Expanding the Repertoire of Target Sites for Zinc Finger Nuclease-mediated Genome Modification

Recent studies have shown that zinc finger nucleases (ZFNs) are powerful reagents for making site-specific genomic modifications. The generic structure of these enzymes includes a ZF DNA-binding domain and nuclease domain (Fn) are separated by an amino acid “linker” and cut genomic DNA at sites that have a generic structure (site1)-(spacer)-(site2) where the “spacer” separates the two binding sites. In this work, we compare the activity of ZFNs with different linkers on target sites with different spacer lengths. We found those nucleases with linkers’ lengths of 2 or 4 amino acid (aa) efficiently cut at target sites with 5 or 6 base pair (bp) spacers, and that those ZFNs with a 5-aa linker length efficiently cut target sites with 6 or 7 bp spacers. In addition, we demonstrate that the Oligomerized Pool ENgineering (OPEN) platform used for making three-fingered ZF proteins (ZFPs) can be modified to incorporate modular assembly fingers (including those recognizing ANNs, CNNs, and TNNs) and we were able to generate nucleases that efficiently cut cognate target sites. The ability to use module fingers in the OPEN platform at target sites of 5–7 bp spacer lengths increases the probability of finding a ZFN target site to 1 in 4 bp. These findings significantly expand the range of sites that can be potentially targeted by these custom-engineered proteins

Quantifying Genome-Editing Outcomes at Endogenous Loci with SMRT Sequencing

Targeted genome editing with engineered nucleases has transformed the ability to introduce precise sequence modifications at almost any site within the genome. A major obstacle to probing the efficiency and consequences of genome editing is that no existing method enables the frequency of different editing events to be simultaneously measured across a cell population at any endogenous genomic locus. We have developed a method for quantifying individual genome-editing outcomes at any site of interest with single-molecule real-time (SMRT) DNA sequencing. We show that this approach can be applied at various loci using multiple engineered nuclease platforms, including transcription-activator-like effector nucleases (TALENs), RNA-guided endonucleases (CRISPR/Cas9), and zinc finger nucleases (ZFNs), and in different cell lines to identify conditions and strategies in which the desired engineering outcome has occurred. This approach offers a technique for studying double-strand break repair, facilitates the evaluation of gene-editing technologies, and permits sensitive quantification of editing outcomes in almost every experimental system used

Author Correction: Gene correction for SCID-X1 in long-term hematopoietic stem cells.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Author Correction: Gene correction for SCID-X1 in long-term hematopoietic stem cells

The original version of this Article omitted the following from the Acknowledgements: “G.B. acknowledges the support from the Cancer Prevention and Research Institute of Texas (RR140081 and RR170721).”This has now been corrected in both the PDF and HTML versions of the Article

Gene correction for SCID-X1 in long-term hematopoietic stem cells

Gene correction in hematopoietic stem cells could be a powerful way to treat monogenic diseases of the blood and immune system. Here the authors develop a strategy using CRISPR-Cas9 and an aAdeno-Associated vVirus(AAV)-delivered IL2RG cDNA to correct X-linked sSevere Ccombined iImmunodeficiency (SCID-X1) with a high success rate

Gene correction for SCID-X1 in long-term hematopoietic stem cells.

Gene correction in human long-term hematopoietic stem cells (LT-HSCs) could be an effective therapy for monogenic diseases of the blood and immune system. Here we describe an approach for X-linked sSevere cCombined iImmunodeficiency (SCID-X1) using targeted integration of a cDNA into the endogenous start codon to functionally correct disease-causing mutations throughout the gene. Using a CRISPR-Cas9/AAV6 based strategy, we achieve up to 20% targeted integration frequencies in LT-HSCs. As measures of the lack of toxicity we observe no evidence of abnormal hematopoiesis following transplantation and no evidence of off-target mutations using a high-fidelity Cas9 as a ribonucleoprotein complex. We achieve high levels of targeting frequencies (median 45%) in CD34+ HSPCs from six SCID-X1 patients and demonstrate rescue of lymphopoietic defect in a patient derived HSPC population in vitro and in vivo. In sum, our study provides specificity, toxicity and efficacy data supportive of clinical development of genome editing to treat SCID-Xl