Combined Gene Editing and RNA Sequencing Enhances Understanding of Gene Expression

Each repetition is followed by short segments of "spacer DNA" from previous exposures to a bacterial virus or plasmid. CRISPRs are found in approximately 40% of sequenced bacteria genomes and 90% of sequenced archaea. CRISPRs are often associated with cas genes that code for proteins related to CRISPRs. Since 2013, the CRISPR/Cas system has been used in research for gene editing (adding, disrupting, or changing the sequence of specific genes) and gene regulation. By delivering the Cas9 enzyme and appropriate guide RNAs (sgRNAs) into a cell, the organism's genome can be cut at any desired location.

The conventional CRISPR/Cas9 system is composed of two parts: the Cas9 enzyme, which cleaves the DNA molecule and specific RNA guides that shepherd the Cas9 protein to the target gene on a DNA strand. Despite its attractiveness as a gene-editing tool, the technique can inadvertently make excessive or unwanted changes in the genome and create off-target mutations, limiting safety and efficacy in therapeutic applications.

RNA-Seq is used to analyze the continually changing cellular transcriptome. Specifically, RNA-Seq facilitates the ability to look at alternative gene spliced transcripts, post-transcriptional modifications, gene fusion, mutations/SNPs, and changes in gene expression. In addition to mRNA transcripts, RNA-Seq can look at different populations of RNA to include total RNA, small RNA, such as miRNA, tRNA, and ribosomal profiling. RNA-Seq can also be used to determine exon/intron boundaries and verify or amend previously annotated 5' and 3' gene boundaries.

The investigators reported in the December 15, 2016, online edition of the journal Cell that they had applied combined CRISP-Seq methodology to probe regulatory circuits of innate immunity. By sampling tens of thousands of perturbed cells in vitro and in mice, they identified interactions and redundancies between developmental and signaling-dependent factors. These included the opposing effects of Cebpb (CCAAT/enhancer-binding protein beta) and Irf8 (Interferon regulatory factor 8) in regulating the monocyte/macrophage versus dendritic cell lineages and differential functions for Rela (Transcription factor p65) and Stat1/2 (Signal transducer and activator of transcription 1/2) in monocyte versus dendritic cell responses to pathogens.

"The advent of CRISPR presented a true leap in the ability to understand and start editing immune circuits, but CRISPR, on its own, is a blunt research tool, since we often have trouble observing or understanding the outcome of this genomic editing," said senior author Dr. Ido Amit, professor of immunology at the Weizmann Institute of Science. "We are hoping that our approach will be the next leap forward, providing, among other things, the ability to engineer immune cells for immunotherapy."

Related Links:
Weizmann Institute of Science