TY - JOUR
T1 - Predictable NHEJ Insertion and Assessment of HDR Editing Strategies in Plants
AU - Molla, Kutubuddin A.
AU - Shih, Justin
AU - Wheatley, Matthew S.
AU - Yang, Yinong
N1 - Funding Information:
KM would like to acknowledge the United States-India Educational Foundation (USIEF), New Delhi and the US Department of State for a Fulbright Nehru Post-Doctoral Fellowship (Award No. 2265/FNPDR/2017). JS has been supported by a pre-doctoral fellowship from USDA/National Institute of Food and Agriculture (2020-67034-31727).
Funding Information:
This work was supported by the NSF Plant Genome Research Program (Grant 1740874) and by USDA/NIFA Hatch Act Appropriations under project PEN04659 and accession 1016432 to YY.
Publisher Copyright:
Copyright © 2022 Molla, Shih, Wheatley and Yang.
PY - 2022
Y1 - 2022
N2 - 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.
AB - 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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U2 - 10.3389/fgeed.2022.825236
DO - 10.3389/fgeed.2022.825236
M3 - Article
C2 - 35481279
AN - SCOPUS:85134201114
SN - 2673-3439
VL - 4
JO - Frontiers in Genome Editing
JF - Frontiers in Genome Editing
M1 - 825236
ER -