CRISPR From A Bacterial Defense System To Gene Editing

Targeted genomic editing using custom nucleases is a general method for inducing precise deletions, insertions, and sequence changes in a wide range of organisms and cell types. For many years, strategies to efficiently induce specific and targeted genome alterations were limited to certain organisms (yeast homologous recombination or mouse recombination) and often required drug-selectable markers or ‘scarring’ sequences.

The introduction of targeted modifications of the genomic sequence into cells and living organisms has become a powerful tool for biological research and a potential avenue for the treatment of genetic diseases. The first method of targeting DSB-mediated nucleases to specific genomic sites were stored by generic linkages with customizable DNA-binding interfaces, such as meganucleases, zinc finger nucleases (ZFNs), and Effector type effector (TALEN). NHEJ can be used effectively from insertion / deletion mutations of different lengths. With targeted nuclease-induced DSBs, the frequencies of these modifications are generally greater than 1% and in some cases greater than 50%; simple screening without selection of drugs resistance marker could identify desired mutations. A crucial first step in targeted genome editing is to create double-stranded DNA (DNA) at the genomic locus to be modified.

More recently, a plate formed on a Streptococcus pyogenes (here in after referred to as Cas9) nuclease associated with CRISPR has been developed; it is one and flexible to depend on the dependence both on the target of nuclease to a DNA sequence is desired. The high efficiency of genome editing avoids the use of additional sequences, drug resistance markers, and additional manipulations to remove them. CRISPR systems are adaptable immune mechanisms used by many bacteria to protect themselves from foreign nucleic acids, such as viruses or plasmids. Type II CRISPR systems incorporate sequences from invading DNA between CRISPR repeat sequences encoded as arrays within the bacterial host genome.

Transcripts from the CRISPR repeat arrays are processed into CRISPR RNAs (crRNAs), each harboring a variable sequence transcribed from the invading DNA, known as the “protospacer” sequence, and part of the CRISPR repeat. Each crRNA hybridizes with a second RNA, known as the trans-activating CRISPR RNA (tracrRNA), and these two RNAs complex with the Cas9 nuclease7. The protospacer-encoded portion of the crRNA directs Cas9 to cleave complementary target-DNA sequences, if they are adjacent to short sequences known as proto- spacer adjacent motifs (PAMs). Protospacer sequences incorporated into the CRISPR locus are not cleaved presumably because they are not next to a PAM sequence.

The type II CRISPR system from S. pyogenes has been adapted for inducing sequence-specific DSBs and targeted genome editing. In the simplest and most widely used form of this system, two components must be introduced into and/or expressed in cells or an organism to perform genome editing: the Cas9 nuclease and a guide RNA (gRNA), consisting of a fusion of a crRNA and a fixed tracrRNA.

Twenty nucleotides at the 5′ end of the gRNA direct Cas9 to a specific target DNA site using standard RNA-DNA complementarity base- pairing rules. These target sites must lie immediately 5′ of a PAM sequence that matches the canonical form 5′-NGG (although recognition at sites with alternate PAM sequences (e.g., 5′-NAG) has also been reported, albeit at less efficient rate. Thus, with this system, Cas9 nuclease activity can be directed to any DNA sequence of the form N -NGG simply by altering the first 20 nt of the gRNA to cor- 20 respond to the target DNA sequence.

Following the initial demonstrations in 2012 that Cas9 could be programmed to cut various DNA sites in vitro, a flurry of papers published in 2013 showed that this platform also functions efficiently in a variety of cells and organisms. Initial proof-of-principle studies showed that Cas9 could be targeted to endogenous genes in bacteria, cultured transformed human cancer cell lines and human pluripotent stem cells in culture, as well as in a whole organism, the zebrafish (J.K.J. and colleagues).

Applications of CRISPR-Cas beyond genome editing

Although observed activation appears to be somewhat less robust than direct mergers with dCas9, this type of configuration could offer additional options and flexibility for the recruitment of multiple effector domains on a promoter, for example by using multiple gRNAs and sites binding of the MS2 envelope protein on each gRNA to recruit many copies of different domains on the same promoter.

In addition, co-expression of fusion of MS2 coat protein with hybrid gRNA and Cas9 was used to recruit activation domains on a gene promoter into human cells. it has been shown that dCas9 fusions with a transcription activation domain (VP64 or p65 nuclear factor kappa subunit B; NF-KB) or a transcriptional representation domain (the domain of Krüppel-associated box (KRAB)) expression of endogenous genes in human and mouse cells. The changes in gene expression induced by these dCas9 fusions in human cells so far appear to be generally lower than those induced by similar transcription factors based on TALE.

Interestingly, dCas9 effectively repressed a bacterial promoter when it was recruited with gRNAs interacting with one of the strands of sequences upstream of the promoter; however, when targeting sites downstream of the transcription start point, only gRNAs that interacted with dCas9-induced non-model induced strand-induced repression. In addition to enabling easy and efficient targeted genome editing, the CRISPR-Cas system can potentially be used to regulate the expression of endogenous genes or to mark specific chromosomal loci in living cells or organisms.

However, multiplex recruitment of dCas9-based activators using between 2 and 10 sGARN targeted on the same promoter may result in significantly higher levels of human gene activation, possibly due to the enhancer synergistic phenomenon.

EGFP-dCas9 fusion has been shown to be useful for visualizing DNA loci containing repetitive sequences, such as telomeres, with single gRNAs or unrepeated loci using gRNA inserted into a 5k region of DNA. The addition of MS2 RNA binding sequences to gRNA does not abolish its ability to target dCas9 on specific DNA sites. In the future, it will be interesting to see if mergers with histone modifiers and proteins involved in the modification of DNA methylation, such as TET (translocation) proteins, can also be used for specific edition of the epigenome. Cas9 also provides a general platform for the recruitment of heterologous effector domains into specific genomic loci, including the alteration of specific histone modifications and the de-methylation of particular cytosine bases in human cells with DNA binding domains TALE.

Future directions

First, methods to broaden the targeting range of RNA-guided Cas9 will be important for inducing specific HDR or NHEJ events, as well as for the implementation of multiplex strategies, including nickases in pairs. Alternatively, the construction of inducible forms of Cas9 and / or gRNA could provide a means to regulate the active concentration of these reagents in the cell and thus improve the ratio of the effects on the target and on the target.

Second, there is an urgent need for the field to develop unbiased strategies to comprehensively assess the non-targeted effects of Cas9 nucleases or matched nickases in any genome of interest. Although the untargeted effects of Cas9 remain to be defined at the genome level, much progress has already been made to improve specificity, and further progress is likely to occur rapidly, given the intensity of research efforts in this domain. Other gRNAs: Cas9 platforms with different PAM sequences isolated from Streptococcus thermophilus, Neisseria meningitidis and Treponema denticola were also characterized and the identification of several of these systems from other species could further improve the targeting range of the platform.

A related challenge will be to develop methods to express gRNAs or Cas9 nuclease specific to a tissue, cell type, or stage of development. This limitation is particularly problematic when using HDR to induce point mutation changes (as opposed to insertions) in the protospacer portion of the target site; Alleles that have been successfully modified in this way can still be effectively cut again and then mutagenized by NHEJ, reducing the yield of properly edited sequences. In addition, although paired tru-gRNAs and nickases can reduce non-targeted effects, it is likely that further improvements will be needed, particularly for therapeutic applications.