CRISPR/Cas9 for Human Genome Engineering and Disease Research

Abstract

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) system, a versatile RNA-guided DNA targeting platform, has been revolutionizing our ability to modify, manipulate, and visualize the human genome, which greatly advances both biological research and therapeutics development. Here, we review the current development of CRISPR/Cas9 technologies for gene editing, transcription regulation, genome imaging, and epigenetic modification. We discuss the broad application of this system to the study of functional genomics, especially genome-wide genetic screening, and to therapeutics development, including establishing disease models, correcting defective genetic mutations, and treating diseases.

Keywords: gene editing; gene regulation; gene therapy; genetic screening; human diseases.

Figure 1

Overview of CRISPR/Cas9 applications. This system has been adapted and developed for gene editing, transcription regulation, chromosome imaging, and epigenetic modification. Gene editing is based on the nuclease activity of Cas9, whereas the three other applications use the catalytic, nuclease-deactivated form of Cas9 (dCas9). Fusing dCas9 to various effector domains enables the sequence-specific recruitment of transcription regulators for gene regulation, fluorescent proteins for genome imaging, and epigenetic modifiers for epigenetic modification.

Figure 2

CRISPR/Cas9 systems for gene editing and gene regulation. (a) Gene editing based on Cas9 nuclease activity. Cas9 cleaves the target DNA and creates double-strand breaks (DSBs), which can be repaired by the endogenous DNA repair mechanism. Two mechanisms are usually deployed by the cells: nonhomologous end joining (NHEJ) and homology-directed repair (HDR). NHEJ usually leads to small insertions or deletions (indels), whereas HDR usually results in the recombination of the donor DNA into the DSB site. (b) Transcriptional repression mediated by the nuclease-deactivated form of Cas9 (dCas9). When binding to the coding sequence, dCas9 can block the progression of RNA polymerase, thereby inhibiting transcription. Tethering a transcription repressor, such as KRAB, to dCas9 could further enhance the transcription repression. (c) Transcription activation mediated by dCas9. Transcription activation can be achieved by recruiting transcription activators to the CRISPR complex. The five illustrated approaches to recruiting various copies and kinds of transcription activators have different levels of activation potency.

Figure 3

Approaches for unbiased genome-wide measurement of double-strand breaks (DSBs) and off-target effects. Next-generation sequencing has greatly facilitated unbiased detection of DSBs in the genome. However, depending on the experimental needs, the upstream DSB labeling and capture and sample preparation for library construction can be very different. Four approaches for capturing DSBs in the genome are shown here: genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-Seq); high-throughput, genome-wide translocation sequencing (HTGTS); breaks labeling, enrichments on streptavidin, and next-generation sequencing (BLESS); and digested genome sequencing (Digenome-Seq). The steps in light-brown boxes are events that occur in the live cells; those in light-blue boxes are cell-free events after DNA extraction. Additional abbreviations: dsODN, double-stranded oligodeoxynucleotide; gDNA, genomic DNA; PCR, polymerase chain reaction; sgRNA, single guide RNA.