensity for off-target activity of Streptococcus pyogenes Cas9 (SpCas9) has been considerably decreased by rationally engineered variants with increased fidelity (eSpCas9; SpCas9-HF1). However, a subset of targets still g…
View article: A convenient method to pre-screen candidate guide RNAs for CRISPR/Cas9 gene editing by NHEJ-mediated integration of a ‘self-cleaving’ GFP-expression plasmid
A convenient method to pre-screen candidate guide RNAs for CRISPR/Cas9 gene editing by NHEJ-mediated integration of a ‘self-cleaving’ GFP-expression plasmid Open
The efficacies of guide RNAs (gRNAs), the short RNA molecules \nthat bind to and determine the sequence specificity of the \nStreptococcus pyogenes Cas9 nuclease, to mediate DNA cleavage \nvary dramatically. Thus, the selection of appropri…
View article: Additional file 4: of Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage
Additional file 4: of Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage Open
Statistics. (XLSX 54 kb)
View article: Additional file 2: of Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage
Additional file 2: of Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage Open
Primer, sequence, and deep sequencing data. (XLSX 36 kb)
View article: Cpf1 nucleases demonstrate robust activity to induce DNA modification by exploiting homology directed repair pathways in mammalian cells
Cpf1 nucleases demonstrate robust activity to induce DNA modification by exploiting homology directed repair pathways in mammalian cells Open