Author Correction: Predictable and precise template-free CRISPR editing of pathogenic variants.

In this Article, a data processing error affected Fig. 3e and Extended Data Table 2; these errors have been corrected online.

Predictable and precise template-free CRISPR editing of pathogenic variants.

Following Cas9 cleavage, DNA repair without a donor template is generally considered stochastic, heterogeneous and impractical beyond gene disruption. Here, we show that template-free Cas9 editing is predictable and capable of precise repair to a predicted genotype, enabling correction of disease-associated mutations in humans. We constructed a library of 2,000 Cas9 guide RNAs paired with DNA target sites and trained inDelphi, a machine learning model that predicts genotypes and frequencies of 1- to 60-base-pair deletions and 1-base-pair insertions with high accuracy (r = 0.87) in five human and mouse cell lines. inDelphi predicts that 5-11% of Cas9 guide RNAs targeting the human genome are 'precise-50', yielding a single genotype comprising greater than or equal to 50% of all major editing products. We experimentally confirmed precise-50 insertions and deletions in 195 human disease-relevant alleles, including correction in primary patient-derived fibroblasts of pathogenic alleles to wild-type genotype for Hermansky-Pudlak syndrome and Menkes disease. This study establishes an approach for precise, template-free genome editing.

Distinct requirements of linker DNA and transcriptional activators in promoting SAGA-mediated nucleosome acetylation.

The Spt-Ada-Gcn5 acetyltransferase (SAGA) family of transcriptional coactivators are prototypical nucleosome acetyltransferase complexes that regulate multiple steps in gene transcription. The size and complexity of both the SAGA enzyme and the chromatin substrate provide numerous opportunities for regulating the acetylation process. To better probe this regulation, here we developed a bead-based nucleosome acetylation assay to characterize the binding interactions and kinetics of acetylation with different nucleosomal substrates and the full SAGA complex purified from budding yeast (Saccharomyces cerevisiae). We found that SAGA-mediated nucleosome acetylation is stimulated up to 9-fold by DNA flanking the nucleosome, both by facilitating the binding of SAGA and by accelerating acetylation turnover. This stimulation required that flanking DNA is present on both sides of the nucleosome and that one side is >15 bp long. The Gal4-VP16 transcriptional activator fusion protein could also augment nucleosome acetylation up to 5-fold. However, contrary to our expectations, this stimulation did not appear to occur by stabilizing the binding of SAGA toward nucleosomes containing an activator-binding site. Instead, increased acetylation turnover by SAGA stimulated nucleosome acetylation. These results suggest that the Gal4-VP16 transcriptional activator directly stimulates acetylation via a dual interaction with both flanking DNA and SAGA. Altogether, these findings uncover several critical mechanisms of SAGA regulation by chromatin substrates.

Mechanisms of stimulation of SAGA-mediated nucleosome acetylation by a transcriptional activator.

Eukaryotic gene expression requires the coordination of multiple factors to overcome the repressive nature of chromatin. However, the mechanistic details of this coordination are not well understood. The SAGA family of transcriptional coactivators interacts with DNA-binding activators to establish regions of hyperacetylation. We have previously shown that, contrary to the prevailing model in which activator protein increases SAGA affinity for nucleosome substrate, the Gal4-VP16 activator model system augments the rate of acetylation turnover for the SAGA complex from budding yeast. To better understand how this stimulation occurs, we have identified necessary components using both kinetics assays and binding interactions studies. We find that Gal4-VP16-mediated stimulation requires activator binding to DNA flanking the nucleosome, as it cannot be reproduced in trans by activator protein alone or by exogenous DNA containing the activator binding site in combination with the activator protein. Further, activator-mediated stimulation requires subunits outside of the histone acetylation (HAT) module, with the Tra1 subunit being responsible for the majority of the stimulation. Interestingly, for the HAT module alone, nucleosome acetylation is inhibited by activator proteins due to non-specific binding of the activator to the nucleosomes. This inhibition is not observed for the yeast ADA complex, a small complex comprised mostly of the HAT module, suggesting that subunits outside of the HAT module in both it and SAGA can overcome non-specific activator binding to nucleosomes. However, this activity appears distinct from activator-mediated stimulation, as ADA complex acetylation is not stimulated by Gal4-VP16.

Structure, High Affinity, and Negative Cooperativity of the Escherichia coli Holo-(Acyl Carrier Protein):Holo-(Acyl Carrier Protein) Synthase Complex.

The Escherichia coli holo-(acyl carrier protein) synthase (ACPS) catalyzes the coenzyme A-dependent activation of apo-ACPP to generate holo-(acyl carrier protein) (holo-ACPP) in an early step of fatty acid biosynthesis. E. coli ACPS is sufficiently different from the human fatty acid synthase to justify the development of novel ACPS-targeting antibiotics. Models of E. coli ACPS in unliganded and holo-ACPP-bound forms solved by X-ray crystallography to 2.05and 4.10Å, respectively, revealed that ACPS bound three product holo-ACPP molecules to form a 3:3 hexamer. Solution NMR spectroscopy experiments validated the ACPS binding interface on holo-ACPP using chemical shift perturbations and by determining the relative orientation of holo-ACPP to ACPS by fitting residual dipolar couplings. The binding interface is organized to arrange contacts between positively charged ACPS residues and the holo-ACPP phosphopantetheine moiety, indicating product contains more stabilizing interactions than expected in the enzyme:substrate complex. Indeed, holo-ACPP bound the enzyme with greater affinity than the substrate, apo-ACPP, and with negative cooperativity. The first equivalent of holo-ACPP bound with a KD=62±13nM, followed by the binding of two more equivalents of holo-ACPP with KD=1.2±0.2µM. Cooperativity was not observed for apo-ACPP which bound with KD=2.4±0.1µM. Strong product binding and high levels of holo-ACPP in the cell identify a potential regulatory role of ACPS in fatty acid biosynthesis.