Systematic optimization of Cas12a base editors in wheat and maize using the ITER platform.

BACKGROUND: Testing an ever-increasing number of CRISPR components is challenging when developing new genome engineering tools. Plant biotechnology has few high-throughput options to perform iterative design-build-test-learn cycles of gene-editing reagents. To bridge this gap, we develop ITER (Iterative Testing of Editing Reagents) based on 96-well arrayed protoplast transfections and high-content imaging. RESULTS: We validate ITER in wheat and maize protoplasts using Cas9 cytosine and adenine base editors (ABEs), allowing one optimization cycle - from design to results - within 3 weeks. Given that previous LbCas12a-ABEs have low or no activity in plants, we use ITER to develop an optimized LbCas12a-ABE. We show that sequential improvement of five components - NLS, crRNA, LbCas12a, adenine deaminase, and linker - leads to a remarkable increase in activity from almost undetectable levels to 40% on an extrachromosomal GFP reporter. We confirm the activity of LbCas12a-ABE at endogenous targets in protoplasts and obtain base-edited plants in up to 55% of stable wheat transformants and the edits are transmitted to T1 progeny. We leverage these improvements to develop a highly mutagenic LbCas12a nuclease and a LbCas12a-CBE demonstrating that the optimizations can be broadly applied to the Cas12a toolbox. CONCLUSION: Our data show that ITER is a sensitive, versatile, and high-throughput platform that can be harnessed to accelerate the development of genome editing technologies in plants. We use ITER to create an efficient Cas12a-ABE by iteratively testing a large panel of vector components. ITER will likely be useful to create and optimize genome editing reagents in a wide range of plant species.

Using WormBase ParaSite: An Integrated Platform for Exploring Helminth Genomic Data.

WormBase ParaSite ( parasite.wormbase.org ) is a comprehensive resource for the genomes of parasitic nematodes and flatworms (helminths). It currently includes genomic data for over 100 helminth species, adding value by way of consistent functional annotation, gene comparative analysis and gene expression analysis. We provide several ways of exploring the data including a choice of genome browsers, genome and gene summary pages, text and sequence searching, a query wizard, bulk downloads, and programmatic interfaces. WormBase ParaSite is released three to six times per year, and is developed in collaboration with WormBase ( www.wormbase.org ) and Ensembl Genomes ( www.ensemblgenomes.org ).

WormBase ParaSite - a comprehensive resource for helminth genomics.

The number of publicly available parasitic worm genome sequences has increased dramatically in the past three years, and research interest in helminth functional genomics is now quickly gathering pace in response to the foundation that has been laid by these collective efforts. A systematic approach to the organisation, curation, analysis and presentation of these data is clearly vital for maximising the utility of these data to researchers. We have developed a portal called WormBase ParaSite (http://parasite.wormbase.org) for interrogating helminth genomes on a large scale. Data from over 100 nematode and platyhelminth species are integrated, adding value by way of systematic and consistent functional annotation (e.g. protein domains and Gene Ontology terms), gene expression analysis (e.g. alignment of life-stage specific transcriptome data sets), and comparative analysis (e.g. orthologues and paralogues). We provide several ways of exploring the data, including genome browsers, genome and gene summary pages, text search, sequence search, a query wizard, bulk downloads, and programmatic interfaces. In this review, we provide an overview of the back-end infrastructure and analysis behind WormBase ParaSite, and the displays and tools available to users for interrogating helminth genomic data.