scitubeproGene editing is a process of introducing a modification into a gene of a living organism. It can be a deletion, insertion or replacement of a particular gene. This gene editing process has a wide variety of applications in different fields including basic biological research, development of biotechnology products, and correction of genetic defects and mutations associated with different diseases.
There are two types of genome editing systems currently available, namely protein-guided system and RNA-guided system. CRISPR/Cas system is a RNA guided genetic engineering tool that uses a CRISPR sequence of DNA and its associated protein to modify any DNA sequence found in anywhere in a particular genome. This concept was first proposed by Jennifer Doudna and Emmanuelle Charpentier in 2012. Now, CRISPR has become the most popular gene editing tool among the researchers in fields such as human biology, agriculture, and microbiology.
What is CRISPR/Cas?
The CRISPR-Cas system is originally discovered in prokaryotes such as bacteria and archaea as an adaptive immunity against foreign genetic material of pathogens
CRISPR stands for Clustered Regularly Interspersed Short Palindromic Repeats. This repeating sequences are separated by non-repeating sequence called spacer sequence. Spacer sequence comes from infectious genetic material. Thus, CRISPR array consists of repeating and non-repeating sequences. Cas locus is accompanied with CRISPR array.
Figure01: Diagram of the CRISPR prokaryotic antiviral defence mechanism (https://images.app.goo.gl/7RGXH8kqvHxjoXJr5)
Bacteria exert an immunity via this CRISPR system by uptaking small DNA particles of invasive genetic material such as virus, phages. those viral DNA pieces get integrated to CRISPR locus. After acquiring foreign DNA, CRISPR transcribes into large precursor RNA and then it gets chopped into small CrRNA(CRISPR-RNA) as Cas (CRISPR-associated) protein load to the loop of CrRNA which is encoded by repeat sequences, RNA- Cas nuclease protein complex is formed. During subsequent encounter of particular pathogen, Cr-spacer will recognize a match within the target DNA sequence present in pathogen and disrupt it by Cas nuclease cleavage.
CRISPR/Cas systems can be divided into 3 major types based on different Cas protein present. Type II system is the basis for current genome engineering applications as it is the simplest version among other bacterial CRISPR/Cas systems. It contains Cas nuclease called Cas9 which is present in Streptococcus pyogenes. Therefore, the system is named as CRISPR/Cas9.
Gene editing using CRISPR-Cas9
This system requires 3 key elements for targeted genome cleavage.
1. Homing device: single gRNA (guide RNA)
gRNA is a chimera of CrRNA and TracrRNA (trans-activating CrRNA). TracrRNA is the part that bind to Cas nuclease while CrRNA bind to target DNA.
CrRNA and TracrRNA are separately encoded in natural bacterial system. Hence, the scientists work on to fuse these two genes in genetic engineering to make editing process easy.
2. PAM (Protospacer adjacent motif)
PAM (Protospacer adjacent motif) is a specific sequence of DNA of between 2 to 5 nucleotides depending upon the bacteria which produce Cas9. In here, PAM with sequence NGG specific to Streptococcus pyogenes. PAM is located in non-complementary strand to CrRNA in target DNA. This helps to lock Cas nuclease in the target site as PAM is recognized by specific amino acids in Cas9. Locking of Cas helps to unwind target sequence and facilitate CrRNA base pairing.
3. Endonuclease
Endonuclease: Cas9, uses CrRNA sequence as the guide to recognize and cleave specific strands of DNA that are complementary to the 20bps CrRNA.
Working like genetic scissors, the Cas9 nuclease ( containing 2 cleavage domains) introduces a double strand break (DSB) on both strands of the targeted sequence of DNA. DSB by Cas9 occurs in both strands 3 basepairs upstream of PAM.
The repair of DSBs occurs via two repair pathways. Homology Directed Repair (HDR) pathway where homologus sequence of DNA is used as donor template. Donor template can be delivered into host cell along with CrRNA containing vector or using a separate vector. HDR theoretically allows genetic changes as precise as single base-pair. Non-Homologous End Joining(NHEJ) pathway where the ends of DSB are joined together can often result in random indels (deletions or insertions) at the repair site disrupting or altering gene functionality. HDR creates gene knock-ins whereas NHEJ creates gene knock-outs.
How CRISPR/Cas9 works?
By cloning designed 20bps CrRNA to pCas-Guide vector is generated with all elements required for targeted genomic editing. In here, double stranded DNA transcribing the CrRNA is cloned to the vector.
Figure 02:Overview of pCas-Guide construct (https://images.app.goo.gl/h8rRw8nMEAmW3yqN6)
pCas-Guide (all-in one vector) expresses human codon-optimized Cas9. The restriction sites, BamH I and BsmB I can be used to clone enomic target sequence which encodes for CrRNA.
Figure 03: Involvement of CRISPR/Cas9 system in targeted gene editing (https://images.app.goo.gl/j5iHLDnU5bX89XcNA)
The construct contains strong U6 promoter, gRNA scaffold and terminator signal which are necessary for gRNA expression inside target cell. Multiple origins are in vector to drive its replication inside bacterial and mammalian cells separately. Separate selectable marker genes (Ampicillin resistant and Puromycin resistant) are there for transformant selection inside bacterial and mammalian cells.
The cloned vector is transferred into bacterial cells and allowed to amplify. Then, the vector is isolated and verified the clone by sequencing. The mammalian cells are transfected with recombinant vector and donor template. Using antibiotic selection marker, transfected cells are identified and clonal expression of cells are allowed. Cells are lysed and performed western blotting using appropriate antibody to detect whether desired protein is expressed or not.
Advantages of CRISPR-Cas9
The most important Advantage of CRISPR-Cas9 over other genome editing technologies is its simplicity and efficiency. gRNA can be designed readily and cheaply to target nearly any sequence in genome specifically. Since CRISPR can introduce modifications directly into developing embryo by micro-injecting RNAs encoding Cas protein and gRNA, CRISPR-Cas9 reduces the time required to modify target genes. And also this system can introduce the mutations in multiple genes at same time by injecting multiple gRNAs.
Applications of CRISPR/Cas
CRISPR/Cas can knock-out target gene and simultaneously knock-in functional cassette. And also conditional knock-out of genes specific for particular tissue or cell can be performed to study its function. This system is used to introduce pre-designed mutations like desired SNP, insertions, deletions, tagging to a particular gene. This technology is applied for safe-harbor insertion of transgenes as this provides total control for insertion site, orientation and copy number compared to other transgene insertion techniques. In addition to DNA cleavage, CRISPR/Cas9 tools exhibit functions of transcription control with the use of dCas9 which lacks endonuclease activity. CRISPR interference (CRISPRi) knock-down gene expression whereas CRISPR activation (CRISPRa) activates gene expression.
Limitations of CRISPR/Cas
The most important complication is the off-target effect. A mutation can be introduced at non-specific loci with similar but not identical homology to target site. To minimize off-targets, the specificity of the system is improved by using double nicking strategy which utilizes Cas9 nickase activity instead Cas9.
If the delivery of the system takes long time, it will lead to mosaicism producing a mosaic organism. The other problem occured is, the healing of nuclease cut site by NHEJ pathway can produce cohorts of mice with different mutations from same targeting construct. So, genome sequencing is necessary to be performed to verify the nature and the position of specific mutation
This amazing CRISPR tool makes gene editing easier and faster than ever and offers endless possibilities beyond its applications in all sorts of genetic changes. This account on CRISPR just discusses on the basics of CRISPR/Cas9 system. But, many more things regarding the tremendous potential CRISPR technology in gene editing is to be understood. Eventhough some limitations of the CRISPR/Cas9 system limit its widespread use, different strategies are being developed to improve its effectiveness for editing human, animal, and plant cells. As well, thinking as much as possible is a must before applying CRISPR technology in any field of genetic engineering
“CRISPR-Cas9 can deliver huge benefits to humanity, but of course we need to handle it responsibly. Interventions into the human germline, for instance, which would influence the genome of future generations, is something that I and most of my colleagues refuse to do.” – Max Planck Research, 2015
Author: Sanuri Gamanayake
B.Sc. (Special degree in BioChemistry and Molecular Biology) – Undergraduate
Faculty of Science University of Colombo
References:
1. Barrangou R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science, 315 (5819) (2007), pp. 1709-1712 2. Chylinski K., Le Rhun A., Charpentier E.,The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems. RNA Biol., 10 (5) (2013), pp. 726-737 3. Hsu, Patrick D., Lander E., and Zhang F., Development and Applications of CRISPR-Cas9 for Genome Engineering. Cell, 2014 Volume 157, Issue 6, 1262 - 1278 4. Barrangou R, Doudna JA. Applications of CRISPR technologies in research and beyond. Nat Biotechnol. 2016;34(9):933–941
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