The multiplexed CRISPR targeting platforms

Abstract

The discovery and engineering of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) in the past several years have revolutionized biomedical research. The CRISPR technology showed great potential to advance detection, prevention, and treatment of human diseases in the near future. Compared to previous developed genome editing approaches, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), the CRISPR-based systems have numerous advantages. One example is that the CRISPR systems can be easily adopted to efficiently target multiple genes simultaneously. Several strategies and toolboxes have been developed to achieve multiplexed targeting using the CRISPR systems. In this short review, we will discuss the principle, approach, and application of these strategies.

Figure 1.. Natural and engineered CRISPR systems.

(A) Schematic of a natural CRISPR pathway. Foreign DNA is captured and inserted between repeats in a CRISPR locus in a bacteria genome. CRISPR array is transcribed and then processed into multiple crRNAs, each carrying a single spacer sequence and a repeat sequence. The crRNA forms a complex with Cas9 and crRNA and directs the complex to the foreign DNA carrying the same sequence as the spacer. Cas9 then generates a double strand break on the foreign DNA. (B) Schematic of an engineered Cas9 genome editing tool. It comprises a Cas9 endonuclease and a single guide RNA (sgRNA). Cas9 and sgRNA form a complex. The guide sequence on sgRNA directs the complex to the target site. Cas9 generates a double strand break, which is repaired via the error-prone non-homologous end-joining (NHEJ), leaving a small insertion or deletion (Indel) at the target site. Indels in protein coding region cause reading frame shift and early termination of protein translation, resulting in loss of protein expression.

Figure 2.. CRISPR-based applications with nuclease-deficient Cas9 (dCas9).

dCas9 serves as a sequence-directed genome target platform. Fusing dCas9 to different functional domains allows for a wide range of applications, including transcriptional up-regulation (with transcriptional activators) and down-regulation (with transcriptional repressors) (A), adding (with epigenetic writers) or removing (with epigenetic erasers) epigenetic marks (B), and visualization of specific genomic regions (C).

Figure 3.. Strategies for multiplexed CRISPR/Cas9-based genome editing.

(A) In vivo pre-assembled RNP of Cas9 and multiple sgRNAs. (B) Introduction of Cas9 mRNA and multiple sgRNAs simultaneously. (C) Introduction of multiple single-sgRNA delivery plasmids simultaneously. (D) Delivery of Cas9, tracrRNA, and a crRNA array with a single plasmid. (E) Delivery of Cas9 and multiple sgRNA expression cassettes with a single plasmid. (F) Delivery of an artificial multi-sgRNA precursor, Cas9, and Csy4 with a single plasmid. sgRNAs are flanked by Csy4 binding sequences. Csy4 digests the precursor to release individual sgRNAs. (G) Delivery of an artificial multi-sgRNA precursor and Cas9 with a single plasmid. sgRNAs are flanked by HH and HDV ribozymes. The ribozymes digest the precursor to release individual sgRNAs. (H) Delivery of an artificial multi-sgRNA precursor and Cas9 with a single plasmid. sgRNAs are flanked by tRNAs. The endogenous tRNA processing machinery digests the precursor to release individual sgRNAs.

Figure 4.. Two Golden Gate assembly-based cloning strategies.

(A) Schematic of a two-step strategy. Guide sequences are first cloned into single sgRNA plasmids. Each sgRNA expression cassette is amplified by primers carrying type IIs enzyme binding and a series of digesting sequences. Type IIs enzyme digestion produces overhangs that allow for assembly of multiple fragments in a designed order. (B) Schematic of a single-step strategy. Primers carrying guide sequences are used to amplify RNA scaffold and promoter sequences from specially designed vectors (Lenti-multi-Guide or Lenti-multi-CRISPR, Addgene #85401 or 85402). BsmBI digestion produces a series of overhangs that allow for assembly of multiple fragments in a designed order.