The next generation of CRISPR-Cas technologies and applications

Abstract

The prokaryote-derived CRISPR-Cas genome editing systems have transformed our ability to manipulate, detect, image and annotate specific DNA and RNA sequences in living cells of diverse species. The ease of use and robustness of this technology have revolutionized genome editing for research ranging from fundamental science to translational medicine. Initial successes have inspired efforts to discover new systems for targeting and manipulating nucleic acids, including those from Cas9, Cas12, Cascade and Cas13 orthologues. Genome editing by CRISPR-Cas can utilize non-homologous end joining and homology-directed repair for DNA repair, as well as single-base editing enzymes. In addition to targeting DNA, CRISPR-Cas-based RNA-targeting tools are being developed for research, medicine and diagnostics. Nuclease-inactive and RNA-targeting Cas proteins have been fused to a plethora of effector proteins to regulate gene expression, epigenetic modifications and chromatin interactions. Collectively, the new advances are considerably improving our understanding of biological processes and are propelling CRISPR-Cas-based tools towards clinical use in gene and cell therapies.

Figure 1.. Overview of the main CRISPR–Cas gene editing tools.

a ∣ CRISPR-associated endonuclease 9 (Cas9) proteins rely on RNA guidance for targeting specificity. In engineered CRISPR–Cas9 systems, Cas9 interacts with the backbone of the guide RNA (gRNA). Complementary pairing of the spacer portion of the gRNA to a DNA target sequence positioned next to a 5’ protospacer adjacent motif (PAM) results in generation of a blunt DNA double-strand break by the two Cas9 nuclease domains, RuvC and HNH^-. b ∣ Cas12a nucleases recognize DNA target sequences with complementarity to the crRNA spacer positioned next to a 3’ PAM. Target recognition results in generation of a staggered DNA double-strand break by a RuvC domain and a putative nuclease (Nuc) domain. c ∣ Cascade is a multimeric complex that targets DNA that has complementarity to the spacer portion of a crRNA and that is positioned next to a 3’ PAM^-. Following target recognition, Cascade recruits Cas3 to generate a single-strand nick, which is followed by 3’ to 5’ degradation of the targeted DNA^,,,.

Figure 2.. Genome editing strategies.

Nucleases generate targeted DNA double-strand breaks (DSBs), which can be repaired by different repair pathways. a ∣ Non-homologous end joining (NHEJ)-mediated repair is error-prone and induces small insertion or deletion mutations (indels). Large, targeted deletions can be produced through repair between two DSBs produced by simultaneously targeting nucleases to two genomic sites. Alternatively, homology-independent targeted integrations (HITI) can be directed to a single cut site by providing donor DNA that is independently targeted for cutting. b ∣ The homology-directed repair (HDR) pathway can be utilized for genome editing by providing either double-strand or single-strand oligodeoxynucleotide (ssODN) donor templates that contain homology arms to the cut target site. Single nucleotide alterations or insertion of larger sequences can be mediated by introducing variations into the donor template, which may also consist of plasmid DNA, viral DNA or long single-stranded DNA. Following HDR, silent mutations — also referred to as blocking mutations (B) — that prevent subsequent target site recognition by the nucleases and formation of NHEJ-mediated indels, can be incorporated into the donor template along with the intended alterations. c ∣ For single nucleotide C→T (or G→A) conversion, Cas9 nickase has been fused to cytidine deaminases such as APOBEC1. For increased base editing efficiency, two uracil glycosylase inhibitors (UGI) have been fused to a base editor for preventing cellular base excision repair.

Figure 3.. RNA targeting tools and their applications.

a ∣ Streptococcus pyogenes Cas9 was repurposed to target RNA (RCas9) by providing it a matching guide RNA (gRNA) and a complementary PAM-presenting oligonucleotide (PAMmer). Cas9 orthologs such as Staphylococcus aureus Cas9 and Campylobacter jejuni Cas9, can target RNA in the absence of a PAMmer, thereby demonstrating PAM-independent RNA cleavage. Cas13 proteins are RNA-guided RNA-targeting nucleases, some requiring recognition of a protospacer flanking sequence (PFS), that generate cuts along target and non-target RNA molecules using two HEPN domains, which are nucleotide-binding domains with RNA cutting activity. b ∣ Similar to catalytically deficient Cas9 (dCas9), dCas13 maintains the capacity to bind the targeted RNA. For RNA visualization and tracking purposes, a fluorescent protein can be fused to the dCas protein and co-localize with an array of crRNAs or gRNAs^,. Adenosine deaminase RNA specific (ADAR) enzymes can be fused to dCas for RNA A→I base editing to correct disease-relevant mutations. To promote alternative splicing, dCas13 can be targeted to bind splicing regulating cis elements. c ∣ Cas13 can be used for targeted RNA degradation in eukaryotic cells for applications such as targeting viral RNA or toxic RNAs that contain microsatellite repeat expansions. Francisella novicida Cas9 has been repurposed in eukaryotic cells to target the RNA genome of hepatitis C virus.

Figure 4.. Targeted gene regulation and other applications.

a ∣ For transcription repression, catalytically-deficient Cas9 (dCas9) alone or dCas9 fused to effectors such as the transcription repression domain of Krϋppel-associated box domain (KRAB) can be targeted to promoters, 5’ untranslated region (5’ UTR) enhancers^,-. Transcription activation can be targeted by fusing dCas9 to transcription activation domains such as VP64: VP64–dCas9–VP64 activated the expression of the neuronal transcription-factor genes Brn2, Ascl1 and Myt1l and thus directed the conversion of primary mouse embryonic fibroblasts into neuronal cells. Similarly, dCas9 was fused to the catalytic domain of methylcytosine dioxygenase TET1 and targeted to the FMR1 gene, to reverse the hypermethylation and silencing of the gene, which is associated with Fragile X syndrome. b ∣ Inducible Cas9-based systems allow dynamic control of gene targeting. For example, chemical induction by rapamycin of the dimerization of split dCas9 fused to the rapamycin-binding domains of FKBP and FRB activates target-gene expression. Alternatively, light-inducible dimerization of the cytochrome proteins CRY2 and CIBN can be used in photoactivatable systems. Combinations of inducible dCas9-ortholog-based systems can be used for dynamic manipulation of multiple targets simultaneously. For example, dimerization of Streptococcus pyogenes dCas9 (dSpCas9)–KRAB by the addition of abscisic acid (ABA) can repress one gene while dimerization of Staphylococcus aureus dCas9 (dSaCas9)–VP64–p65–Rta (VPR) by the addition of gibberellin can lead to activation of another gene. c ∣ CRISPR–dCas9 tools can monitor or manipulate chromatin interactions that regulate gene expression. The fusion of dCas9 to the peroxidase APEX2 can be used to biotinylate proteins in the vicinity of a targeted genomic locus; the proteins are then identified using mass spectrometry. Distal loci can be brought into proximity using chromatin loop reorganization with CRISPR–dCas9 (CLOuD9). In the CLOuD9 system, dSpCas9 and dSaCas9 are fused to the dimerizing, ABA-binding proteins PYL1 and ABI1. ABA induces targeted protein dimerization and chromatin looping, which can be reversed following its removal to restore the endogenous chromatin conformation.