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

Abstract

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.

Not Only Editing: A Cas-Cade of CRISPR/Cas-Based Tools for Functional Genomics in Plants and Animals

“…After incubation with DNA, the target-bound dCas9 is isolated through immunomagnetic precipitation, target DNA is then purified and analyzed through allele-specific qPCR resulting in a 21-fold enrichment. CATE-seq ( CRISPR-assisted targeted enrichment-sequencing ) [ 106 ] is a highly sensitive alternative approach able to reach over a 3000-fold enrichment of the target sequences. Such protocol consists of the fragmentation and subsequent specific adaptor ligation to sample DNA, targets are then bound by dCas9 and purified for allele-specific PCR or library preparation.…”

Not Only Editing: A Cas-Cade of CRISPR/Cas-Based Tools for Functional Genomics in Plants and Animals

“…After incubation with DNA, the target-bound dCas9 is isolated through immunomagnetic precipitation, target DNA is then purified and analyzed through allelespecific qPCR resulting in a 21-fold enrichment. CATE-seq targeted enrichment-sequencing) [106] is a highly sensitive alternative approach able to reach over a 3000-fold enrichment of the target sequences. Such protocol consists of the fragmentation and subsequent specific adaptor ligation to sample DNA, targets are then bound by dCas9 and purified for allele-specific PCR or library preparation.…”

Cas12a-Capture: A Novel, Low-Cost, and Scalable Method for Targeted Sequencing

“…These systems leverage targetspecific guide RNAs (gRNAs) to direct Cas9 family endonucleases to specific genomic loci. Several methods have taken advantage of CRISPR-based cutting for capture of genomic regions followed by nanopore (4)(5)(6)(7)(8)(9) or Illumina sequencing (9)(10)(11)(12)(13)(14)(15). However, some of these rely on laborious size selection steps or require specialized equipment or reagents (8,(13)(14)(15).…”

“…The copyright holder for this preprint this version posted November 20, 2020. ; https://doi.org/10.1101/2020.11. 18.388876 doi: bioRxiv preprint ribonucleoprotein (RNP) affinity to target DNA by pulling down DNA-bound RNP (10)(11)(12). Recently, a method relying on direct adapter ligation to Cas9 cleavage sites, followed by long-read sequencing with nanopore technology has been developed to enable detection of structural variation as well as single nucleotide variants (SNVs) (5)(6)(7).…”