Background

Highlights

• CRISPR-Cas9 is a powerful tool to modulate transcription in wide range of cell types.
• An expanding set of CRISPR-based transcription effectors is available.
• Gene networks can be efficiently probed and modified for biotechnology applications.^[1]

CRISPR-Cas9 has recently emerged as a promising system for multiplexed genome editing as well as epigenome and transcriptome perturbation. Due to its specificity, ease of use and highly modular programmable nature, it has been widely adopted for a variety of applications such as genome editing, transcriptional inhibition and activation, genetic screening, DNA localization imaging, and many more. In this review, we will discuss non-editing applications of CRISPR-Cas9 for transcriptome perturbation, metabolic engineering, and synthetic biology.^[1]

Since the early days of genetic engineering there has been a need for control of gene expression. Naturally occurring transcription factors (TFs) have traditionally been used to achieve this goal (reviewed in ^[2]). However, their limited DNA binding sequence space required installing specific sequences within the transcription regulatory elements of the target genes. This can be technically difficult and may have unintended consequences on gene expression. Zinc fingers (ZFs) and transcription activator-like effectors (TALEs) were developed to overcome the fixed binding sequence requirements of native TFs. However, both ZFs and TALEs have significant limitations. ZFs have complicated design criteria and large highly repetitive TALE genes are difficult to synthesize and clone (reviewed in ^[3][4]). These challenges have recently been overcome using CRISPR-Cas9 based TFs. The biochemical properties of CRISPR-Cas9 based TFs that enable such flexibility and describe their applications to synthetic gene circuit design and multi-plexed perturbation of native gene networks.^[1]

Many bacteria and archaea possess an adaptive immune system consisting of repetitive genetic elements known as clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins. Similar to RNAi pathways in eukaryotes, CRISPR–Cas systems require small RNAs for sequence-specific detection and degradation of complementary nucleic acids. Cas5 and Cas6 enzymes have evolved to specifically recognize and process CRISPR-derived transcripts into functional small RNAs used as guides by interference complexes. Our detailed understanding of these proteins has led to the development of several useful Cas6-based biotechnological methods. The structures, functions, mechanisms, and applications of the enzymes responsible for CRISPR RNA (crRNA) processing, highlighting a fascinating family of endonucleases with exquisite RNA recognition and cleavage activities are reviewed.^[5]

Transcriptional regulation with CRISPR-Cas9

Figure 3. Overview of Cas9 nuclease and dCas9-based transcription factors. (a) Wild-type Cas9 endonuclease guided by crRNA:tracrRNA to a specific site in DNA creates a double-stranded DNA break. (b) dCas9, nuclease deactivated mutant of Cas9, is an RNA programmable DNA binding protein. It can act as a steric repressor of transcription in prokaryotes and eukaryotes. sgRNA is an artificial chimeric molecule consisting of crRNA and tracrRNA molecules connected with a short loop. (c) dCas9 fusion with various transcription effectors can be used to repress or activate transcription. (d) Effector domains can be recruited by sgRNA in addition to dCas9 for enhanced activity. (e) sgRNA can be modified with specific protein binding hairpins to concurrently recruit repressor or activator domains in the same cell.^[1]

Cas9 is a key protein of bacterial Type II CRISPR adaptive immune system (reviewed in ^[6]). (SpyCas9; PDB entry 4zt0^[7]). In its native context, Cas9 is an RNA-guided endonuclease that is responsible for targeted degradation of the invading foreign DNA–plasmids and phages. Cas9 is directed to its DNA targets by forming a ribonucleoprotein complex with two small non-coding RNAs: CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA) (Figure 3a, see also above Examples of 3D structures of CRISPR RNA (crRNA)). By elegant engineering, (PDB entry 4zt9^[7]) that too efficiently directs Cas9 protein to DNA targets encoded within the guide sequence of sgRNA ^[8] (see also above Examples of 3D structures of single guide RNA (sgRNA)). The , termed the guide sequence, adjacent to a ^[8][9]. Despite this, a ^{[8][10][11][12]}, more so within the 5’ proximal position of the guide sequence.

• ^[7] (4cmp^[13]).
• (4zt0)^[7].
• (4oo8)^[14].
• .

• (5f9r)^[15].
• .
• - place of accurate, precise, and programmable DNA cleavage.
• (sgRNA is not shown).

Cas9 nuclease can be converted into (PDB entry 4zt9), an RNA-programmable DNA-binding protein, by (Figure 3b) ^[7][1][8][9].

In the simplest case, dCas9 can repress transcription by sterically interfering with transcription initiation or elongationby being targeted to the gene of interest with a properly chosen sgRNA ^{[8][9][10][11][16][17][18][19][20]}. The repression strength is strongly dependent on the position with respect to the target promoter as well as the nature of promoter itself ^{[10][11][16][17]}. In prokaryotes, repression of up to 1000-fold was achieved when targeting dCas9 to either DNA strand within a promoter or to the non-template DNA strand downstream. However, in eukaryotic cells such steric repression is weaker: only up to 2-fold and 20-fold repression was observed with natural promoters in mammalian and yeast cells correspondingly^[10][18][19]. As a notable exception, synthetic promoters specifically constructed for direct repression by dCas9 can be repressed up to 100-fold in mammalian cells^[20].

See aslo

• Cas9
• Endonuclease