Novel precision genetic technologies such as CRISPR/Cas9 genome editing technology offer novel avenues to a better understanding the mechanisms of diseases. Using CRISPR/Cas9 we are able to precisely modify the mouse or the human genome by creating knockout or a specific single nucleotide change to enable the study of the function of the gene of interest. The generation of these models lies on the ability of Cas9 to create a double strand break in the DNA and the repair to occur via the error prone Non-Homologous End Joining (NHEJ) or the precise Homology direct Repair (HDR) mechanisms. A large body of work has been recently dedicated to either improve the technology to generate efficiently knockout or knock-in mouse models (point mutations, tags or floxed alleles). The rapid pace of the technology development has generated a lot of excitement but also some disappointment over the lack of reproducibility of the experiments. This led to a substantial loss of research time and funding. This short presentation will give an overview over the initiatives undertaken to tackle irreproducible research and we will discuss how to address these problems in the field of gene editing technology.

Learning objectives:

1. Generation of knock-in mouse model using CRISPR/Cas9
2. How to design robust and reproducible experiments

Banana (Musa spp.) is one of the major staple food crops grown in over 140 countries in the subtropics and tropics with annual global production of around 148 million metric, feeding about 500 million people. It is a valuable food security and cash crop as it can be cultivated in diverse environments and produces fruits throughout the year in favourable weather conditions. Its production is largely constrained by diseases and pests in addition to other factors like declining soil fertility, narrow genetic diversity in germplasm, and inadequate availability of clean planting material. There is need to develop banana varieties with multiple and durable resistance to combat biotic stresses. Modern breeding tools, including genome editing, can be applied for the improvement of banana bypassing the natural bottlenecks of traditional breeding. Intensive efforts using genetic modification have been made to develop improved banana varieties with resistance to biotic stresses. Genome editing, an emerging powerful tool, can be applied for developing durable resistance to diseases. CRISPR/Cas9-based genome editing has been lately established for banana, paving the way for functional genomics allowing identification of genes associated with disease resistant trait, which could be used for the improvement of banana. The genome editing research at IITA for improvement of banana will be presented during this seminar.

CRISPR based gene editing has proven to be an incredibly powerful tool for studying functions of individual proteins, understanding the role of multi-gene pathways, and even knocking out protein function genome-wide for drug candidate screening. With rapidly increasing amounts of information, reagent formats, and downstream applications, there is a lot to consider before beginning a CRISPR experiment.
Key questions to address include: What to consider when choosing a representative biological system? Which reagents are most suitable for your experimental aim? What techniques and equipment will be required for end-point data analysis?

In this webinar we will discuss:

• tips for choosing a cell line/type and the reagent formats best suited for that system
• methods for optimizing reagent delivery
• ways of enriching for edited cells
• techniques for analyzing post-edited cells

Learning objectives:

1. Identify key criteria necessary for determining the ideal cell line and reagent format for your gene editing experiment.

2. Outline techniques and equipment required for end-point data analysis

CRISPR has evolved into a collection of technologies for basic research with new applications for precise gene editing, screening and chromosomal detection. With recent advances in CRISPR, therapeutic applications now extend not only to humans but also to animal health and the environment. This presentation will feature original research from Merck R&D, as well as a variety of Merck CRISPR reagents for use in screening and editing of mammalian cell lines and editing of non-mammalian model organisms.

Learning Objectives:

1. Using CRISPR reagents in mammalian cell lines and non-mammalian models

2. Using CRISPR reagents for screening applications

3. Apply real life examples of how CRISPR gene editing can improve the environment

CRISPR-Cas9 based gene editing is a powerful tool which can induce heritable mutations at desired genomic loci without leaving transgenic trace in plants. Here we constructed a robust, multiplex CRISPR/Cas9 system, including 12 intermediate sgRNA vectors and 6 binary vectors (https://www.addgene.org/Yao-Guang_Liu/), to accommodate diversified demanding in plants. We further developed an integrated CRISPR-GE web-based toolkit (http://skl.scau.edu.cn/) to fulfill bio-informatic processing and analysis requirements while using CRISPR. This CRISPR vector system has been applied in many crops including rice. As an example, we created glutinous rice with the transgene cassette segregated within two generations using our vector. This high efficiency, and user-friendly CRISPR platform are benefiting plant research and crop improvements.  

Learning objectives:

1. Introducing the multiplex CRISPR-Cas9 vector system
2. CRISPR-GE web based toolkit for designing CRISPR experiment and analyzing mutants in plant

CRISPR/Cas9 technology has become very popular in the last 5 years and has profoundly changed our way to do genetically engineered animals. I will present the latest development in genome editing at PHENOMIN-ICS.  From  the easy high throughput KO production for the International Mouse Phenotyping consortium  (https://www.mousephenotype.org/) to the generation of complex models, different examples (from simple point mutations to conditional knock-outs) will be shown and discussed.  The benefits of oocyte electroporation, which is much more efficient than microinjection and has allowed to reduce the number of animal needed for all experiments, will be presented. The pertinence of the use of CRISPR base editing (cytidine deaminase) for specific mutations was assessed and the results of our pilot experiment will be shown. And finally, the advantages of the use of embryonic stem cells (with the help of CRISPR) for genetic modifications larger than small knock-ins will be presented.

Insects are the largest and most diverse arthropod group. Many insect species are agricultural pests or vectors of numerous diseases. In fact, the mosquito is the deadliest animal on the earth killing around 725,000 people per year. Traditional methods of insect population control relying on insecticides are not effective and harmful to ecological habitats. In 2003, Austin Burt suggested that gene drives would revolutionize insect population control. According to the standard Mendelian inheritance, one copy of any gene has a 50% chance in being transmitted to the offspring. A single copy of a gene drive can promote its transmission to 95% of offspring. This super-Mendelian inheritance results in the spread of a gene drive and linked cargo genes into a local population, even when their carriers are not completely fit. The recent discovery of CRISPR/Cas9 technology simplified engineering synthetic gene drives for diverse animal species. Nowadays, synthetic gene drives have been engineered in laboratory mosquitoes for population repression or replacement. In other words, the release of gene drive mosquitoes into a natural population could block transmission of a mosquito-borne disease such as malaria, dengue fever, zika, etc. by suppressing mosquito numbers or by replacing wild mosquitos for lab-engineered disease refractory counterparts. However, many gene drives induce resistance mutations that accumulate and eventually suppress the spread of gene drives. To address these concerns, the CRISPR/Cas9 technology was leveraged to develop novel systems, such as resistance-proof gene drives and precision guided Sterile Insect Technique (pgSIT). In my presentation, I will explain how gene drives force their inheritance, and discuss the current progress and future applications of CRISPR/Cas9 technology for insect population control.

Learning objectives:
1. Comprehend how CRISPR/Cas9-mediated gene drive works
2. Learn how and why CRISPR/Cas9-mediated gene drive fails