CRISPR-mediated isolation of specific megabase segments of genomic DNA

Bennett-Baker, Pamela E.; Mueller, Jacob L.

doi:10.1093/nar/gkx749

Cited by 51 publications

(42 citation statements)

References 35 publications

(38 reference statements)

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“…Those identified in two different mutant lines -3R1 and 1R1 -were 391 confirmed by whole genome sequencing. It is thus likely that many of the cryopreserved lines not yet 392 recovered and made homozygous harbor additional mutations to those described here from the initial 393 analysis ( within a relatively small region should use other methods to identify rearrangements between guide 395 sites, such as targeted long-read sequencing (Bennett-Baker and Mueller, 2017). 396…”

Section: Excessive Developmental Neuronal Apoptosis Occurs In Mutantsmentioning

confidence: 98%

CRISPR/Cas9 interrogation of the mouse Pcdhg gene cluster reveals a crucial isoform-specific role for Pcdhgc4

Garrett

Bösch

Steffen

et al. 2019

Preprint

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14The mammalian Pcdhg gene cluster encodes a family of 22 cell adhesion molecules, the gamma-15 Protocadherins (γ-Pcdhs), critical for neuronal survival and neural circuit formation. The extent to which 16 isoform diversity-a γ-Pcdh hallmark-is required for their functions remains unclear. We used a 17 CRISPR/Cas9 approach to reduce isoform diversity, targeting each Pcdhg variable exon with pooled 18 sgRNAs to generate an allelic series of 26 mouse lines with 1 to 21 isoforms disrupted via discrete indels 19 at guide sites and/or larger deletions/rearrangements. Analysis of 5 mutant lines indicates that 20 postnatal viability and neuronal survival do not require isoform diversity. Surprisingly, as it is the only γ-21Pcdh that cannot independently engage in homophilic trans-interactions, we find that γC4, encoded by 22Pcdhgc4, is the only critical isoform. Because the human orthologue is the only PCDHG gene constrained 23 in humans, our results indicate a conserved γC4 function that likely involves distinct molecular 24 mechanisms. 25 26 mammalian analogue to fly Dscam1, although their diversity is generated by differential isoform 38 expression via promoter choice rather than alternative splicing (Zipursky and Sanes, 2010). 39

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Section: Excessive Developmental Neuronal Apoptosis Occurs In Mutantsmentioning

confidence: 98%

CRISPR/Cas9 interrogation of the mouse Pcdhg gene cluster reveals a crucial isoform-specific role for Pcdhgc4

Garrett

Bösch

Steffen

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…Several CRISPR-based methods for targeted sequencing have been developed, including STR-Seq (49), CRISPR-DS (50), and CRISPR-Cap (51). Especially, specific megabase segments of genomic DNA can be enriched by CRISPRmediated isolation (52). However, these methods used Cas9-sgRNA to cut out target DNA fragments from gDNAs.…”

Section: Discussionmentioning

confidence: 99%

“…However, these methods used Cas9-sgRNA to cut out target DNA fragments from gDNAs. The excised small or large target fragments were then isolated by hybridizing with probes coupled in flowcell (49), or performing size selection using SPRI beads (50) or pulse field gel electrophoresis (PFGE) (52). Only in the CRISPR-Cap method, the excised target DNA fragments were isolated with the biotinylated sgRNAs using streptavidin-coupled magnetic beads (51).…”

Section: Discussionmentioning

confidence: 99%

CRISPR-assisted targeted enrichment-sequencing (CATE-seq)

Xia

Zhang

et al. 2019

Preprint

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The current targeted sequencing of genome is mainly dependent on various hybridization-based methods. However, the hybridization-based methods are still limited by the intrinsic shortcomings of nucleic acid hybridization. This study developed a new CRISPR-based targeted sequencing technique, CRISPR-assisted targeted enrichment-sequencing (CATE-seq). In this technique, the input genomic DNA (gDNA) was firstly bound by a complex of dCas9 and capture sgRNA (csgRNA). The DNA-dCas9-csgRNA complex was then captured on magnetic beads through an easy room-temperature annealing between a short universal capture sequence (24 bp) at the 3′ end of csgRNA and capture oligonucleotide coupled on magnetic beads. The enriched DNAs were finally analyzed by next generation sequencing. Using this technique, three different scales of targeted enrichments were successfully performed, including enriching 35 target exons of 6 genes from 6 gDNA samples with 54 csgRNAs, 339 target exons of 186 genes from 9 gDNA samples with 367 csgRNAs, and 2031 target exons of 451 genes from 2 gDNA samples with 2302 csgRNAs. This technique has several significant advantages over the current hybridization-based methods, including high simplicity, specificity, sensitivity, throughput, and scalability.

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“…The experiment is carried out on the E. coli dataset with a series of relatively low coverage (10×, 15× and 20×) and a number of randomly mutated SNPs (10,100,1000 and 10000 SNPs) on the genomic region covering the first 2.5Mbp. Here we choose 2.5Mbp because this length is roughly the upper bound of the targeted sequencing reported so far using the CATCH (Cas9-assisted targeting of chromosome segments) technology (Bennett-Baker and Mueller, 2017).…”

Section: Case Studymentioning

confidence: 99%

Novel algorithms for efficient subsequence searching and mapping in nanopore raw signals towards targeted sequencing

Ren

Wang

Gao

2018

Preprint

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Genome diagnostics have gradually become a prevailing routine for human healthcare. With the advances in understanding the causal genes for many human diseases, targeted sequencing provides a rapid, costefficient and focused option for clinical applications, such as SNP detection and haplotype classification, in a specific genomic region. Although nanopore sequencing offers a perfect tool for targeted sequencing because of its mobility, PCR-freeness, and long read properties, it poses a challenging computational problem of how to efficiently and accurately search and map genomic subsequences of interest in a pool of nanopore reads (or raw signals). Due to its relatively low sequencing accuracy, there is no reliable solution to this problem, especially at low sequencing coverage. Here, we propose a brand new signal-based subsequence inquiry pipeline as well as two novel algorithms to tackle this problem. The proposed algorithms follow the principle of subsequence dynamic time warping and directly operate on the electrical current signals, without loss of information in basecalling. Therefore, the proposed algorithms can serve as a tool for sequence inquiry in targeted sequencing. Two novel criteria are offered for the consequent signal quality analysis and data classification. Comprehensive experiments on real-world nanopore datasets show the efficiency and effectiveness of the proposed algorithms. We further demonstrate the potential applications of the proposed algorithms in two typical tasks in nanoporebased targeted sequencing: SNP detection under low sequencing coverage, and haplotype classification under low sequencing accuracy.

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CRISPR-mediated isolation of specific megabase segments of genomic DNA

Cited by 51 publications

References 35 publications

CRISPR/Cas9 interrogation of the mouse Pcdhg gene cluster reveals a crucial isoform-specific role for Pcdhgc4

CRISPR/Cas9 interrogation of the mouse Pcdhg gene cluster reveals a crucial isoform-specific role for Pcdhgc4

CRISPR-assisted targeted enrichment-sequencing (CATE-seq)

Novel algorithms for efficient subsequence searching and mapping in nanopore raw signals towards targeted sequencing

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