Using CRISPR to understand and manipulate gene regulation

Abstract

Understanding how genes are expressed in the correct cell types and at the correct level is a key goal of developmental biology research. Gene regulation has traditionally been approached largely through observational methods, whereas perturbational approaches have lacked precision. CRISPR-Cas9 has begun to transform the study of gene regulation, allowing for precise manipulation of genomic sequences, epigenetic functionalization and gene expression. CRISPR-Cas9 technology has already led to the discovery of new paradigms in gene regulation and, as new CRISPR-based tools and methods continue to be developed, promises to transform our knowledge of the gene regulatory code and our ability to manipulate cell fate. Here, we discuss the current and future application of the emerging CRISPR toolbox toward predicting gene regulatory network behavior, improving stem cell disease modeling, dissecting the epigenetic code, reprogramming cell fate and treating diseases of gene dysregulation.

Keywords: CRISPR screening; CRISPR-Cas9; Disease modeling; Epigenetics; Gene regulation.

Fig. 1.

Cas9 nuclease repair outcomes. Cas9-nuclease, which is guided to the target DNA region by a single guide RNA (sgRNA) because of complementarity between the sgRNA and the target DNA, creates double-stranded DNA breaks (DSBs). DSBs are imperfectly fixed by endogenous host DNA repair mechanisms such as non-homologous end joining (NHEJ) and microhomology-mediated end joining (MMEJ), creating short insertions/deletions (indels) around the cleavage site. If a DSB is created in the presence of an external DNA fragment bearing homology arms flanking the DSB site, this external DNA can integrate into the targeted position of the genome through cellular homology-directed repair (HDR). CRISPR-induced repair outcomes for any given sgRNA are predictable by the algorithm inDelphi (http://indelphi.giffordlab.mit.edu/; Shen et al., 2018), enabling selection of sgRNA designs that maximize desirable edited products.

Fig. 2.

The CRISPR toolbox. (A) Strategic CRISPR target designs enable a range of desired genome edits. One single guide RNA (sgRNA) targeting the coding region of a gene of interest can be used to induce a frameshift and thus a truncated nonfunctional protein product. Use of a pair of sgRNAs can excise an entire region of interest. Tiling sgRNA libraries can be used in pooled screens to map functional regions within coding and non-coding DNA. CRISPR-based tools with amino acid modifications and/or effector peptide/protein additions have been developed to perform distinct editing functions. (B,C) CRISPR activation (CRISPRa) (B) and CRISPR interference (CRISPRi) (C) enable epigenetic modification through use of a nuclease-deficient Cas9 (dCas9) enzyme in which two amino acids (D10A and H840A) in catalytic domains (RuvC and HNH) are mutated. In CRISPRi and CRISPRa, dCas9 is attached to transcriptional silencer or epigenetic activators domains, respectively, enabling modulation of the expression of target genes when recruited to the promoter or enhancer region. Various versions of CRISPRi and CRISPRa tools have been devised. First generation CRISPRa tools used direct attachment of a single activation domain such as the Herpes simplex tandem activator protein VP64. Among second-generation CRISPR activators, VPR entails attachment of three distinct activation domains [VP64, p65 and Epstein-Barr virus R transactivator (Rta)] to dCas9, SunTag entails indirect attachment of 10 VP64 peptides to dCas9 through binding of a single chain variable fragment (scFv) antibody to a tail of dCas9-fused peptide ligands, and SAM combines a direct dCas9-VP64 and recruitment of 2 activator domains [p65 and heat shock factor 1 (HSF1)] to sgRNA-embedded MS2 RNA aptamers (2× MS2). (D) Prime editors and base editors are capable of precise genome editing without DSBs. Prime editors catalyze short sequence replacements, insertions or deletions through use of a Cas9-nickase fused to a reverse transcriptase (RT) enzyme. The sgRNA used by prime editors is extended with a sequence that acts as an RT primer and template, stimulating DNA synthesis of the nicked DNA strand, which is integrated through cellular DNA repair mechanisms. Base editors are composed of a Cas9-nickase fused to a nucleotide deaminase enzyme, which catalyzes specific base transitions (C to T for cytidine deaminases and A to G with TadA adenine deaminase). MCP, MS2 coat protein; pegRNA, prime editing guide RNA.

Fig. 3.

Applications of the CRISPR toolbox to elucidate gene regulation. (A) CRISPR-based gene loss-of-function screens, through permanent or inducible mutation strategies, have eased the investigation of genetic underpinnings during different stages of cellular development. A generic screen paradigm involves the use of a pooled single guide RNA (sgRNA) library, transduced into cells using lentivirus, to target all or a focused set of genes/genomic elements of interest. Relative representation of each sgRNA can be quantified before and after phenotypic selection using techniques such as cell survival/proliferation under defined conditions or flow cytometry-based cell sorting, followed by sgRNA quantification methods including next-generation sequencing or single-cell RNA-sequencing, to investigate genes/elements involved in developmental processes. (B-D) Cas9 enzyme variants have facilitated elucidating gene regulation. Cas9 can be employed for single gene/element mutation, deletion and tiling of gene regulatory sequences. dCas9 fused with a transcriptional effector domain has been used to enhance or repress regulatory regions to understand their involvement in gene regulation and target epigenetic factors and modifications such as histone modifications, CpG methylation and enhancer transcripts to dissect their role in regulating gene expression. (E) The effect of 3D chromosomal architecture on gene expression can also be investigated with CRISPR tools that bring targeted chromosomal regions into proximity or allow recruitment to distinct sub-nuclear regions.