The New State of the Art: Cas9 for Gene Activation and Repression

Abstract

CRISPR-Cas9 technology has rapidly changed the landscape for how biologists and bioengineers study and manipulate the genome. Derived from the bacterial adaptive immune system, CRISPR-Cas9 has been coopted and repurposed for a variety of new functions, including the activation or repression of gene expression (termed CRISPRa or CRISPRi, respectively). This represents an exciting alternative to previously used repression or activation technologies such as RNA interference (RNAi) or the use of gene overexpression vectors. We have only just begun exploring the possibilities that CRISPR technology offers for gene regulation and the control of cell identity and behavior. In this review, we describe the recent advances of CRISPR-Cas9 technology for gene regulation and outline advantages and disadvantages of CRISPRa and CRISPRi (CRISPRa/i) relative to alternative technologies.

Molecular phenotyping of infection-associated small non-coding RNAs

“…RNA interference (RNAi) screens taking advantage of endogenous RNA-based regulatory pathways in eukaryotes have been available for more than a decade, and have enabled functional genomics on a previously impossible scale. However, there have been concerns about both offtarget effects, as well as uneven knock-down efficiency [72,73]. The emergence of CRISPR-Cas9 technology for eukaryotic genome editing has led to the development of this tool for the purposes of whole-genome forward genetic screens [74,75].…”

“…The emergence of CRISPR-Cas9 technology for eukaryotic genome editing has led to the development of this tool for the purposes of whole-genome forward genetic screens [74,75]. CRISPR-based technologies are rapidly developing, and variants using catalytically dead Cas9 (dCas9) tethered to factors that either activate or inhibit transcription (the so-called CRISPRa and CRISPRi) have been introduced to facilitate manipulation of the transcriptome and screening [73,75]. Progress with CRISPR tools in bacterial systems has moved more slowly, but proof-of-concept CRISPRa and CRISPRi has been demonstrated [76], suggesting genomewide screens could be developed in prokaryotes.…”

A Multipurpose Toolkit to Enable Advanced Genome Engineering in Plants

“…Increased specificity has been achieved using paired TALEN or CRISPR/Cas9 nickases , truncated gRNAs (Fu et al, 2014), or dimeric CRISPR/Cas9 nucleases (Guilinger et al, 2014;Tsai et al, 2014). Other diverse applications have emerged, including targeted regulation of gene expression, targeted epigenetic modification, or site-specific base editing through deamination (La Russa and Qi, 2015;Kungulovski and Jeltsch, 2016;Zong et al, 2017;Shimatani et al, 2017).…”

Cellular Therapies: Gene Editing and Next-Gen CAR T Cells

“…Libraries can therefore be used to activate (CRISPRa) or inhibit (CRISPRi) gene transcription in human cells (Perez-Pinera et al 2013 ;Larson et al 2013 ;Qi et al 2013 ;Maeder et al 2013 ;Gilbert et al 2014 ). CRISPR/Cas libraries demonstrate much higher levels of effectiveness and reliability with respect to both loss-of-function and gain-of-function screenings, than other methods such as RNAi (La Russa and Qi 2015 ). One CRISPR library structure used tethered engineered transcriptional activating complexes for improved function.…”