Genome Editing: the CRISPR/Cas9 system

Genome Editing

Genetic modification is the intended modulation of gene expression in specific cells or organisms to treat pathological disorders. The introduction of exogenous nucleic acids such as DNA, messenger RNA (mRNA), small interfering RNA (siRNA), micro RNA (miRNA) or antisense nucleotides settles the basis of gene-based therapy. Given the plurality of size and charge of these macromolecules, OZ Biosciences has developed numerous versatile transfection reagents to mediate their delivery. 

A very recent technology named “Genome editing” or “genome engineering” gives now the investigators ability to precisely and efficiently introduces a variety of genetic alterations into mammalian cells (deletion, insertion…). During the past decade, zinc finger nucleases (ZFNs) and transcription activator-like effector nuclease (TALENs) illustrate the rapid development and innovation in genome-editing technologies. ZFNs and TALENs defined a new powerful class of tools that redefined the limits of biological research until the very recent discovery of the clustered regularly interspaced short palindromic repeat (CRISPR) arrays and their CRISPR associated (Cas) proteins.

With more than 10 years of expertise in the development of transfection reagents, OZ Biosciences offers tailored transfection solutions for CRISPR/Cas9 technology.

 

How does CRISPR/Cas9 work?

CRISPR/Cas9 system is originated from bacteria in which it provides acquired immunity against invading foreign DNA via RNA-guided cleavage [1]. The bacteria collect “protospacers”, short segments of foreign DNA (e.g. from bacteriophages) and integrate them into their genome. Sequences from CRISPR genomic loci are then transcribed into short CRISPR RNA (crRNA) that anneal to transactivating crRNA (tracrRNAs) to destroy any DNA sequence matching the protospacers. After transcription and processing, crRNA first complexes with Cas9 and tracrRNA and then bind target sequence onto DNA. An R-loop forms and both strands of DNA are cut. crRNA is used as a guide while Cas9 acts as an endonuclease to cleave the DNA (figure 1).

Figure 1. The CRISPR-Cas9 nuclease programmed with sgRNA. Upon binding the sgRNA guide (tracrRNA-crRNA) specifically targets a short DNA sequence-tag (PAM) and unzips DNA complementary to the sgRNA. sgRNA–target DNA heteroduplex, triggering R-loop formation results in a further structural rearrangement: Recognition (REC) and Nuclease lobes (NUC) undergo rotation to fully enclose the DNA target sequence. Two nuclease domains (RuvC, HNH) each nicking one DNA strand, generate a double-strand break. Structurally, REC domain interacts with the sgRNA, while NUC lobe drives interaction with the PAM and target DNA.

 

Genome Editing with CRISPR/Cas9

In 2013, four groups demonstrated that CRISPR/Cas9 associated with guide RNA can be utilized for gene editing [2-5]. Based on the type II CRISPR/Cas9 mechanism, researchers created a single guide RNA (sgRNA), a chimeric form of crRNA and tracrRNA which is able to bind to a specific dsDNA sequence. This resulted in double strand breaks (DSB) at target site with: (1) a 20-bp sequence matching the protospacer of the guide RNA and (2) a protospacer-adjacent motif (PAM) 3 bp downstream NGG sequence. CRISPR/Cas9-mediated genome editing thus depends on the generation of DSB and subsequent cellular DNA repair process. The presence of DSB in the DNA generated by CRISPR/Cas9 leads to activation of cellular DNA repair processes, including non-homologous end-joining (NHEJ)-mediated error prone DNA repair and homology-directed repair (HDR)-mediated error-free DNA repair. Insertions and deletion mutations at target site generated by NHEJ and HDR allow disrupting or abolishing the function of a target gene. Moreover, modifications in this system can also be used to silence gene, insert new exogenous DNA or to block RNA transcription. 

Various Cas9-based applications:

• Indel mutations,
• Specific sequence insertion or replacement
• Large deletion or genomic rearrangement (inversions or translocation)
• Fusion to an activation domain :

o   Gene Activation

o   Other modifications (histone modification, DNA methylation, fluorescent protein)

o   Imaging location of genomic locus.

 

CRISPR/Cas9 advantages over ZFNs and TALENs

CRISPR/Cas9 can be easily adapted to virtually any genomic sequence by changing the 20-bp protospacer of the guide RNA; the Cas9 protein component remaining unchanged. This ease of use presents a main advantage over ZFNs and TALENs in generating genome-wide libraries or multiplexing guide RNA into the same cells.

• ZFNs and TALENs are built on protein-guided DNA cleavage that needs complex protein engineering.
• CRISPR/Cas9 only needs a short guide RNA for DNA targeting.
• CRISPR/Cas9 allows using several gRNA with different target sites: simultaneously genomic modifications at multiple independent sites (Cong et al, 2013).
• Accelerates the generation of transgenic animals with multiple gene mutations [6].

CRISPR/Cas9 system presents a versatile and reliable genome editing tool to facilitate a large variety of targeting genome applications. CRISPR/Cas9 components comprise endonuclease and sgRNA that can be delivered into cells under several forms. For each form we can propose a solution. 

 

OZBIOSCIENCES’ Tools for CRISPR/Cas9 system

For generation of cellular models, Cas9 and the appropriately designed sgRNA (a chimeric RNA containing all essential crRNA and tracRNA components) can be easily introduced into the target cells. The type II CRISPR/Cas system only needs a single Cas protein that can be expressed into target cells by: (1) plasmid transfection^**, (2) direct delivery of the active Cas9 endonuclease, (3) transfection of mRNA enconding for Cas9 or (4) by viral vectors transduction.

OZ Biosciences offers solutions transfecting any kind of cell with every form of Cas9 (figure 2).

 ^**most CRISPR/Cas9 system also expresses the guide RNA from a plasmid using a RNA Polymerase III promoter such as the U6 promoter.

Figure 2. For each CRISPR/Cas9 application a specific transfection reagent is provided by OZBiosciences.

Bibliographic references

1. Wiedenheft B, et al. RNA-guided genetic silencing systems in bacteria and archaea. Nature 482, 331-338.

2. Cong L, et al. Multiplex genome engineering using CRISPR/Cas systems. Science. 2013;339 (6121):819-823.

3. Mali P, et al. RNA-guided genome engineering via Cas9. Science. 2013;339 (6121):823-826.

4. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 2012;337 (6096):816-821.

5. Cho SW, Kim S, Kim JM, Kim JS.  Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol. 2013;31(3):230-232.

6. Wang H, et al. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell. 2013;153 (4):910-8.