Digital Droplet PCR and IDAA for the Detection of CRISPR Indel Edits in the Malaria Species
            <i>Anopheles Stephensi</i>

A Cas9/guide RNA-based gene drive strain, AgNosCd-1, was developed to deliver antiparasite effector molecules to the malaria vector mosquito, Anopheles gambiae. The drive system targets the cardinal gene ortholog producing a red-eye phenotype. Drive can achieve 98 to 100% in both sexes and full introduction was observed in small cage trials within 6 to 10 generations following a single release of gene-drive males. No genetic load resulting from the integrated transgenes impaired drive performance in the trials. Potential drive-resistant target-site alleles arise at a frequency <0.1, and five of the most prevalent polymorphisms in the guide RNA target site in collections of colonized and wild-derived African mosquitoes do not prevent cleavage in vitro by the Cas9/guide RNA complex. Only one predicted off-target site is cleavable in vitro, with negligible deletions observed in vivo. AgNosCd-1 meets key performance criteria of a target product profile and can be a valuable component of a field-ready strain for mosquito population modification to control malaria transmission.

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“…Digital droplet PCR drop-off assay reactions were performed as described in Carballar-Lejarazú et al ( 37 ).…”

Section: Methodsmentioning

confidence: 99%

Next-generation gene drive for population modification of the malaria vector mosquito, Anopheles gambiae

Carballar-Lejarazú

Ogaugwu

Tushar

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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176

216

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“…Since IDAA is based solely on fragment analysis, it can analyse editing outcomes in very complex polyploid/multi-allelic genomes, where the complexity would preclude sequencing-based indel analysis. Recent comparative analyses showed that IDAA, targeted NGS and digital droplet PCR assays provide very similar editing profiles for pools of cells with respect to size and frequency of indels ( 116 , 140 , 142 ). Thus, IDAA can provide quantitative determination of indels in complex editing profiles with single-base discrimination.…”

Section: Introductionmentioning

confidence: 99%

INDEL detection, the ‘Achilles heel’ of precise genome editing: a survey of methods for accurate profiling of gene editing induced indels

Bennett

Petersen

Johansen

et al. 2020

Nucleic Acids Research

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Advances in genome editing technologies have enabled manipulation of genomes at the single base level. These technologies are based on programmable nucleases (PNs) that include meganucleases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated 9 (Cas9) nucleases and have given researchers the ability to delete, insert or replace genomic DNA in cells, tissues and whole organisms. The great flexibility in re-designing the genomic target specificity of PNs has vastly expanded the scope of gene editing applications in life science, and shows great promise for development of the next generation gene therapies. PN technologies share the principle of inducing a DNA double-strand break (DSB) at a user-specified site in the genome, followed by cellular repair of the induced DSB. PN-elicited DSBs are mainly repaired by the non-homologous end joining (NHEJ) and the microhomology-mediated end joining (MMEJ) pathways, which can elicit a variety of small insertion or deletion (indel) mutations. If indels are elicited in a protein coding sequence and shift the reading frame, targeted gene knock out (KO) can readily be achieved using either of the available PNs. Despite the ease by which gene inactivation in principle can be achieved, in practice, successful KO is not only determined by the efficiency of NHEJ and MMEJ repair; it also depends on the design and properties of the PN utilized, delivery format chosen, the preferred indel repair outcomes at the targeted site, the chromatin state of the target site and the relative activities of the repair pathways in the edited cells. These variables preclude accurate prediction of the nature and frequency of PN induced indels. A key step of any gene KO experiment therefore becomes the detection, characterization and quantification of the indel(s) induced at the targeted genomic site in cells, tissues or whole organisms. In this survey, we briefly review naturally occurring indels and their detection. Next, we review the methods that have been developed for detection of PN-induced indels. We briefly outline the experimental steps and describe the pros and cons of the various methods to help users decide a suitable method for their editing application. We highlight recent advances that enable accurate and sensitive quantification of indel events in cells regardless of their genome complexity, turning a complex pool of different indel events into informative indel profiles. Finally, we review what has been learned about PN-elicited indel formation through the use of the new methods and how this insight is helping to further advance the genome editing field.

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“…The translation of Cas9-mediated gene editing using adult injections have been demonstrated to efficiently support targeted mutagenesis in several species ( Chaverra-Rodriguez et al, 2018 ; Chaverra‐Rodriguez et al, 2020 ; Heu et al, 2020 ; Macias et al, 2020 ). This is supported by robust and affordable PCR and sequencing techniques ( Ran et al, 2013 ; Bell et al, 2014 ; Carballar-Lejarazú et al, 2020 ). Expansion of this will include both translation to more vector species and engineering the techniques for DNA and RNA delivery for heritable transgenesis and RNA interference methods.…”

Section: The General Impetus To Expand Genetic Engineering For Molecumentioning

confidence: 99%

Leaning Into the Bite: The piRNA Pathway as an Exemplar for the Genetic Engineering Need in Mosquitoes

Macias

Palatini

Bonizzoni

et al. 2021

Front. Cell. Infect. Microbiol.

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The piRNA pathway is a specialized small RNA interference that in mosquitoes is mechanistically distant from analogous biology in the Drosophila model. Current genetic engineering methods, such as targeted genome manipulation, have a high potential to tease out the functional complexity of this intricate molecular pathway. However, progress in utilizing these methods in arthropod vectors has been geared mostly toward the development of new vector control strategies rather than to study cellular functions. Herein we propose that genetic engineering methods will be essential to uncover the full functionality of PIWI/piRNA biology in mosquitoes and that extending the applications of genetic engineering on other aspects of mosquito biology will grant access to a much larger pool of knowledge in disease vectors that is just out of reach. We discuss motivations for and impediments to expanding the utility of genetic engineering to study the underlying biology and disease transmission and describe specific areas where efforts can be placed to achieve the full potential for genetic engineering in basic biology in mosquito vectors. Such efforts will generate a refreshed intellectual source of novel approaches to disease control and strong support for the effective use of approaches currently in development.

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Digital Droplet PCR and IDAA for the Detection of CRISPR Indel Edits in the Malaria Species Anopheles Stephensi

Cited by 11 publications

References 21 publications

Next-generation gene drive for population modification of the malaria vector mosquito, Anopheles gambiae

Next-generation gene drive for population modification of the malaria vector mosquito, Anopheles gambiae

INDEL detection, the ‘Achilles heel’ of precise genome editing: a survey of methods for accurate profiling of gene editing induced indels

Leaning Into the Bite: The piRNA Pathway as an Exemplar for the Genetic Engineering Need in Mosquitoes

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