Multiplexed conditional genome editing with Cas12a in Drosophila

Aug 27, 2020Proceedings of the National Academy of Sciences of the United States of America

Using Cas12a to edit multiple genes at once in fruit flies under specific conditions

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Abstract

Cas12a can mediate robust gene editing in vivo, outperforming Cas9 in identifying essential genes.

Cas12a operates effectively at 29 °C but shows low activity at 18 °C, indicating that gene editing can be modulated by temperature.
The platform enables the use of compact crRNA arrays, making multiplex genome engineering simpler compared to traditional Cas9 systems.
Conditional expression of LbCas12a allows for precise control of gene editing across various tissues in a multicellular organism.
A variant of LbCas12a with a D156R mutation exhibits significantly higher activity than the standard Cas9 system.

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CRISPR-Cas genome engineering has revolutionized biomedical research by enabling targeted genome modification with unprecedented ease. In the popular model organism, gene editing has so far relied exclusively on the prototypical CRISPR nuclease Cas9. Additional CRISPR systems could expand the genomic target space, offer additional modes of regulation, and enable the independent manipulation of genes in different cells of the same animal. Here we describe a platform for efficient Cas12a gene editing inWe show that Cas12a from, but notspec., can mediate robust gene editing in vivo. In combination with most CRISPR RNAs (crRNAs), LbCas12a activity is high at 29 °C, but low at 18 °C, enabling modulation of gene editing by temperature. LbCas12a can directly utilize compact crRNA arrays that are substantially easier to construct than Cas9 single-guide RNA arrays, facilitating multiplex genome engineering. Furthermore, we show that conditional expression of LbCas12a is sufficient to mediate tightly controlled gene editing in a variety of tissues, allowing detailed analysis of gene function in a multicellular organism. We also test a variant of LbCas12a with a D156R point mutation and show that it has substantially higher activity and outperforms a state-of-the-art Cas9 system in identifying essential genes. Cas12a gene editing expands the genome-engineering toolbox inand will be a powerful method for the functional annotation of the genome. This work also presents a fully genetically encoded Cas12a system in an animal, laying out principles for the development of similar systems in other genetically tractable organisms for multiplexed conditional genome engineering. Drosophila melanogaster Drosophila Lachnospiraceae bacterium Acidaminococcus Drosophila

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Increase in Gene Editing Efficiency

Number of crRNAs resulting in efficient gene editing at 29 °C.

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Lethality with Cas12a+

Number of essential genes targeted resulting in lethality with Cas12a+ at 29 °C.

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Multiplexed conditional genome editing with Cas12a in Drosophila

Abstract

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