Genetics Synthetic Biology

Most Frequently Asked Questions about CRISPR?

CRISPR-Cas9 Editing of the Genome

In this post, let me answer some of the most frequently asked questions related to CRISPR technology!

What is Gene-editing?

Gene-editing or genome editing is a new method of genetic engineering that allows scientists to more precisely than ever before editing the genome of almost any organism. Gene-editing plays a vital role in a wide range of sector including medicines, agriculture, food, energy and more.

What is CRISPR?

CRISPR is a new gene-editing tool that can edit or modify DNA easily and more precisely. It is also known as CRISPR/CAS gene editing tool or CRISPR/CAS9.

CRISPR has the ability to either turn off a gene (by making it non-functional) or turn on a gene or insert a new one.

It is derived from the prokaryotes, where it plays a role in its defense system, providing them with adaptive immunity. Also, whenever a virus invades bacteria, it takes foreign DNA or RNA pieces and integrating it in its own genome.

These bacteria or archaea will become active, transcribe the foreign pieces in their genome and with the help of it, it will check the foreign genome, based on complementarity. When the same virus infects the bacteria, it will hybridize with the foreign genome, giving the signal to the CRISPR system to cleave it, hence stopping viral infection.

 

CRISPR FAQs
Credit: Vox

What CRISPR stands for? and Why?

CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats.

However, the question is what does it mean? What does cluster regularly interspaced repeats mean?

CRISPR loci are those regions in prokaryote genomes, where they integrate foreign genome pieces, but they do not do it randomly. There is a sequential way for it. The parts are incorporated between short palindromic repeats.

Short palindromic repeats, means those repeated parts of DNA, that can be read the same from both sides, for example, Dad, if you read it from the right side, or left side, it is identical.

Moreover, these palindromic repeats are repeated at regular intervals, meaning there are precisely defined interspaces between two consecutive palindromic repeats.

Foreign genome pieces or fragments are stored between these regularly short interspaced palindromic repeats.

That is why it is called clustered regularly interspaced short palindromic repeats.

What are the Components of CRISPR/CAS System?

CRISPR cas9 system includes the following components:

sgRNA or Synthetic guide RNA

A synthetic RNA which is a combination of crRNA and tracrRNA. It guides the Cas nuclease towards the target.

CRISPR Spacer

CRISPR spacer sequence is the sequence which is complementary to the target sequence, and it attached with sgRNA. And after its annealing with the target, then cleaving occurs.

CAS Proteins

CRISPR associated proteins, are a group of proteins, which carry out the primary function of CRISPR – that is cleaving.

There are several CRISPR CAS proteins, i.e. Cas9 protein is used in CRISPR/cas9 system, and it is a nuclease which cleaves DNA. Similarly, Cas13 protein which cleaves RNA. Other include Cas1 and Cas2.

PAM Sequence

CRISPR Pam sequence is a string of three bases, which are needed to be present near the target sequence. They act as a recognition site for the CRISPR system. After its recognition, spacer sequence binds to the target sequence, followed by the nuclease activity of Cas protein.

What is CRISPR cas9?

When Cas9 protein is used in CRISPR gene-editing tool, then it is called CRISPR Cas9.

Cas9 is a DNA nuclease enzyme in nature. It means that CRISPR Cas9 will be used in those experiments which only target DNA.

What is CRISPR cas13?

When CAS13 protein is used in CRISPR gene-editing tool, it is called CRISPR CAS13.

Cas13 is an RNA nuclease, so CRISPR Cas13 acts only on RNA targets.

 

Explore More: How CRISPR discovered mostly by accident?

What other types of Gene-editing are there?

Other gene-editing techniques include;

  • Zinc Finger Nuclease (ZFN), an engineered artificial restriction enzyme that can be used to target specific sequences.
  • Transcription Activator-like effector-based nuclease (TALEN), a restriction enzyme that can be engineered to cleave the target sequences.
  • Meganuclease, an endonuclease that can be engineered to cut, replace or modify specific target DNA sequences.

How CRISPR is different from other gene editing tools?

As compared to other genome editing tools, CRISPR- Cas is used as an alternative because it is more effective and customized. As the CRISPR system can cut DNA strands, it does not require the attachment of segregate cleaving enzymes as other genome editing tools do.

Also, a contrast to other tools, it only needs the redesign/construction of CrRNA to change the specificity of target DNA. Simultaneously, CRISPR can target multiples genes which are another advantage.

What is CRISPR Pam?

In the CRISPR system, Protospacer adjacent motif (PAM) are three nucleotide bases that are present on or near the target sequence. The CRISPR system recognizes the Pam sequences. It signals the CRISPR/Cas for cleavage.

What is the CRISPR patent?

As CRISPR is a revolutionary technique, therefore many Institutions, organizations, and researchers have filed for patents covering several aspects of CRISPR.

There are several patents about CRISPR, but the most famous application for patent is of Broad Institute and University of Berkeley, California. The broad patent was under Feng Zhang and UC, Berkeley patent was under Jennifer Doudna and Emmanuelle Charpentier.

They both had a legal war on the ownership of the patent because both institutes targeted CRISPR Cas9. Broad patent focused on the use of CRISPR CAS9 on Eukaryotes. While UC, Berkeley patent focuses on the use of CRISPR CAS9 in a cell-free system.

What is CRISPR technology? or CRISPR Applications?

CRISPR technology is a novel gene-editing technology that truly revolutionized the field of biotechnology and life sciences.

It is most sought out gene-editing technique because it is cheap, fast, effective, efficiently designed. Also, it has low chances of mistakes/off-target effects as compared to other techniques.

What is CRISPR food?

The food that is modified using CRISPR technology. Due to increase in population, global warming, malnutrition, we need to have Food altered via CRISPR, to overcome these problems. For example, soon we will have a fungal resistant banana, gluten-free wheat, anti-browning mushrooms, disease-resistant crops and more.

What is the CRISPR protocol?

The CRISPR protocols are the comprehensive collection of detailed and peer-reviewed protocols focused on CRISPR/Cas applications, different approaches for guide RNA design. Also, delivery mechanisms of Cas effector proteins and/or guide RNAs into cells and organisms. Moreover, as well as protocols that allow further investigation of the biological role and mechanisms of CRISPR/Cas systems.

What is a CRISPR kit?

CRISPR kit is a set of CRISPR components, lab equipment, Agar media and other important materials needed to perform a successful CRISPR experiment. These CRISPR kits are available commercially.

What are CRISPR babies?

Babies that are genetically modified via CRISPR are called CRISPR babies. There is only one claim regarding CRISPR babies. He Jiankui, a Chinese researcher, claimed that he had created CRISPR babies. He knocked out a gene called CCR5, to make them resistant to HIV Aids.

Nonetheless, he is heavily criticized for his actions. But soon, we will have CRISPR babies to cure several deadly genetic diseases like Huntington’s disease, Duchene muscular dystrophy, cystic fibrosis, phenylketonuria, etc.

Explore More: What Chinese CRISPR Babies mean for the Scientific World?

What is some CRISPR journal/publication?

Some of the CRISPR journals are as follow:

  • Nature
  • Trends in genetics
  • The CRISPR Journal
  • Genome Research
  • UC Berkeley news
  • MIT news

What is the CRISPR stock?

CRISPR stock is a term used for CRISPR associated shares and equities held by different CRISPR companies in the international stock market.

What are a few CRISPR companies?

Some of the famous CRISPR companies are:

CRISPR Therapeutics

Established in 2013
Headquarters: Zug, Switzerland

CRISPR Therapeutics’ mission is to develop transformative gene-based medicines for patients with serious diseases. Their therapeutic approach is to cure diseases at the molecular level using the breakthrough gene editing technology called CRISPR/Cas9.

Caribou Bioscience

Established in 2011
Headquarters: Berkeley, CA, United States

Caribou Biosciences, Inc. develops cellular engineering and analysis solutions based on CRISPR technologies. The company offers CRISPR-Cas gene editing technology with applications in human and animal therapeutics. Moreover, new disease models, genomics, bioproduction cell lines, and fermentation strains, and plants with enhanced traits.

Editas Medicine

Established in 2013
Headquarters Cambridge, MA, United States

Editas aims to develop therapies based on CRISPR–Cas9 gene editing technology.

Intellia Therapeutics

Established: 2014
Headquarters: Cambridge, MA, United States

Intellia Therapeutics, Inc., a gene editing company, focuses on the development of therapeutics utilizing a biological tool known as the CRISPR/Cas9 system. The company develops in vivo programs focused on liver diseases, including transthyretin amyloidosis. Also, alpha-1 antitrypsin deficiency, hepatitis B virus, and inborn errors of metabolism programs.

Other CRISPR Companies include Horizon discovery group, Sangamo Therapeutics, Inscripta, Precision Biosciences and more.

Explore More: Scientists find new and smaller CRISPR gene editor: CasX

 

Images Sources: Flickr, Vox

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