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Predictable NHEJ Insertion and Assessment of HDR Editing Strategies in Plants

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Title Predictable NHEJ Insertion and Assessment of HDR Editing Strategies in Plants
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Creator Kutubuddin A. Molla, Justin Shih, Matthew S. Wheatley and Yinong Yang
 
Subject Genome editing, NHEJ, Prediction, Homology Directed Repair
 
Description Not Available
Canonical CRISPR-Cas9 genome editing technique has profoundly impacted the fields of plant biology, biotechnology, and crop improvement. Since non-homologous end joining (NHEJ) is usually considered to generate random indels, its high efficiency mutation is generally not pertinent to precise editing. Homology-directed repair (HDR) can mediate precise editing with supplied donor DNA, but it suffers from extreme low efficiency in higher plants. Therefore, precision editing in plants will be facilitated by the ability to predict NHEJ repair outcome and to improve HDR efficiency. Here, we report that NHEJ-mediated single nucleotide insertion at different rice genes is predictable based on DNA sequences at the target loci. Three mutation prediction tools (inDelphi, FORECasT, and SPROUT) have been validated in the rice plant system. We also evaluated the chimeric guide RNA (cgRNA) and Cas9-Retron precISe Parallel Editing via homologY (CRISPEY) strategies to facilitate donor template supply for improving HDR efficiency in Nicotiana benthamiana and rice. However, neither cgRNA nor CRISPEY improved plant HDR editing efficiency in this study. Interestingly, our data indicate that tethering of 200–250 nucleotides long sequence to either 5′ or 3′ ends of guide RNA did not significantly affect Cas9 cleavage activity.
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Date 2022-03-26T04:32:38Z
2022-03-26T04:32:38Z
2022-03-16
 
Type Book
 
Identifier Molla KA, Shih J, Wheatley MS and Yang Y (2022) Predictable NHEJ Insertion and Assessment of HDR Editing Strategies in Plants. Front. Genome Ed. 4:825236. doi: 10.3389/fgeed.2022.825236
https://www.frontiersin.org/articles/10.3389/fgeed.2022.825236/full
http://krishi.icar.gov.in/jspui/handle/123456789/70620
 
Language English
 
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Publisher Not Available