Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing

The type-V CRISPR effector Cas12i, with its smaller size, short crRNA guiding, and self-processing features, is a potentially versatile genome editing tool. By screening Cas12i proteins from a metagenomic database, we identified a natural variant with high activity in mammalian cells, named as xCas12i. We further engineered the PAM-interacting, REC, and RuvC domains for enhanced cleavage activity and specificity. This variant, named as high-fidelity Cas12Max, exhibited robust genome editing activity and minimal off-target activity with a broad 5'-TN recognition profile. With the fusion of deaminase TadA8e and further optimization of xCas12i, the base editor dCas12i-Tad8e also showed the high editing efficiency. This study provides highly efficient and specific tools for gene therapy.

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“…Note that two groups have recently reported improved Cas12i2 using similar strategies in this study 31, 32 .…”

Section: Discussionsupporting

confidence: 62%

An engineered xCas12i with high activity, high specificity and broad PAM range

Zhang

Kong

Xue

et al. 2022

Preprint

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“…Our near-saturation screen revealed a large pool of point mutations with strong effects that enhanced the efficiency of LbCas12a (Additional file 2 : Table S1) and further demonstrated that combining selected mutations can be sufficient to generate a potent nuclease, such as LbCas12a-RVQ. This trend appears to hold true for other type V nucleases, such as Cas12i2 [ 28 – 30 ], CasΦ/Cas12j2 [ 31 , 32 ], and Cas14 [ 33 ]. It is intriguing to ask why the natural evolution of these nucleases in bacteria did not reach supreme DNA binding affinity.…”

Section: Discussionmentioning

confidence: 80%

Boosting genome editing efficiency in human cells and plants with novel LbCas12a variants

Zhang

et al. 2023

Genome Biol

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Background 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. Results To improve the editing efficiency of LbCas12a, we conduct saturation mutagenesis in E. coli and identify 1977 positive point mutations of LbCas12a. We selectively assess the editing efficiency of 56 LbCas12a variants in human cells, identifying an optimal LbCas12a variant (RVQ: G146R/R182V/E795Q) with the most robust editing activity. We further test LbCas12a-RV, LbCas12a-RRV, and LbCas12a-RVQ in plants and find LbCas12a-RV has robust editing activity in rice and tomato protoplasts. Interestingly, LbCas12a-RRV, resulting from the stacking of RV and D156R, displays improved editing efficiency in stably transformed rice and poplar plants, leading to up to 100% editing efficiency in T0 plants of both plant species. Moreover, this high-efficiency editing occurs even at the non-canonical TTV PAM sites. Conclusions 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.

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“…However, native Cas12i2 is an inefficient editor due to weak binding to the target DNA. Semirational engineering for stronger DNA binding converted cas12i2 into a robust, highly specific, and therefore promising editing tool . In contrast to Cas12i2, Cas12i1, instead of introducing a DSB into the target DNA molecule, predominantly nicks the nontarget strand, which could create additional opportunities for highly specific genome editing …”

Section: Harnessing Class 2 Crispr Diversity For Applications In Geno...mentioning

confidence: 99%

“…138 In contrast to Cas12i2, Cas12i1, instead of introducing a DSB into the target DNA molecule, predominantly nicks the nontarget strand, 57 which could create additional opportunities for highly specific genome editing. 138 Cas12j, a Cas12 variant that ranges from 700 to 800 amino acids in size and has been found exclusively in bacteriophages, so far has not been used for endogenous genome editing, but mutants that rapidly cleave the target have been engineered and could be promising as editors. 139 Certain Cas12j proteins have been used with reporters in mammalian cells for indel formation.…”

Section: Applications In Genome Engineering and Beyondmentioning

confidence: 99%

Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox

2023

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CRISPR systems mediate adaptive immunity in bacteria and archaea through diverse effector mechanisms and have been repurposed for versatile applications in therapeutics and diagnostics thanks to their facile reprogramming with RNA guides. RNA-guided CRISPR-Cas targeting and interference are mediated by effectors that are either components of multisubunit complexes in class 1 systems or multidomain single-effector proteins in class 2. The compact class 2 CRISPR systems have been broadly adopted for multiple applications, especially genome editing, leading to a transformation of the molecular biology and biotechnology toolkit. The diversity of class 2 effector enzymes, initially limited to the Cas9 nuclease, was substantially expanded via computational genome and metagenome mining to include numerous variants of Cas12 and Cas13, providing substrates for the development of versatile, orthogonal molecular tools. Characterization of these diverse CRISPR effectors uncovered many new features, including distinct protospacer adjacent motifs (PAMs) that expand the targeting space, improved editing specificity, RNA rather than DNA targeting, smaller crRNAs, staggered and blunt end cuts, miniature enzymes, promiscuous RNA and DNA cleavage, etc. These unique properties enabled multiple applications, such as harnessing the promiscuous RNase activity of the type VI effector, Cas13, for supersensitive nucleic acid detection. class 1 CRISPR systems have been adopted for genome editing, as well, despite the challenge of expressing and delivering the multiprotein class 1 effectors. The rich diversity of CRISPR enzymes led to rapid maturation of the genome editing toolbox, with capabilities such as gene knockout, base editing, prime editing, gene insertion, DNA imaging, epigenetic modulation, transcriptional modulation, and RNA editing. Combined with rational design and engineering of the effector proteins and associated RNAs, the natural diversity of CRISPR and related bacterial RNA-guided systems provides a vast resource for expanding the repertoire of tools for molecular biology and biotechnology.

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Engineered Cas12i2 is a versatile high-efficiency platform for therapeutic genome editing

Cited by 19 publications

References 39 publications

An engineered xCas12i with high activity, high specificity and broad PAM range

An engineered xCas12i with high activity, high specificity and broad PAM range

Boosting genome editing efficiency in human cells and plants with novel LbCas12a variants

Discovery of Diverse CRISPR-Cas Systems and Expansion of the Genome Engineering Toolbox

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