CRISPR/Cas9 System: A Bacterial Tailor for Genomic Engineering

Abstract

Microbes use diverse defence strategies that allow them to withstand exposure to a variety of genome invaders such as bacteriophages and plasmids. One such defence strategy is the use of RNA guided endonuclease called CRISPR-associated (Cas) 9 protein. The Cas9 protein, derived from type II CRISPR/Cas system, has been adapted as a versatile tool for genome targeting and engineering due to its simplicity and high efficiency over the earlier tools such as ZFNs and TALENs. With recent advancements, CRISPR/Cas9 technology has emerged as a revolutionary tool for modulating the genome in living cells and inspires innovative translational applications in different fields. In this paper we review the developments and its potential uses in the CRISPR/Cas9 technology as well as recent advancements in genome engineering using CRISPR/Cas9.

Figure 1

An overview of type II CRISPR/Cas immunity. The CRISPR/Cas system provides the adaptive immunity to prokaryotes against the foreign DNA in three phases. (A) Adaptation: during the adaptation phase, the Cas1-Cas2 complex selects the new spacer (red) and integrates it into the leader-proximal end of CRISPR locus. (B) crRNA biogenesis: in this phase, the CRISPR locus is transcribed into pre-crRNA that forms duplexes with tracr-RNAs with repeat-anti-repeat interaction followed by recognition and cleavage by RNase III into mature crRNA in the presence of Cas9. (C) Interference: during this phase, the mature crRNA/tracr RNA hybrid that remains bound to Cas9 acts as a guide for Cas9 to recognize and degrade the foreign DNA upon subsequent infection.

Figure 2

An overview of CRISPR/Cas9 mediated genome editing. The sgRNA that comprises a single strand RNA guides the Cas9 protein to the target DNA site with a sequence complementary to the 5′ end of sgRNA. The PAM dependent recognition of target DNA sequence by Cas9 initiates the DNA cleavage at a specific site 3 bp upstream of the PAM. The double-strand break generated by Cas9 can be repaired by either NHEJ or HDR. The NHEJ repair often results in indel mutation and inactivation of the gene while the HDR allows the high-fidelity precise genome editing when supplied with donor template.

Figure 3

Zinc Finger Nuclease (ZFN). ZFN (discovered by Chandrasegaran and his team in 1996) are sequence-specific chimeric proteins containing DNA binding domain fused to nonspecific cleavage domain (derived from type II restriction enzyme FOKI) [–20]. DNA binding domain consists of 3-6 Cys₂-His₂ tandemly arranged zinc finger repeats that recognize 9-18 bp sequences (3 bp by each ZFN unit) [21]. Each finger contains approximately 30 amino acids with one α helix and two β strands [19]. The chimeric ZFN are engineered to assemble in pairs and enable efficient and precise genetic modifications by inducing DSBs. In addition to dimerization of FokI nuclease domains, ZFN requires correct spacer sequence (5-6 bp) and orientation of chimeric nucleases for the cleavage of dsDNA [22, 23]. Despite wide applications, the major challenge was to increase the specificity since it was cleaving off-targets that had sequence homology to on-target making ZNF cytotoxic [24]. Custom designed ZNF are prepared by altering DNA binding domain and catalytic domain through mutagenesis and modular assembly of precharacterized ZNF [25]. Till date it has been used for genome editing in mice [26], insects [27], zebrafish [28], and humans (embryonic cells and induced pluripotent stem cells) [29]. Transcription activator-like effector Nuclease (TALEN). TALENS derived from the plant pathogen Xanthomonas sp. are virulence proteins which consist of DNA binding domain and FOK1 nuclease domain. These domains act as dimers and bind to the opposite strands of DNA, separated by a spacer sequence, and create a double-stranded break. DNA binding domain consists of 33-35 amino acid repeats and is arranged in tandem [30, 31]. These repeats are similar except for two highly variable amino acids at positions 12 and 13 called repeat variable di-residue (RVD) which are responsible for specific base recognition and engineering of these bases in repeats [32]. A total four of RVD modules can recognize each of the bases guanine (G), adenine (A), cytosine (C), and thymine (T) and each module is able to function independently. The target sequence must contain thymine (T) at the 5′ end for recognition by TALENS and the spacer sequence should be of 12-20 bp between the dimers [31]. Compared to ZNF, TALENS possess reduced cytotoxicity, are high on targeting efficiency, and are easy to design. However, its high molecular weight makes it difficult for the delivery in the nucleus. AAV vectors are generally used for the delivery of TALENs due to their low immunogenicity and less oncogenic risk [33]. TALENS have been applied for gene disruption in Drosophila [34], C. elegans [35], Arabidopsis [36], Zebrafish [37], and human embryonic stem cells [38].

Figure 4

Cas9-sgRNA-DNA complex. Cas9 protein is an architecture of multiple domains: RecI, RecII, Bridge helix, PAM-interacting, RuvC, and HNH, which accommodates negatively charged sgRNA-DNA heteroduplex. The complex triggers the cleavage of target DNA when sufficient RNA-DNA complementarity is available for the activation of HNH and RuvC nuclease domains.

Figure 5

Strategies for improving the CRISPR/Cas9 specificity. (a) A pair of sgRNAs guiding Cas9 nickases to bind and nick the opposite DNA strands complementary to gRNA sequences. (b) Fusion of catalytically “dead” Cas9 with dimerization-dependent FokI nuclease domains. (c) Altering the gRNA to truncated gRNA (truRNA) with only17-18 nucleotides. (d) gRNA with two additional guanine nucleotides at the 5′- end to form 5′-GGX20 sgRNA. Engineered variants of Cas9: (e) SPCas9-HF1, (f) eSpCas9, and (g) HypaCas9 (in silico mutants were generated from RCSB PDB-4008 using Discovery Studio).

Figure 6

Genome-wide methods for the detection of off-target sites caused by Cas9 nuclease. (A) dCas9-Chip-seq: in dCas9-Chip-seq, the sgRNA and catalytically dead Cas9 (dCas9) plasmid are transfected into the cells. dCas9 proteins bound to DNA are immunoprecipitated after the cross-linking and shearing. The immunoprecipitated dCas9-associated DNA is analyzed by HTGS. (B) GUIDE-seq: in GUIDE-seq, the DSBs generated by RGN in living cells are tagged by integration of a blunt, short, double-stranded oligodeoxynucleotide (dsODN) followed by unbiased tag amplification and high-throughput sequencing for mapping the off-target cleavage sites. Integration sites are identified by LAM-PCR and high-throughput sequencing. (C) IDLV capture: after the transfection of Cas9-sgRNA complexes, the IDLV particles are delivered to get integrated into the RGN induced DSBs. Integration sites are identified by LAM-PCR and high-throughput sequencing. (D) BLESS: in BLESS, the RGN induced DSBs are ligated with sequencing adapters followed by fragment enrichment and amplification for high-throughput sequencing. (E) HTGST: in HTGST, the RGN generated unknown “prey” sequences are captured by known “bait” sequences by end joining repair of DSBs. The captured bait sequences are subjected to LAM-PCR followed by high-throughput sequencing.

Figure 7

Different formats for gene editing using CRISPR/Cas9 in cultured cells: CRISPR/Cas9 components are delivered in DNA, RNA, or RNP formats. Delivery as RGN-ribonucleoproteins (RNPs) improves efficiency and specificity and sidesteps other limitations associated with the use of plasmids.

Figure 8

Overview of Cas9 approach for transcriptional activation and repression of gene/s. (a) RNA guided transcription activation can be achieved by fusing the dCas9 with VP64. (b) Transcription activation by chimeric sgRNA, capable of recruiting activation domains (MS2-VP64). (c) Transcriptional activation by the dCas9-p300 system. (d) SAM based transcriptional activation of endogenous genes. The SAM complex consists of dCas9-VP64 complex, sgRNA with two MS2 RNA aptamers, and MS2 RNA-binding protein (MCP) fused to activators p65 and HSF1. (e) Fusion of dcas9 with KRAB repressor can be used to achieve transcriptional repression. (f) Cas9 acts as a repressor by sterically blocking the transcription machinery. (g) Cas9 acts as a repressor by blocking transcription elongation.