| Autor: |
LaFave, Matthew C., Gaurav K. Varshney, Wuhong Pei, Idol, Jennifer, Xu, Lisha, Gallardo, Viviana, Carrington, Blake, Bishop, Kevin, MaryPat Jones, Mingyu Li, Harper, Ursula, Huang, Sunny C., Wenbiao Chen, Sood, Raman, Ledin, Johan, Burgess, Shawn M. |
| Rok vydání: |
2014 |
| DOI: |
10.6084/m9.figshare.1251196.v1 |
| Popis: |
Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) technology is an efficient means of creating targeted deletion or insertion variants (DIVs). Our lab has taken advantage of this to develop a genome engineering pipeline that remains efficient at scale, which puts saturation mutagenesis within reach. The pipeline involves inducing DIVs with CRISPR/Cas9, amplifying the targeted regions via PCR, and subjecting the amplicons to high-throughput sequencing. A key component of this process is the ampliconDIVider software, which identifies and characterizes DIVs at the nucleotide level. The program recovers DIVs within user-defined regions, parses samples based on DNA barcodes, and uses the sam2pairwise program to rapidly visualize DIVs as pairwise alignments. We applied this approach in Danio rerio, and identified 678 unique DIV alleles induced by 114 CRISPR targets in 58 genes. Contrary to previous reports, we detected no evidence that the primary sequence of the CRISPR affected mutagenesis efficiency, other than that the rate was reduced by the addition of extra guanines to the 5’ end of the target. ampliconDIVider is flexible, and is not limited to the context of this pipeline, nor to DIVs induced specifically by CRISPRs. |
| Databáze: |
OpenAIRE |
| Externí odkaz: |
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