Chimeric RNA: DNA TracrRNA Improves Homology-Directed Repair In Vitro and In Vivo.

Nearly 90% of human pathogenic mutations are caused by small genetic variations, and methods to correct these errors efficiently are critically important. One way to make small DNA changes is providing a single-stranded oligo deoxynucleotide (ssODN) containing an alteration coupled with a targeted double-strand break (DSB) at the target locus in the genome. Coupling an ssODN donor with a CRISPR-Cas9-mediated DSB is one of the most streamlined approaches to introduce small changes. However, in many systems, this approach is inefficient and introduces imprecise repair at the genetic junctions. We herein report a technology that uses spatiotemporal localization of an ssODN with CRISPR-Cas9 to improve gene alteration. We show that by fusing an ssODN template to the trans-activating RNA (tracrRNA), we recover precise genetic alterations, with increased integration and precision in vitro and in vivo. Finally, we show that this technology can be used to enhance gene conversion with other gene editing tools such as transcription activator like effector nucleases.

Unlocking loxP to Track Genome Editing In Vivo.

The development of CRISPR-associated proteins, such as Cas9, has led to increased accessibility and ease of use in genome editing. However, additional tools are needed to quantify and identify successful genome editing events in living animals. We developed a method to rapidly quantify and monitor gene editing activity non-invasively in living animals that also facilitates confocal microscopy and nucleotide level analyses. Here we report a new CRISPR "fingerprinting" approach to activating luciferase and fluorescent proteins in mice as a function of gene editing. This system is based on experience with our prior cre recombinase (cre)-detector system and is designed for Cas editors able to target loxP including gRNAs for SaCas9 and ErCas12a. These CRISPRs cut specifically within loxP, an approach that is a departure from previous gene editing in vivo activity detection techniques that targeted adjacent stop sequences. In this sensor paradigm, CRISPR activity was monitored non-invasively in living cre reporter mice (FVB.129S6(B6)-Gt(ROSA)26Sortm1(Luc)Kael/J and Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J, which will be referred to as LSL-luciferase and mT/mG throughout the paper) after intramuscular or intravenous hydrodynamic plasmid injections, demonstrating utility in two diverse organ systems. The same genome-editing event was examined at the cellular level in specific tissues by confocal microscopy to determine the identity and frequency of successfully genome-edited cells. Further, SaCas9 induced targeted editing at efficiencies that were comparable to cre, demonstrating high effective delivery and activity in a whole animal. This work establishes genome editing tools and models to track CRISPR editing in vivo non-invasively and to fingerprint the identity of targeted cells. This approach also enables similar utility for any of the thousands of previously generated loxP animal models.

Suppression-Replacement KCNQ1 Gene Therapy for Type 1 Long QT Syndrome.

BACKGROUND: Type 1 long QT syndrome (LQT1) is caused by loss-of-function variants in the KCNQ1-encoded Kv7.1 potassium channel α-subunit that is essential for cardiac repolarization, providing the slow delayed rectifier current. No current therapies target the molecular cause of LQT1. METHODS: A dual-component suppression-and-replacement (SupRep) KCNQ1 gene therapy was created by cloning a KCNQ1 short hairpin RNA and a short hairpin RNA-immune KCNQ1 cDNA modified with synonymous variants in the short hairpin RNA target site, into a single construct. The ability of KCNQ1-SupRep gene therapy to suppress and replace LQT1-causative variants in KCNQ1 was evaluated by means of heterologous expression in TSA201 cells. For a human in vitro cardiac model, induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) were generated from 4 patients with LQT1 (KCNQ1-Y171X, -V254M, -I567S, and -A344A/spl) and an unrelated healthy control. CRISPR-Cas9 corrected isogenic control iPSC-CMs were made for 2 LQT1 lines (correction of KCNQ1-V254M and KCNQ1-A344A/spl). FluoVolt voltage dye was used to measure the cardiac action potential duration (APD) in iPSC-CMs treated with KCNQ1-SupRep. RESULTS: In TSA201 cells, KCNQ1-SupRep achieved mutation-independent suppression of wild-type KCNQ1 and 3 LQT1-causative variants (KCNQ1-Y171X, -V254M, and -I567S) with simultaneous replacement of short hairpin RNA-immune KCNQ1 as measured by allele-specific quantitative reverse transcription polymerase chain reaction and Western blot. Using FluoVolt voltage dye to measure the cardiac APD in the 4 LQT1 patient-derived iPSC-CMs, treatment with KCNQ1-SupRep resulted in shortening of the pathologically prolonged APD at both 90% and 50% repolarization, resulting in APD values similar to those of the 2 isogenic controls. CONCLUSIONS: This study provides the first proof-of-principle gene therapy for complete correction of long QT syndrome. As a dual-component gene therapy vector, KCNQ1-SupRep successfully suppressed and replaced KCNQ1 to normal wild-type levels. In TSA201 cells, cotransfection of LQT1-causative variants and KCNQ1-SupRep caused mutation-independent suppression and replacement of KCNQ1. In LQT1 iPSC-CMs, KCNQ1-SupRep gene therapy shortened the APD, thereby eliminating the pathognomonic feature of LQT1.

Expanding the CRISPR Toolbox with ErCas12a in Zebrafish and Human Cells.

CRISPR and CRISPR-Cas effector proteins enable the targeting of DNA double-strand breaks to defined loci based on a variable length RNA guide specific to each effector. The guide RNAs are generally similar in size and form, consisting of a ∼20 nucleotide sequence complementary to the DNA target and an RNA secondary structure recognized by the effector. However, the effector proteins vary in protospacer adjacent motif requirements, nuclease activities, and DNA binding kinetics. Recently, ErCas12a, a new member of the Cas12a family, was identified in Eubacterium rectale. Here, we report the first characterization of ErCas12a activity in zebrafish and expand on previously reported activity in human cells. Using a fluorescent reporter system, we show that CRISPR-ErCas12a elicits strand annealing mediated DNA repair more efficiently than CRISPR-Cas9. Further, using our previously reported gene targeting method that utilizes short homology, GeneWeld, we demonstrate the use of CRISPR-ErCas12a to integrate reporter alleles into the genomes of both zebrafish and human cells. Together, this work provides methods for deploying an additional CRISPR-Cas system, thus increasing the flexibility researchers have in applying genome engineering technologies.

Precision gene editing technology and applications in nephrology.

The expanding field of precision gene editing is empowering researchers to directly modify DNA. Gene editing is made possible using synonymous technologies: a DNA-binding platform to molecularly locate user-selected genomic sequences and an associated biochemical activity that serves as a functional editor. The advent of accessible DNA-targeting molecular systems, such as zinc-finger nucleases, transcription activator-like effectors (TALEs) and CRISPR-Cas9 gene editing systems, has unlocked the ability to target nearly any DNA sequence with nucleotide-level precision. Progress has also been made in harnessing endogenous DNA repair machineries, such as non-homologous end joining, homology-directed repair and microhomology-mediated end joining, to functionally manipulate genetic sequences. As understanding of how DNA damage results in deletions, insertions and modifications increases, the genome becomes more predictably mutable. DNA-binding platforms such as TALEs and CRISPR can also be used to make locus-specific epigenetic changes and to transcriptionally enhance or suppress genes. Although many challenges remain, the application of precision gene editing technology in the field of nephrology has enabled the generation of new animal models of disease as well as advances in the development of novel therapeutic approaches such as gene therapy and xenotransplantation.