An official website of the United States government
The Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein system (CRISPR/Cas) has recently become the most powerful tool available for genome engineering in various organisms. With efficient and proper expression of multiple guide RNAs (gRNAs), the CRISPR/Cas system is particularly suitable for multiplex genome editing. During the past several years, different CRISPR/Cas expression strategies, such as two-component transcriptional unit, single transcriptional unit, and bidirectional promoter systems, have been developed to efficiently express gRNAs as well as Cas nucleases. Significant progress has been made to optimize gRNA production using different types of promoters and RNA processing strategies such as ribozymes, endogenous RNases, and exogenous endoribonuclease (Csy4). Besides being constitutively and ubiquitously expressed, inducible and spa- tiotemporal regulations of gRNA expression have been demonstrated using inducible, tissue-specific, and/or synthetic promoters for specific research purposes. Most recently, the emergence of CRISPR/Cas ribonucleoprotein delivery methods, such as engineered nanoparticles, further revolutionized trans- gene-free and multiplex genome editing. In this review, we discuss current strategies and future per- spectives for efficient expression and engineering of gRNAs with a goal to facilitate CRISPR/Cas-based multiplex genome editing.
more »
« less
Applications of CRISPR/Cas13-Based RNA Editing in Plants
The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) system is widely used as a genome-editing tool in various organisms, including plants, to elucidate the fundamental understanding of gene function, disease diagnostics, and crop improvement. Among the CRISPR/Cas systems, Cas9 is one of the widely used nucleases for DNA modifications, but manipulation of RNA at the post-transcriptional level is limited. The recently identified type VI CRISPR/Cas systems provide a platform for precise RNA manipulation without permanent changes to the genome. Several studies reported efficient application of Cas13 in RNA studies, such as viral interference, RNA knockdown, and RNA detection in various organisms. Cas13 was also used to produce virus resistance in plants, as most plant viruses are RNA viruses. However, the application of CRISPR/Cas13 to studies of plant RNA biology is still in its infancy. This review discusses the current and prospective applications of CRISPR/Cas13-based RNA editing technologies in plants.
more »
« less
- PAR ID:
- 10390260
- Date Published:
- Journal Name:
- Cells
- Volume:
- 11
- Issue:
- 17
- ISSN:
- 2073-4409
- Page Range / eLocation ID:
- 2665
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
Cas12a, also known as Cpf1, is a highly versatile CRISPR-Cas enzyme that has been widely used in genome editing. Unlike its well-known counterpart, Cas9, Cas12a has unique features that make it a highly efficient genome editing tool at AT-rich genomic regions. To enrich the CRISPR-Cas12a plant genome editing toolbox, we explored 17 novel Cas12a orthologs for their genome editing capabilities in plants. Out of them, Ev1Cas12a and Hs1Cas12a showed efficient multiplexed genome editing in rice and tomato protoplasts. Notably, Hs1Cas12a exhibited greater tolerance to lower temperatures. Moreover, Hs1Cas12a generated up to 87.5% biallelic editing in rice T0plants. Both Ev1Cas12a and Hs1Cas12a achieved effective editing in poplar T0plants, with up to 100% of plants edited, albeit with high chimerism. Taken together, the efficient genome editing demonstrated by Ev1Cas12a and Hs1Cas12a in both monocot and dicot plants highlights their potential as promising genome editing tools in plant species and beyond.more » « less
-
null (Ed.)Abstract CRISPR–Cas is an anti-viral mechanism of prokaryotes that has been widely adopted for genome editing. To make CRISPR–Cas genome editing more controllable and safer to use, anti-CRISPR proteins have been recently exploited to prevent excessive/prolonged Cas nuclease cleavage. Anti-CRISPR (Acr) proteins are encoded by (pro)phages/(pro)viruses, and have the ability to inhibit their host's CRISPR–Cas systems. We have built an online database AcrDB (http://bcb.unl.edu/AcrDB) by scanning ∼19 000 genomes of prokaryotes and viruses with AcrFinder, a recently developed Acr-Aca (Acr-associated regulator) operon prediction program. Proteins in Acr-Aca operons were further processed by two machine learning-based programs (AcRanker and PaCRISPR) to obtain numerical scores/ranks. Compared to other anti-CRISPR databases, AcrDB has the following unique features: (i) It is a genome-scale database with the largest collection of data (39 799 Acr-Aca operons containing Aca or Acr homologs); (ii) It offers a user-friendly web interface with various functions for browsing, graphically viewing, searching, and batch downloading Acr-Aca operons; (iii) It focuses on the genomic context of Acr and Aca candidates instead of individual Acr protein family and (iv) It collects data with three independent programs each having a unique data mining algorithm for cross validation. AcrDB will be a valuable resource to the anti-CRISPR research community.more » « less
-
Abstract CRISPR/Cas systems have been widely used for genome engineering in many plant species, while their potentials have remained largely untapped in fruit crops, particularly in pear, due to the high levels of genomic heterozygosity and difficulties in tissue culture and stable transformation. To date, only few reports on application of CRISPR/Cas9 system in pear have been documented with a very low editing efficiency. Here, we report a highly efficient CRISPR toolbox for loss-of-function and gain-of-function research in pear. We compared four different CRISPR/Cas9 expression systems for loss-of-function analysis and identified a potent system that showed nearly 100% editing efficiency for multi-site mutagenesis. To expand targeting scope, we further tested different CRISPR/Cas12a and Cas12b systems in pear for the first time, albeit with low editing efficiency. In addition, we established a CRISPR activation (CRISPRa) system for multiplexed gene activation in pear calli for gain-of-function analysis. Furthermore, we successfully engineered the anthocyanin and lignin biosynthesis pathways using both CRISPR/Cas9 and CRISPRa systems in pear calli. Taken together, we build a highly efficient CRISPR toolbox for genome editing and gene regulation, paving the way for functional genomics studies as well as molecular breeding in pear.more » « less
-
Abstract Among CRISPR-Cas genome editing systems,Streptococcus pyogenesCas9 (SpCas9), sourced from a human pathogen, is the most widely used. Here, through in silico data mining, we have established an efficient plant genome engineering system using CRISPR-Cas9 from probioticLactobacillus rhamnosus. We have confirmed the predicted 5’-NGAAA-3’ PAM via a bacterial PAM depletion assay and showcased its exceptional editing efficiency in rice, wheat, tomato, and Larix cells, surpassing LbCas12a, SpCas9-NG, and SpRY when targeting the identical sequences. In stable rice lines, LrCas9 facilitates multiplexed gene knockout through coding sequence editing and achieves gene knockdown via targeted promoter deletion, demonstrating high specificity. We have also developed LrCas9-derived cytosine and adenine base editors, expanding base editing capabilities. Finally, by harnessing LrCas9’s A/T-rich PAM targeting preference, we have created efficient CRISPR interference and activation systems in plants. Together, our work establishes CRISPR-LrCas9 as an efficient and user-friendly genome engineering tool for diverse applications in crops and beyond.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.3390/cells11172665
