Genome editing: propelling the next generation of crop improvement

Climate change and population size records threaten food security. Therefore, the call for a more sustainable and efficient crop production has never been more urgent. Traditional plant breeding was one of the first successful approaches to expand cultivation areas and crop yield. Later, biotechnological tools and their products, such as genetically modified organisms containing exogenous DNA, further broadened the limits of agricultural results, yet bringing huge financial, bureaucratic, and public rejection hurdles. In the 90s, scientific advances brought the opportunity to drive mutations using engineered nucleases, and since 2013 CRISPR-Cas has emerged as the most practical toolkit to edit genomes. One of the most striking possibilities is to generate edited and non-transgenic plants. In this review, we present the working mechanism behind CRISPR-induced mutations and pinpoint the latest techniques developed, as well as its myriad of applications in agriculture. The enhancing scope of CRISPR ranges from introducing traits of agronomic interest – such as herbicide resistance, resistance/tolerance to biotic and abiotic stresses, and quality and durability of products – to accelerating plant breeding processes, including haploid induction, generating male-sterile lines, fixating hybrid vigor, and overcoming self-incompatibility. We also discuss regulatory issues surrounding edited plants and derived products around the world, challenges that must be overcome, and future prospects to harness all the potential of this amazing tool to guarantee the new crop production revolution.

CRISPR/Cas9 gene editing for genodermatoses: progress and perspectives

Genodermatoses constitute a clinically heterogeneous group of devastating genetic skin disorders. Currently, therapy options are largely limited to symptomatic treatments and although significant advances have been made in ex vivo gene therapy strategies, various limitations remain. However, the recent technical transformation of the genome editing field promises to overcome the hurdles associated with conventional gene addition approaches. In this review, we discuss the need for developing novel treatments and describe the current status of gene editing for genodermatoses, focusing on a severe blistering disease called epidermolysis bullosa (EB), for which significant progress has been made. Initial research utilized engineered nucleases such as transcription activator-like effector nucleases and meganucleases. However, over the last few years, clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) have upstaged older generation gene editing tools. We examine different strategies for CRISPR/Cas9 application that can be employed depending on the type and position of the mutation as well as the mode of its inheritance. Promising developments in the field of base editing opens new avenues for precise correction of single base substitutions, common in EB and other genodermatoses. We also address the potential limitations and challenges such as safety concerns and delivery efficiency. This review gives an insight into the future of gene editing technologies for genodermatoses.

A therapeutic genome editing primer for cardiologist

Genome editing, or genome engineering is a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of a living organism using engineered nucleases, or molecular scissors. Genome editing is being rapidly adopted into all fields of biomedical research, including the cardiovascular field, where it has facilitated a greater understanding of lipid metabolism, electrophysiology, cardiomyopathies, and other cardiovascular disorders, has helped to create a wider variety of cellular and animal models, and has opened the door to a new class of therapies. In this review, we discuss the applications of in vivo genome-editing therapies for cardiovascular disorder.

Versatile and robust genome editing with Streptococcus thermophilus CRISPR1-Cas9

Targeting definite genomic locations using CRISPR-Cas systems requires a set of enzymes with unique protospacer adjacent motif (PAM) compatibilities. To expand this repertoire, we engineered nucleases, cytosine base editors, and adenine base editors from the archetypal Streptococcus thermophilus CRISPR1-Cas9 (St1Cas9) system. We found that St1Cas9 strain variants enable targeting to five distinct A-rich PAMs and provide structural basis for their specificities. The small size of this ortholog enables expression of the holoenzyme from a single adeno-associated viral vector for in vivo editing applications. Delivery of St1Cas9 to the neonatal liver efficiently rewired metabolic pathways, leading to phenotypic rescue in a mouse model of hereditary tyrosinemia. These robust enzymes expand and complement current editing platforms available for tailoring mammalian genomes.

199 Efficient Knock-out of Ovine β-Lactoglobulin (BLG) Gene and Knock-in of Recombinant Human Factor IX (rhFIX) Under BLG Native Regulatory Sequences in Somatic Cells and Zygotes Using TALEN Nuclease

Site-specific genetic engineering is a valuable tool for pharmaceutical research and development of biomedical models. Despite engineered nucleases allow targeted gene edition in a rather simple fashion; few reports are available so far on specific gene knock-in (KI) combined with engineered nucleases in domestic species. Here, we evaluated the possibility of inducing specific KI of cDNAs coding for proteins of pharmaceutical interest under the control of milk native promoter sequences, taking advantage of the TALEN system, both in ovine somatic cells and in zygotes. We designed 2 TALENs, targeting exons 1 and 5 of ovine β-lactoglobulin gene (BLG), respectively, and a homologous recombination vector (pHR), carrying recombinant human factor IX (rhFIX) flanked by homology arms contiguous to the TALEN target sites. In an initial set of experiments, 5 × 105 to 1 × 106 ovine fibroblasts were transfected with 1 μg of each TALEN mRNA, with or without 50 ng μL−1 pHR. The feasibility of inducing knock-out (KO) was confirmed by Cel1 assay. The deletion of the genomic region between TALEN target sites and the occurrence of HR in cell lysates were assessed by PCR. Also, 576 individual colonies were picked up and analyzed by PCR. The deletion of the region between TALEN target sites was achieved with 7.8% efficiency (45/576). The incidence of HR in cells was 0.5% (3/576), as detected by PCR. In order to evaluate the system in zygotes, laparoscopic AI was performed on synchronized and superovulated ewes. Zygotes were recovered 16 h after AI and cytoplasmically injected with (1) 5 ng μL−1 TALEN mix (2.5 ng μL−1 oaBLG T1.1 + 2.5 ng μL−1 oaBLG T5.1) (5TM); (2) 5 ng μL−1 TALEN mix + 25 ng μL−1 pHR-hFIX plasmid (5TM+25pRH); or (3) 15 ng μL−1 TALEN mix (7.5 ng μL−1 oaBLG T1.1 + 7.5 ng μL−1 oaBLG T5.1) + 50 ng μL−1 pHR-hFIX (15TM+50pRH). A non-injected control (NIC) was also included. Embryo analysis was conducted on whole-genome amplified DNA from blastocysts, followed by PCR and sequencing. Non-parametric Fisher test was applied to detect significant differences among treatments. Although blastocyst rates for NIC and 5TM did not statistically differ, 5TM+25pRH and 15TM+50pRH groups resulted in lower blastocysts rates than the NIC [P < 0.05; 13/17 (76%), 6/15 (40%), 4/15 (26%) and 2/14 (14%) for NIC, 5TM, 5TM+25pRH and 15TM+50pRH respectively]. It was possible to detect the PCR product compatible with deletion of the entire region among TALEN target sites in 6/6 blastocysts (100%) from the group 5TM, 3/4 blastocysts (75%) from the group 5TM+25pRH and 2/2 (100%) blastocysts from the group 15TM+50pRH. HR was detected in 1/2 (50%) blastocysts injected with 15TM+50pRH and in 1/4 (25%) blastocysts injected with 5TM+25pRH, by PCR and sequencing of the PCR products. Our results indicate that TALEN combined with homologous recombination constitutes a powerful platform for the production of proteins of pharmaceutical interest under native regulatory sequences in the milk of genetically modified animals.

Therapeutic genome editing with engineered nucleases

SummaryTargeted genome editing with designer nucleases, such as zinc finger nucleases, TALE nucleases, and CRISPR-Cas nucleases, has heralded a new era in gene therapy. Genetic disorders, which have not been amenable to conventional gene-addition-type gene therapy approaches, such as disorders with dominant inheritance or diseases caused by mutations in tightly regulated genes, can now be treated by precise genome surgery. Moreover, engineered nucleases enable novel genetic interventions to fight infectious diseases or to improve cancer immunotherapies. Here, we review the development of the different classes of programmable nucleases, discuss the challenges and improvements in translating gene editing into clinical use, and give an outlook on what applications can expect to enter the clinic in the near future.