Journal of Genetics and Genomics

Abstract:
The CRISPR-Cas revolution is taking place in virtually all fields of life sciences. Harnessing DNA cleavage with the CRISPR-Cas9 system of Streptococcus pyogenes has proven to be extraordinarily simple and efficient, relying only on the design of a synthetic single guide RNA (sgRNA) and its co-expression with Cas9. Here, we review the progress in the design of sgRNA from the original dual RNA guide for S. pyogenes and Staphylococcus aureus Cas9 (SpCas9 and SaCas9). New assays for genome-wide identification of off-targets have provided important insights into the issue of cleavage specificity in vivo. At the same time, the on-target activity of thousands of guides has been determined. These data have led to numerous online tools that facilitate the selection of guide RNAs in target sequences. It appears that for most basic research applications, cleavage activity can be maximized and off-targets minimized by carefully choosing guide RNAs based on computational predictions. Moreover, recent studies of Cas proteins have further improved the flexibility and precision of the CRISPR-Cas toolkit for genome editing. Inspired by the crystal structure of the complex of sgRNA-SpCas9 bound to target DNA, several variants of SpCas9 have recently been engineered, either with novel protospacer adjacent motifs (PAMs) or with drastically reduced off-targets. Novel Cas9 and Cas9-like proteins called Cpf1 have also been characterized from other bacteria and will benefit from the insights obtained from SpCas9. Genome editing with CRISPR-Cas9 may also progress with better understanding and control of cellular DNA repair pathways activated after Cas9-induced DNA cleavage.

Targeted Gene Manipulation in Plants Using the CRISPR/Cas Technology

Dandan Zhang, Zhenxiang Li, Jian-Feng Li

2016, 43(5): 251-262. doi: 10.1016/j.jgg.2016.03.001

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Abstract:
The CRISPR/Cas technology is emerging as a revolutionary genome editing tool in diverse organisms including plants, and has quickly evolved into a suite of versatile tools for sequence-specific gene manipulations beyond genome editing. Here, we review the most recent applications of the CRISPR/Cas toolkit in plants and also discuss key factors for improving CRISPR/Cas performance and strategies for reducing the off-target effects. Novel technical breakthroughs in mammalian research regarding the CRISPR/Cas toolkit will also be incorporated into this review in hope to stimulate prospective users from the plant research community to fully explore the potential of these technologies.

Genome Editing: From Drosophila to Non-Model Insects and Beyond

Yueping Huang, Zhiping Liu, Yikang S. Rong

2016, 43(5): 263-272. doi: 10.1016/j.jgg.2016.04.007

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Abstract:
Insect is the largest group of animals on land. Many insect species inflict economical and health losses to humans. Yet many more benefit us by helping to maintain balances in our ecosystem. The benefits that insects offer remain largely untapped, justifying our continuing efforts to develop tools to better understand their biology and to better manage their activities. Here we focus on reviewing the progresses made in the development of genome engineering tools for model insects. Instead of detailed descriptions of the molecular mechanisms underlying each technical advance, we focus our discussion on the logistics for implementing similar tools in non-model insects. Since none of the tools were developed specific for insects, similar approaches can be applied to other non-model organisms.

CRISPR Double Cutting through the Labyrinthine Architecture of 3D Genomes

Haiyan Huang, Qiang Wu

2016, 43(5): 273-288. doi: 10.1016/j.jgg.2016.03.006

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Abstract:
The genomes are organized into ordered and hierarchical topological structures in interphase nuclei. Within discrete territories of each chromosome, topologically associated domains (TADs) play important roles in various nuclear processes such as gene regulation. Inside TADs separated by relatively constitutive boundaries, distal elements regulate their gene targets through specific chromatin-looping contacts such as long-distance enhancer-promoter interactions. High-throughput sequencing studies have revealed millions of potential regulatory DNA elements, which are much more abundant than the mere ∼20,000 genes they control. The recently emerged CRISPR-Cas9 genome editing technologies have enabled efficient and precise genetic and epigenetic manipulations of genomes. The multiplexed and high-throughput CRISPR capabilities facilitate the discovery and dissection of gene regulatory elements. Here, we describe the applications of CRISPR for genome, epigenome, and 3D genome editing, focusing on CRISPR DNA-fragment editing with Cas9 and a pair of sgRNAs to investigate topological folding of chromatin TADs and developmental gene regulation.

Spermatogenic Cell-Specific Gene Mutation in Mice via CRISPR-Cas9

Meizhu Bai, Dan Liang, Yinghua Wang, Qing Li, Yuxuan Wu, Jinsong Li

2016, 43(5): 289-296. doi: 10.1016/j.jgg.2016.02.003

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Abstract:
Tissue-specific knockout technology enables the analysis of the gene function in specific tissues in adult mammals. However, conventional strategy for producing tissue-specific knockout mice is a time- and labor-consuming process, restricting rapid study of the gene function in vivo. CRISPR-Cas9 system from bacteria is a simple and efficient gene-editing technique, which has enabled rapid generation of gene knockout lines in mouse by direct injection of CRISPR-Cas9 into zygotes. Here, we demonstrate CRISPR-Cas9-mediated spermatogenic cell-specific disruption of Scp3 gene in testes in one step. We first generated transgenic mice by pronuclear injection of a plasmid containing Hspa2 promoter driving Cas9 expression and showed Cas9 specific expression in spermatogenic cells. We then produced transgenic mice carrying Hspa2 promoter driven Cas9 and constitutive expressed sgRNA targeting Scp3 gene. Male founders were infertile due to developmental arrest of spermatogenic cells while female founders could produce progeny normally. Consistently, male progeny from female founders were infertile and females could transmit the transgenes to the next generation. Our study establishes a CRISPR-Cas9-based one-step strategy to analyze the gene function in adult tissues by a temporal-spatial pattern.

TALEN-Mediated Homologous Recombination Produces Site-Directed DNA Base Change and Herbicide-Resistant Rice

Ting Li, Bo Liu, Chih Ying Chen, Bing Yang

2016, 43(5): 297-305. doi: 10.1016/j.jgg.2016.03.005

Abstract (128) HTML PDF (2)

Abstract:
Over the last decades, much endeavor has been made to advance genome editing technology due to its promising role in both basic and synthetic biology. The breakthrough has been made in recent years with the advent of sequence-specific endonucleases, especially zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPRs) guided nucleases (e.g., Cas9). In higher eukaryotic organisms, site-directed mutagenesis usually can be achieved through non-homologous end-joining (NHEJ) repair to the DNA double-strand breaks (DSBs) caused by the exogenously applied nucleases. However, site-specific gene replacement or genuine genome editing through homologous recombination (HR) repair to DSBs remains a challenge. As a proof of concept gene replacement through TALEN-based HR in rice (Oryza sativa), we successfully produced double point mutations in rice acetolactate synthase gene (OsALS) and generated herbicide resistant rice lines by using TALENs and donor DNA carrying the desired mutations. After ballistic delivery into rice calli of TALEN construct and donor DNA, nine HR events with different genotypes ofOsALS were obtained in T₀ generation at the efficiency of 1.4%–6.3% from three experiments. The HR-mediated gene edits were heritable to the progeny of T₁ generation. The edited T₁ plants were as morphologically normal as the control plants while displayed strong herbicide resistance. The results demonstrate the feasibility of TALEN-mediated genome editing in rice and provide useful information for further genome editing by other nuclease-based genome editing platforms.

A Zebrafish Model of 5q-Syndrome Using CRISPR/Cas9 Targeting RPS14 Reveals a p53-Independent and p53-Dependent Mechanism of Erythroid Failure

Jason Ear, Jessica Hsueh, Melinda Nguyen, QingHua Zhang, Victoria Sung, Rajesh Chopra, Kathleen M. Sakamoto, Shuo Lin

2016, 43(5): 307-318. doi: 10.1016/j.jgg.2016.03.007

Abstract (56) HTML PDF (1)

Abstract:
5q-syndrome is a distinct form of myelodysplastic syndrome (MDS) where a deletion on chromosome 5 is the underlying cause. MDS is characterized by bone marrow failures, including macrocytic anemia. Genetic mapping and studies using various models support the notion that ribosomal protein S14 (RPS14) is the candidate gene for the erythroid failure. Targeted disruption of RPS14 causes an increase in p53 activity and p53-mediated apoptosis, similar to what is observed with other ribosomal proteins. However, due to the higher risk for cancer development in patients with ribosome deficiency, targeting the p53 pathway is not a viable treatment option. To better understand the pathology of RPS14 deficiency in 5q-deletion, we generated a zebrafish model harboring a mutation in the RPS14 gene. This model mirrors the anemic phenotype seen in 5q-syndrome. Moreover, the anemia is due to a late-stage erythropoietic defect, where the erythropoietic defect is initially p53-independent and then becomes p53-dependent. Finally, we demonstrate the versatility of this model to test various pharmacological agents, such as RAP-011, L-leucine, and dexamethasone in order to identify molecules that can reverse the anemic phenotype.

Delivery of Cas9 Protein into Mouse Zygotes through a Series of Electroporation Dramatically Increases the Efficiency of Model Creation

Wenbo Wang, Peter M. Kutny, Shannon L. Byers, Charles J. Longstaff, Michael J. DaCosta, Changhong Pang, Yingfan Zhang, Robert A. Taft, Frank W. Buaas, Haoyi Wang

2016, 43(5): 319-327. doi: 10.1016/j.jgg.2016.02.004

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Abstract:
Previously we established Zygote Electroporation of Nucleases (ZEN) technology as an efficient and high-throughput way to generate genetically modified mouse models. However, there were significant variations of the targeting efficiency among different genomic loci using our previously published protocol. In this study, we improved the ZEN technology by delivering Cas9 protein into mouse zygotes through a series of electroporation. Using this approach, we were able to introduce precise nucleotide substitutions, large segment deletion and short segment insertion into targeted loci with high efficiency.

Generation of a Double KO Mouse by Simultaneous Targeting of the Neighboring Genes Tmem176a and Tmem176b Using CRISPR/Cas9: Key Steps from Design to Genotyping

Aurélie Lemoine, Gaëlle Chauveau-Le Friec, Francina Langa, Cédric Louvet

2016, 43(5): 329-340. doi: 10.1016/j.jgg.2016.04.004

Abstract (111) HTML PDF (7)

Abstract:
The CRISPR/Cas9 system has been tailored to a revolutionary genetic tool because of its remarkable simplicity and efficacy. While complex genome editing in the mouse since the 1990s has been dominated by the use of embryonic stem (ES) cells, CRISPR/Cas9 now offers a versatile and fast approach to precisely modify virtually any DNA regions directly in mouse zygotes. Yet, this relative simplicity does not preclude a conscientious preparatory work that is often neglected when initiating a project. Here, we describe the key steps leading to successful generation of a double knockout (KO) mouse by simultaneously targeting two homolog genes, Tmem176a and Tmem176b, which are located in the same genomic locus. Additionally, we show that similar efficiency can be obtained in a mixed genetic background or directly in the C57BL/6 inbred strain. Thus, presented as a detailed case study that should be helpful to the non-specialists, we focus on the genotyping strategy to anticipate the various possibilities.

A Rapid and Cost-Effective Method for Genotyping Genome-Edited Animals: A Heteroduplex Mobility Assay Using Microfluidic Capillary Electrophoresis

Vanessa Chenouard, Lucas Brusselle, Jean-Marie Heslan, Séverine Remy, Séverine Ménoret, Claire Usal, Laure-Hélène Ouisse, Tuan Huy NGuyen, Ignacio Anegon, Laurent Tesson

2016, 43(5): 341-348. doi: 10.1016/j.jgg.2016.04.005

Abstract (65) HTML PDF (1)

Abstract:
The recent emergence and application of engineered endonucleases have led to the development of genome editing tools capable of rapidly implementing various targeted genome editions in a wide range of species. Moreover, these novel tools have become easier to use and have resulted in a great increase of applications. Whilst gene knockout (KO) or knockin (KI) animal models are relatively easy to achieve, there is a bottleneck in the detection and analysis of these mutations. Although several methods exist to detect these targeted mutations, we developed a heteroduplex mobility assay on an automated microfluidic capillary electrophoresis system named HMA-CE in order to accelerate the genotyping process. The HMA-CE method uses a simple PCR amplification of genomic DNA (gDNA) followed by an automated capillary electrophoresis step which reveals a heteroduplexes (HD) signature for each mutation. This allows efficient discrimination of wild-type and genome-edited animals down to the single base pair level.

2016 Vol. 43, No. 5