Figure 2. Expanding applications with CRISPR variants and combined use of conditional genetic manipulation techniques.

(A) Alternative ways of genetic modification with CRISPR variants. The sticky-end DNA fragments generated by staggered cutting of Cpf1 allow a precise insertion of donor DNA in the proper orientation into the genome via non-homology-directed repair mechanisms such as NHEJ. C2c2 is a RNA-guided RNA-targeting CRISPR effector that can be programmed to knock down specific mRNAs by cleaving single-stranded RNA targets carrying complementary protospacers. (B) In vivo genome and epigenome editing by adeno-associated viruses (AAV). The SaCas9 and its single guide RNA expression cassette can be packaged into a single AAV delivery vehicle for efficient and specific in vivo genome editing. In dual AAV vectors system, dSaCas9-based chromatin modifiers can be used for multiplex epigenome editing by co-transduction of a dSaCas9-VP64 vector and an expression vector with three U6-sgRNA cassettes in tandem. (C) CRISPR/dCas9-based photoactivatable targeted epigenome engineering. In response to blue light irradiation, fusing of sgRNA-dCas9-CIB1 with the light-sensitive cryptochrome 2 (CRY2) bearing VP64 induces targeted gene activation through VP64 transactivation domain. VP64 co-localizes with dCas9 via CRY2-CIBN interactions and induce transcription only in the presence of blue light. Gene activation is reversible through simple removal of illumination. (D) CRISPR/dCas9-based AND logic gate genetic circuits. CRISPR/dCas9-based AND gate circuits integrate cellular information from two promoters as inputs and activate the output gene only when both inputs are active in the tested disease-relevant cell/tissue types. One promoter (tissue-specific) drives the transcription of dCas9-VP64 mRNA and another promoter is linked to the transcription of sgRNA targeting a specific gene. The expression of sgRNA is mediated by two hammerhead ribozymes placed at both ends of sgRNA. When the primary transcript is generated, it undergoes self-catalyzed cleavage to release the designed sgRNA. The effector (target gene) can be expressed only when both dCas9-VP64 protein and sgRNA are presented. (E) CRISPRainbow and CRISPR-Multicolor for studying nuclear architecture and higher order chromatin organization. Different fluorescent-tagged dCas9-sgRNAs (red, green and blue) allows multiplexed labeling of chromatin loci for tracking gene and chromatin dynamics by spatial and temporal visualizing endogenous genomic loci in live cells. Co-localization of red, green and blue fluorescents indicates a close physical interaction between topologically associated domains (TADs) from different chromosomes. In the case of studying cis/trans-acting regulatory elements within a TAD, CRISPRainbow and CRISPR-Multicolor allow tracking dynamics interaction of enhancers or silencers with a specific promoter for transcriptional regulation. When the dCas9-Blue labeled enhancer close in contact with the dCas9-Red labeled promoter, gene A is activated and pink fluorescent can be visualized. Similarly, when the dCas9-Green labeled silencer close in contact with the dCas9-Red labeled promoter, gene B is activated and yellow fluorescent can be visualized.