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Abstract 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.

CRISPR/Cas9 genome editing has been used extensively in a wide variety of plant species. Creation of loss-of-function alleles, promoter variants and mutant collections are a few of the many uses of genome editing. In a typical workflow for sexually reproducing species, plants are generated that contain an integrated CRISPR/Cas9 transgene. After editing of the gene of interest, T-DNA null segregants can be identified in the next generation that contain only the desired edit. However, maintained presence of the CRISPR/Cas9 transgene and continued editing in the subsequent generations offer a range of applications for model plants and crops. In this review, we define transgenerational gene editing (TGE) as the continued editing of CRISPR/Cas9 after a genetic cross. We discuss the concept of TGE, summarize the current main applications, and highlight special cases to illustrate the importance of TGE for plant genome editing research and breeding.

Advances in high-throughput sequencing have led to many new technologies for assessing genomes and population diversity. In spite of this, inexpensive and technically simple methods for efficiently pinpointing the location of transgenes and other specific sequences in large genomes are lacking. Here we report the development of a modified TA cloning and Illumina sequencing method called high-throughput sequencing to locate unmapped DNA fragments (HtStuf). Transgenic insertion sites were identified and confirmed in nine out of 10 transgenic soybean [ (L.) Merr.] lines, and major rearrangements of the transgene were detected in these lines. Additionally this method was used to map insertions of the introduced DNA transposon, , in four T6 lines derived from a single event. Fifteen of the insertion sites were validated with polymerase chain reaction. Together, these data demonstrate the simplicity and effectiveness of this novel sequencing method.