High-Throughput Silencing Using the CRISPR-Cas9 System: A Review of the Benefits and Challenges

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has been seized upon with a fervor enjoyed previously by small interfering RNA (siRNA) and short hairpin RNA (shRNA) technologies and has enormous potential for high-throughput functional genomics studies. The decision to use this approach must be balanced with respect to adoption of existing platforms versus awaiting the development of more "mature" next-generation systems. Here, experience from siRNA and shRNA screening plays an important role, as issues such as targeting efficiency, pooling strategies, and off-target effects with those technologies are already framing debates in the CRISPR field. CRISPR/Cas can be exploited not only to knockout genes but also to up-or down-regulate gene transcription-in some cases in a multiplex fashion. This provides a powerful tool for studying the interaction among multiple signaling cascades in the same genetic background. Furthermore, the documented success of CRISPR/Cas-mediated gene correction (or the corollary, introduction of disease-specific mutations) provides proof of concept for the rapid generation of isogenic cell lines for highthroughput screening. In this review, the advantages and limitations of CRISPR/Cas are discussed and current and future applications are highlighted. It is envisaged that complementarities between CRISPR, siRNA, and shRNA will ensure that all three technologies remain critical to the success of future functional genomics projects.

Dynamic Heterogeneity of Brachyury in Mouse Epiblast Stem Cells Mediates Distinct Response to Extrinsic Bone Morphogenetic Protein (BMP) Signaling

“…The Id1 gene, a downstream gene of BMP signaling, is essential for the epidermis differentiation (44,45). To investigate whether Snail or Id1 is associated with the responses to BMP4 in EpiSCs, Snail or Id1 was knocked down in T-EpiSCs by using a CRISPR-Cas9-mediated gene silencing strategy (46,47). Two sets of gRNAs (gRNA1 and gRNA2) were used to excise the Id1 or Snail gene, respectively, and the expression of Id1 or Snail could be efficiently reduced with corresponding gRNAs in EpiSCs (Fig.…”

High-Throughput Imaging for the Discovery of Cellular Mechanisms of Disease

“…In pooled screens a large single cell population is infected with libraries of lentiviral vectors expressing the CRISPR/Cas9 components and enrichment or depletion of target genes upon selective pressure is typically measured by next generation sequencing. Compared to arrayed HTI screens, pooled CRISPR/Cas9 screens allows using genome-wide libraries that include many targeting reagents per gene and do not necessitate the use of expensive microplates, transfection reagents, and automated liquid handling equipment [3]. However, pooled CRISPR/Cas9 screens are currently of limited use in HTI approaches as it is often experimentally difficult to identify the affected gene at the single cell level in a population.…”

“…Systematic perturbations using small molecules, RNA interference (RNAi) or Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 are then applied with the goal of identifying and studying factors that control the cellular phenotypes monitored in the readout assay (Fig. 1, Key Figure) [1–3]. High-throughput fluorescence or bright-field microscopes are routinely used to acquire large numbers of images, in some applications up to 10 5 per day (Fig.…”

T-cell therapies for HIV: Preclinical successes and current clinical strategies

“…These complexes in turn digest the foreign DNA elements that are complementary to the CRISPR-RNA, serving as a gene editing tool. The CRISPR/Cas9 system was adapted from Streptococcus pyogenes , with two main components: the guide RNA (gRNA) that complements the genomic sequence being targeted, and the CRISPR-associated Cas9 nuclease that creates a dsDNA break that can be repaired by NHEJ, similar to ZFN gene editing [59, 60]. One advantage over ZFN, is that multiple gRNAs can be employed simultaneously to target multiple genomic loci, allowing for multiple knockdowns in the same cell [61].…”