Systematic optimization of Cas12a base editors in wheat and maize using the ITER platform
bioRxiv (Cold Spring Harbor Laboratory)(2022)
摘要
AbstractThe ever-increasing number of CRISPR components creates a significant burden when developing new genome engineering tools. Plant biotechnology in particular has few high-throughput options to perform iterative design-build-test-learn cycles when creating new gene-editing reagents. We have established ITER (Iterative Testing of Editing Reagents) based on arrayed protoplast transfections and high-content imaging, allowing one optimization cycle – from design to results– within three weeks. We validated ITER in wheat and maize protoplasts using Cas9 cytosine and adenine base editors. Given that previous LbCas12a-ABEs have low or no activity in plants, we used ITER to develop an optimized LbCas12a-ABE. We show that the sequential improvement of five components –NLS, crRNA, LbCas12a, adenine deaminase and linker– led to a remarkable increase in ABE activity from almost undetectable levels to 40% on an extrachromosomal GFP reporter. We confirmed the activity of LbCas12a-ABE at endogenous targets and in stable wheat transformants and leveraged these improvements to develop a highly mutagenic LbCas12a nuclease and LbCas12a-CBE. 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.
更多查看译文
关键词
cas12a base editors,wheat,maize,systematic optimization
AI 理解论文
溯源树
样例
生成溯源树,研究论文发展脉络
Chat Paper
正在生成论文摘要