CRISPR‐Cas9 is widely used for genome editing, but its PAM sequence requirements limit its efficiency. In this study, we explore
Prime editors are reverse transcriptase (RT)-based genome-editing tools that utilize double-strand break (DSB)-free mechanisms to decrease off-target editing in genomes and enhance the efficiency of targeted insertions. The multiple prime editors that have been developed within a short span of time are a testament to the potential of this technique for targeted insertions. This is mainly because of the possibility of generation of all types of mutations including deletions, insertions, transitions, and transversions. Prime editing reverses several bottlenecks of gene editing technologies that limit the biotechnological applicability to produce designer crops. This review evaluates the status and evolution of the prime editing technique in terms of the types of editors available up to prime editor 5 and twin prime editors, and considers the developments in plants in a systematic manner. The various factors affecting prime editing efficiency in plants are discussed in detail, including the effects of temperature, the prime editing guide (peg)RNA, and RT template amongst others. We discuss the current obstructions, key challenges, and available resolutions associated with the technique, and consider future directions and further improvements that are feasible to elevate the efficiency in plants.
more » « less- PAR ID:
- 10541372
- Publisher / Repository:
- Oxford University Press
- Date Published:
- Journal Name:
- Journal of Experimental Botany
- Volume:
- 75
- Issue:
- 17
- ISSN:
- 0022-0957
- Format(s):
- Medium: X Size: p. 5344-5356
- Size(s):
- p. 5344-5356
- Sponsoring Org:
- National Science Foundation
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Summary Faecalibaculum rodentium Cas9 (FrCas9) for plant genome editing, especially in rice. FrCas9 recognizes a concise 5′‐NNTA‐3′ PAM, targeting more abundant palindromic TA sites in plant genomes than the 5′‐NGG‐3′ PAM sites of the most popular SpCas9. FrCas9 shows cleavage activities at all tested 5′‐NNTA‐3′ PAM sites with editing outcomes sharing the same characteristics of a typical CRISPR‐Cas9 system. FrCas9 induces high‐efficiency targeted mutagenesis in stable rice lines, readily generating biallelic mutants with expected phenotypes. We augment FrCas9's ability to generate larger deletions through fusion with the exonuclease, TREX2. TREX2‐FrCas9 generates much larger deletions than FrCas9 without compromise in editing efficiency. We demonstrate TREX2‐FrCas9 as an efficient tool for genetic knockout of a microRNA gene. Furthermore, FrCas9‐derived cytosine base editors (CBEs) and adenine base editors (ABE) are developed to produce targeted C‐to‐T and A‐to‐G base edits in rice plants. Whole‐genome sequencing‐based off‐target analysis suggests that FrCas9 is a highly specific nuclease. Expression of TREX2‐FrCas9 in plants, however, causes detectable guide RNA‐independent off‐target mutations, mostly as single nucleotide variants (SNVs). Together, we have established an efficient CRISPR‐FrCas9 system for targeted mutagenesis, large deletions, C‐to‐T base editing, and A‐to‐G base editing in plants. The simple palindromic TA motif in the PAM makes the CRISPR‐FrCas9 system a promising tool for genome editing in plants with an expanded targeting scope. -
Abstract Efficient and precise targeted insertion holds great promise but remains challenging in plant genome editing. An efficient nonhomologous end-joining-mediated targeted insertion method was recently developed by combining clustered regularly interspaced short palindromic repeat (CRISPR)/Streptococcus pyogenes CRISPR-associated nuclease 9 (SpCas9) gene editing with phosphorothioate modified double-stranded oligodeoxynucleotides (dsODNs). Yet, this approach often leads to imprecise insertions with no control over the insertion direction. Here, we compared the influence of chemical protection of dsODNs on efficiency of targeted insertion. We observed that CRISPR/SpCas9 frequently induced staggered cleavages with 1-nucleotide 5′ overhangs; we also evaluated the effect of donor end structures on the direction and precision of targeted insertions. We demonstrate that chemically protected dsODNs with 1-nucleotide 5′ overhangs significantly improved the precision and direction control of target insertions in all tested CRISPR targeted sites. We applied this method to endogenous gene tagging in green foxtail (Setaria viridis) and engineering of cis-regulatory elements for disease resistance in rice (Oryza sativa). We directionally inserted 2 distinct transcription activator-like effector binding elements into the promoter region of a recessive rice bacterial blight resistance gene with up to 24.4% efficiency. The resulting rice lines harboring heritable insertions exhibited strong resistance to infection by the pathogen Xanthomonas oryzae pv. oryzae in an inducible and strain-specific manner.
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Abstract Among CRISPR-Cas genome editing systems,
Streptococcus pyogenes Cas9 (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. -
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