CRISPR-Cas12a is an RNA-guided, programmable genome editing enzyme found within bacterial adaptive immune pathways. Unlike CRISPR-Cas9, Cas12a uses only a single catalytic site to both cleave target double-stranded DNA (dsDNA) (cis-activity) and indiscriminately degrade single-stranded DNA (ssDNA) (trans-activity). To investigate how the relative potency of cis- versus trans-DNase activity affects Cas12a-mediated genome editing, we first used structure-guided engineering to generate variants of Lachnospiraceae bacterium Cas12a that selectively disrupt trans-activity. The resulting engineered mutant with the biggest differential between cis- and trans-DNase activity in vitro showed minimal genome editing activity in human cells, motivating a second set of experiments using directed evolution to generate additional mutants with robust genome editing activity. Notably, these engineered and evolved mutants had enhanced ability to induce homology-directed repair (HDR) editing by 2–18-fold compared to wild-type Cas12a when using HDR donors containing mismatches with crRNA at the PAM-distal region. Finally, a site-specific reversion mutation produced improved Cas12a (iCas12a) variants with superior genome editing efficiency at genomic sites that are difficult to edit using wild-type Cas12a. This strategy establishes a pipeline for creating improved genome editing tools by combining structural insights with randomization and selection. The available structures of other CRISPR-Cas enzymes will enable this strategy to be applied to improve the efficacy of other genome-editing proteins.
Cas12a (formerly known as Cpf1), the class II type V CRISPR nuclease, has been widely used for genome editing in mammalian cells and plants due to its distinct characteristics from Cas9. Despite being one of the most robust Cas12a nucleases, LbCas12a in general is less efficient than SpCas9 for genome editing in human cells, animals, and plants.
To improve the editing efficiency of LbCas12a, we conduct saturation mutagenesis in
Our results demonstrate that LbCas12a-RVQ is a powerful tool for genome editing in human cells while LbCas12a-RRV confers robust genome editing in plants. Our study reveals the tremendous potential of these LbCas12a variants for advancing precision genome editing applications across a wide range of organisms.
- NSF-PAR ID:
- 10410171
- Publisher / Repository:
- Springer Science + Business Media
- Date Published:
- Journal Name:
- Genome Biology
- Volume:
- 24
- Issue:
- 1
- ISSN:
- 1474-760X
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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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.
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Summary CRISPR‐Cas9 is widely used for genome editing, but its PAM sequence requirements limit its efficiency. In this study, we explore
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.
