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An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms.

ABSTRACT: We previously reported a CRISPR-mediated knock-in strategy into introns of Drosophila genes, generating an attP-FRT-SA-T2A-GAL4-polyA-3XP3-EGFP-FRT-attP transgenic library for multiple uses (Lee et al., 2018a). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration cassette, followed by enzymatic removal of one strand. Using this method, we generated GFP-tagged proteins that mark organelles in S2 cells. We then describe two dsDNA methods using cheap synthesized donors flanked by 100 nt homology arms and gRNA target sites cloned into a plasmid. Upon injection, donor DNA (1 to 5 kb) is released from the plasmid by Cas9. The cassette integrates efficiently and precisely in vivo. The approach is fast, cheap, and scalable.

SUBMITTER: Kanca O

PROVIDER: S-EPMC6855806 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms.

Kanca Oguz O Zirin Jonathan J Garcia-Marques Jorge J Knight Shannon Marie SM Yang-Zhou Donghui D Amador Gabriel G Chung Hyunglok H Zuo Zhongyuan Z Ma Liwen L He Yuchun Y Lin Wen-Wen WW Fang Ying Y Ge Ming M Yamamoto Shinya S Schulze Karen L KL Hu Yanhui Y Spradling Allan C AC Mohr Stephanie E SE Perrimon Norbert N Bellen Hugo J HJ

eLife 20191101

We previously reported a CRISPR-mediated knock-in strategy into introns of <i>Drosophila</i> genes, generating an <i>attP-FRT-SA-T2A-GAL4-polyA-3XP3-EGFP-FRT-attP</i> transgenic library for multiple uses (Lee et al., 2018a). The method relied on double stranded DNA (dsDNA) homology donors with ~1 kb homology arms. Here, we describe three new simpler ways to edit genes in flies. We create single stranded DNA (ssDNA) donors using PCR and add 100 nt of homology on each side of an integration casset ...[more]

PMID: 31674908

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Project description:BackgroundThe assignment of taxonomic attributions to DNA fragments recovered directly from the environment is a vital step in metagenomic data analysis. Assignments can be made using rank-specific classifiers, which assign reads to taxonomic labels from a predetermined level such as named species or strain, or rank-flexible classifiers, which choose an appropriate taxonomic rank for each sequence in a data set. The choice of rank typically depends on the optimal model for a given sequence and on the breadth of taxonomic groups seen in a set of close-to-optimal models. Homology-based (e.g., LCA) and composition-based (e.g., PhyloPythia, TACOA) rank-flexible classifiers have been proposed, but there is at present no hybrid approach that utilizes both homology and composition.ResultsWe first develop a hybrid, rank-specific classifier based on BLAST and Naïve Bayes (NB) that has comparable accuracy and a faster running time than the current best approach, PhymmBL. By substituting LCA for BLAST or allowing the inclusion of suboptimal NB models, we obtain a rank-flexible classifier. This hybrid classifier outperforms established rank-flexible approaches on simulated metagenomic fragments of length 200 bp to 1000 bp and is able to assign taxonomic attributions to a subset of sequences with few misclassifications. We then demonstrate the performance of different classifiers on an enhanced biological phosphorous removal metagenome, illustrating the advantages of rank-flexible classifiers when representative genomes are absent from the set of reference genomes. Application to a glacier ice metagenome demonstrates that similar taxonomic profiles are obtained across a set of classifiers which are increasingly conservative in their classification.ConclusionsOur NB-based classification scheme is faster than the current best composition-based algorithm, Phymm, while providing equally accurate predictions. The rank-flexible variant of NB, which we term ?-NB, is complementary to LCA and can be combined with it to yield conservative prediction sets of very high confidence. The simple parameterization of LCA and ?-NB allows for tuning of the balance between more predictions and increased precision, allowing the user to account for the sensitivity of downstream analyses to misclassified or unclassified sequences.

Dataset Information

An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms.

Publications

An efficient CRISPR-based strategy to insert small and large fragments of DNA using short homology arms.

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