Genetic Engineering Publications - GEG Tech top picks

The scientists recently reported Digenome-seq (digested genome sequencing), a method for in vitro identification of potential off-target sites, and They evaluated the specificity of CRISPR–Cas9 and CRISPR–Cpf1 endonuclease by whole-genome sequencing. Digenome-seq pinpoints the exact location of double-strand break (DSB) sites by recognizing specific patterns of aligned reads. However, the analysis pipeline presented in their previous report required extensive manual interaction and produced several large intermediate files, resulting in a long running time. Here, the scientists present a redesigned analysis tool for Digenome-seq data that runs on web browsers. The core algorithm of the tool is written in C++ and compiled to asm.js (http://asmjs.org/), a preoptimized subset of JavaScript. Users can instantly perform the complete analysis in an ordinary web browser (Supplementary Note 1) with fast execution speed without uploading any data to a server and without local tool installation. In their benchmark, the full analysis for 100 GB of BAM file took 3 h for whole analysis on Intel i5 3570k central processing unit in a single thread.

Here, the scientists report that four to six bases at the 3′ end of the short CRISPR RNA (crRNA) used to program Cpf1 nucleases are insensitive to single base mismatches, but that many of the other bases in this region of the crRNA are highly sensitive to single or double substitutions. Using GUIDE-seq and targeted deep sequencing analyses performed with both Cpf1 nucleases, they were unable to detect off-target cleavage for more than half of 20 different crRNAs. Our results suggest that AsCpf1 and LbCpf1 are highly specific in human cells.