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Expanding the scope of plant genome engineering with Cas12a orthologs and highly multiplexable editing systems.
- Source :
-
Nature communications [Nat Commun] 2021 Mar 29; Vol. 12 (1), pp. 1944. Date of Electronic Publication: 2021 Mar 29. - Publication Year :
- 2021
-
Abstract
- CRISPR-Cas12a is a promising genome editing system for targeting AT-rich genomic regions. Comprehensive genome engineering requires simultaneous targeting of multiple genes at defined locations. Here, to expand the targeting scope of Cas12a, we screen nine Cas12a orthologs that have not been demonstrated in plants, and identify six, ErCas12a, Lb5Cas12a, BsCas12a, Mb2Cas12a, TsCas12a and MbCas12a, that possess high editing activity in rice. Among them, Mb2Cas12a stands out with high editing efficiency and tolerance to low temperature. An engineered Mb2Cas12a-RVRR variant enables editing with more relaxed PAM requirements in rice, yielding two times higher genome coverage than the wild type SpCas9. To enable large-scale genome engineering, we compare 12 multiplexed Cas12a systems and identify a potent system that exhibits nearly 100% biallelic editing efficiency with the ability to target as many as 16 sites in rice. This is the highest level of multiplex edits in plants to date using Cas12a. Two compact single transcript unit CRISPR-Cas12a interference systems are also developed for multi-gene repression in rice and Arabidopsis. This study greatly expands the targeting scope of Cas12a for crop genome engineering.
- Subjects :
- Agrobacterium tumefaciens
Alleles
Arabidopsis metabolism
Bacterial Proteins metabolism
Base Sequence
CRISPR-Associated Protein 9 genetics
CRISPR-Associated Protein 9 metabolism
CRISPR-Associated Proteins metabolism
Clustered Regularly Interspaced Short Palindromic Repeats
Crops, Agricultural
Endodeoxyribonucleases metabolism
Humans
Isoenzymes genetics
Isoenzymes metabolism
Oryza metabolism
Plants, Genetically Modified
RNA, Guide, CRISPR-Cas Systems genetics
RNA, Guide, CRISPR-Cas Systems metabolism
Sequence Alignment
Arabidopsis genetics
Bacterial Proteins genetics
CRISPR-Associated Proteins genetics
CRISPR-Cas Systems
Endodeoxyribonucleases genetics
Gene Editing methods
Genetic Engineering methods
Genome, Plant
Oryza genetics
Subjects
Details
- Language :
- English
- ISSN :
- 2041-1723
- Volume :
- 12
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Nature communications
- Publication Type :
- Academic Journal
- Accession number :
- 33782402
- Full Text :
- https://doi.org/10.1038/s41467-021-22330-w