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Cas9-Based Local Enrichment and Genomics Sequence Revision of Megabase-Sized Shark IgNAR Loci

J Immunol. 2021-12; 
Hongming Dong, Yaolei Zhang, Jiahao Wang, Haitao Xiang, Tianhang Lv, Likun Wei, Shaosen Yang, Xiaopan Liu, Bingzhao Ren, Xiuqing Zhang, Lirong Liu, Jun Cao, Meiniang Wang, Jiahai Shi, Naibo Yang
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Gene Synthesis … Singapore; # Department of Biochemistry, Yong Loo Lin School of Medicine, National University … Sampling and RBC isolation Genetic material was extracted from male shark nucleated RBCs … of CRISPR RNA (crRNA) and a trans-activating CRISPR was completed by GenScript … Get A Quote

摘要

The 0.8-Mb Ig new Ag receptor (IgNAR) region of the whitespotted bamboo shark () is incompletely assembled in Chr_44 of the reference genome. Here we used Cas9-assisted targeting of chromosome segments (CATCH) to enrich the 2 Mb region of the Chr_44 IgNAR loci and sequenced it by PacBio and next-generation sequencing. A fragment >3.13 Mb was isolated intact from the RBCs of sharks. The target was enriched 245.531-fold, and sequences had up to 94% coverage with a 255× mean depth. Compared with the previously published sequences, 20 holes were filled, with a total length of 3508 bp. In addition, we report five potential germline V alleles of IgNAR1 from six sharks that may belong to two clusters of the IgNAR. Ou... More

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