Multiplexed Genome Engineering with Cas12a

Weisbach, Niels R.; Meijs, Ab; Platt, Randall J.

doi:10.1007/978-1-0716-1441-9_11

Cited by 5 publications

(3 citation statements)

References 19 publications

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“…Potentially, more important is the ability of Cas12a to process pre-crRNA, which provides for multiplexing, that is, co-expression of several guides on a single transcript for compact delivery, expression under inducible or state-dependent promoters, or for monitoring of gene expression . Indeed, highly efficient, multiplex, simultaneous genome editing of dozens of targets using Cas12a has been repeatedly demonstrated. ,− Moreover, efficient simultaneous regulation of multiple genes in mammalian cells by multiplexing with inactivated Cas12a has been reported by several groups. − Several studies have reported high specificity of Cas12a, − with an extremely low off-target rate. Although the natural diversity of Cas12a is far lower than that of Cas9, Cas12a’s from different bacteria were found to be optimal for different applications in different in vitro and in vivo systems.…”

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

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

confidence: 99%

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

2023

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“…1,3,5 Furthermore, due to Cas12a nucleases’ ability to process CRISPR arrays, multiplex genome editing is more accessible. 6–8 These properties, along with the favorable precision of Cas12a nuclease platforms, make it an attractive alternative nuclease platform for genome editing applications to the Cas9 system for certain therapeutic and research applications. 9–11…”

Section: Introductionmentioning

confidence: 99%

“…1,3,5 Furthermore, due to Cas12a nucleases' ability to process CRISPR arrays, multiplex genome editing is more accessible. [6][7][8] These properties, along with the favorable precision of Cas12a nuclease platforms, make it an attractive alternative nuclease platform for genome editing applications to the Cas9 system for certain therapeutic and research applications. [9][10][11] AspCas12a and LbaCas12a are the most widely employed Cas12a nucleases in the genome editing field due to their promising editing activity in a number of biological systems including fruit flies, mammalian cells, mouse embryos, zebrafish, and plants.…”

Section: Introductionmentioning

confidence: 99%

Optimization of NLS Composition Improves CRISPR-Cas12a Editing Rates in Human Primary Cells

Luk

Zeng

et al. 2022

Preprint

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Type V CRISPR Cas12a systems are an attractive alternative nuclease platform for specific genome editing applications. However, previous studies demonstrate that there is a gap in overall activity between Cas12a and Cas9 in primary cells. Here we describe optimization to the nuclear localization signal composition and architecture of Cas12a to facilitate highly efficient targeted mutagenesis in mammalian cell lines (HEK293T, Jurkat, and K562 cells) and primary cells (NK cells and CD34+ HSPCs), regardless of Cas12a ortholog. A 3xNLS Cas12a architecture resulted in the most robust editing platform. The improved editing activity of Cas12a in both NK cells and CD34+ HSPCs resulted in pronounced phenotypic changes associated with target gene editing. Lastly, we demonstrated that optimization of the NLS composition and architecture of Cas12a did not decrease the specificity of editing in HEK293T and CD34+ HSPCs. Our new Cas12a NLS variant provides an improved nuclease platform for therapeutic genome editing.

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CRISPR for neuroscientists

Kalamakis

Platt

2023

Neuron

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Multiplexed Genome Engineering with Cas12a

Cited by 5 publications

References 19 publications

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

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

Optimization of NLS Composition Improves CRISPR-Cas12a Editing Rates in Human Primary Cells

CRISPR for neuroscientists

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