Summary:

Jennifer A. Doudna and Emmanuelle Charpentier were awarded the 2020 Nobel Prize for Chemistry for their discovery of a technology, described by the Nobel Committee as “Genetic scissors: a tool for rewriting the code of life”. CRISPR (a wisely chosen acronym for Clustered Regularly Interspaced Short Palindromic Repeats) in combination with the enzyme Cas9 (CRISPR-associated protein 9), is a new breakthrough technology that can target a section of DNA, literally cut out a sequence of interest and insert a new sequence in its stead that is then incorporated during the process of DNA repair. There is no doubt that CRISPR/Cas9 represents a watershed moment in science, and applications for therapeutics and even cures of disorders once thought incurable are on the horizon. The Nobel Committee appropriately describes CRISPR as a “technology has had a revolutionary impact on the life sciences, is contributing to new cancer therapies and may make the dream of curing inherited diseases come true.”

In a publication by Ma et al. (Nature, 2017), researchers were able to take human preimplantation embryos and cut out and replace a disease-causing pathogenic variant, using the CRISPR/Cas9 gene-editing tool.

In anticipation of a fast-paced research trajectory, The American Society of Human Genetics (ASHG) brought together an international workgroup that included representatives from the following organizations

• UK Association of Genetic Nurses and Counsellors
• Canadian Association of Genetic Counsellors
• International Genetic Epidemiology Society
• US National Society of Genetic Counselors

The document was endorsed by

• American Society for Reproductive Medicine
• Asia Pacific Society of Human Genetics
• British Society for Genetic Medicine
• Human Genetics Society of Australasia
• Professional Society of Genetic Counselors in Asia
• Southern African Society for Human Genetics

The following positions are stated in this document:

(1) At this time, given the nature and number of unanswered scientific, ethical, and policy questions, it is inappropriate to perform germline gene editing that culminates in human pregnancy. (2) Currently, there is no reason to prohibit in vitro germline genome editing on human embryos and gametes, with appropriate oversight and consent from donors, to facilitate research on the possible future clinical applications of gene editing. (3) There should be no prohibition on making public funds available to support this research. (4) Future clinical application of human germline genome editing should not proceed unless, at a minimum, there is (a) a compelling medical rationale, (b) an evidence base that supports its clinical use, (c) an ethical justification, and (d) a transparent public process to solicit and incorporate stakeholder input.

What is CRISPR and How Does It Work?

History: Bacteria Defense System

• In 1987, segments of short palindromic repeats were discovered in bacterial DNA
  • These repeats were actually part of an ancient system used by bacteria to defend themselves against viruses
  • Through a process of ‘acquired immunity’, bacteria were found to have ‘spacers’ between the repeats that were actually the same sequences found in threatening viral genomes
  • If a bacteria was found to have a specific viral sequence, they were protected from that particular virus
• Over time, researchers discovered that
  • Cas (CRISPR-associated system) genes were identified near these repeat sequences, producing enzymes that can cut DNA
  • Bacteria can generate RNA that match the spacer sequences
  • The Cas enzymes circulate with these specialized RNA molecules that function as viral genome locators
  • If there is a matching viral sequence, the RNA molecule will lock on and the Cas enzymes will then cut through DNA preventing replication

Now: Genome Engineering and CRISPR/Cas9

• Scientists modified the above system, found in nature, but the overall principles are the same
• If one knows the sequence of interest in a particular gene, an RNA locator can be created that matches that sequence
  • The RNA locator sequence will bind to the sequence of choice
  • The Cas9 enzyme can then cut out the sequence of interest
  • Researchers can replace the missing sequence with another sequence of choice
  • The DNA will undergo repair and incorporate the new sequence
• In the case of the recent paper in Nature, the authors applied CRISPR/Cas9 to embryos with a pathogenic variant (a deletion of just 4 base pairs – GAGT) in the MYBPC3 gene that causes hypertrophic cardiomyopathy
  • The pathogenic variant, inherited from the father, was replaced with a normal sequence from the mother
  • In theory, because hypertrophic cardiomyopathy is an autosomal dominant disorder, this procedure would be curative and a normal embryo could then be transferred to the mother
  • The authors of this research paper do not feel that this technique can be used clinically without further optimization because intrinsic mechanisms used to repair DNA introduce new deletions and insertions
• Tap the icon below to see the CRISPR/Cas9 NIH Graphic

CRISPR/Cas9 NIH Graphic (Credit: NIH)

CRISPR and COVID-19

• In May 2020, the FDA gave emergency approval for the development of a CRISPR based test (under the guidance of Sherlock Biosciences) that can diagnose COVID-19 in around an hour
  • The test utilizes Cas13a (instead of Cas9 as discussed above), to identify the specific RNA sequence that is unique to COVID-19
• In June 2020, researchers at Stanford University and Lawrence Berkeley National Laboratory announced in “Cell” that they created a way to disable COVID-19
  • The paper discusses a new technique called prophylactic antiviral CRISPR in human cells, or PAC-MAN, that is able to knock out the virus by using CRISPR to scramble the genetic code in COVID-19.
  • The researchers also found a way to deliver PAC-MAN to lung cells where COVID-19 does the most damage
  • How it works: PAC-MAN uses a guide RNA to direct Cas13d to target areas of the COVID-19 genome – cleaving the genetic sequences and inactivating the virus

Learn More – Primary Sources:

The ObG Project highly recommends you join the almost 2-million people who have viewed Dr. Jennifer Doudna’s TED talk. As a co-inventor of CRISPR/Cas9, she explains this groundbreaking technology in less than 20 minutes and makes the case for why this research must be done within an ethical framework.

How CRISPR lets us edit our DNA | Jennifer Doudna

Correction of a pathogenic gene mutation in human embryos

ASHG Position Statement: Human Germline Genome Editing

Review: Development and Applications of CRISPR-Cas9 for Genome Engineering

Review: CRISPR-Based Technologies for the Manipulation of Eukaryotic Genomes

Development of CRISPR as an Antiviral Strategy to Combat SARS-CoV-2 and Influenza

Nobel Prize in Chemistry Announcement 2020

Related ObG Topics:

The Genome and Whole Genome Sequencing

The Exome and Whole Exome Sequencing: Prenatal Testing Recommendations

The Chromosome – Structure and Number