- Explain the natural role of CRISPR sequences in bacteria and how this system has been adapted for editing other genomes.
- Describe the roles of homologous recombination or non-homologous end-joining in CRISPR-mediated genome editing.
- Distinguish between somatic and germ-line genome editing.
We learned previously that most mutations occur at random, so while any mutation is possible, until recently scientists had limited and labor-intensive tools to create desired mutations. The discovery and elucidation of the mechanisms of a bacterial defense system changed this reality.
What is CRISPR?
Viruses are infectious particles of genetic material that affect virtually every type of organism, and therefore defense mechanisms against viruses have evolved. In the case of prokaryotes, the CRISPR locus is a region of genomic DNA to which viral genome sequences can be added to serve as a "memory" of previous infections for future defense against the same virus. The name CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats. The short regions (20-50 nucleotides) of viral DNA are the "spacers" and are used to transcribe CRISPR RNAs (crRNAs). The crRNAs interact with an additional RNA (tracrRNA) and a CRISPR-associated (Cas) protein, which cleaves complementary DNA that enters the cell, thus breaking the viral replication cycle.
EXPERIMENT: Do spacer sequences confer bacteriophage resistance?
A wild-type strain of Streptococcus thermophilus was exposed to two types of bacteriophages (phage 858 and phage 2972) and mutant bacteria resistant to each strain were identified. The spacer sequences in the CRISPR locus in the mutants were then determined and compared to the two bacteriophage genome sequences. Mutants with spacer sequences that corresponded to a sequence in the bacteriophage genome were resistant to specifically to that bacteriophage (Barrangou et al. 2007). Even a few mismatched bases did not confer resistance.
Modifying CRISPR/Cas systems for genome editing
The discovery that an RNA could guide an endonuclease to a specific genomic position to cut DNA presented an opportunity to modify this system to edit genomes. Scientists discovered that they could combined the two RNAs (tracrRNA and crRNAs) into a single small guide RNA (sgRNA). The Cas enzyme can be provided as DNA via a plasmid or RNA encoding the protein, which is produced by the cellular gene expression machinery, or directly as purified protein. The Cas9 protein does require a consensus sequence to interact with DNA (5' NGG 3'), but this motif is relatively common, allowing targeting of most loci.
But how can creating double-stranded breaks in genome benefit an organism? The answer lies in cellular DNA repair. Double-stranded breaks are typically repaired by non-homologous end-joining (NHEJ) or homologous recombination. NHEJ frequently makes small insertions or deletions, which could for example, disrupt the coding sequence of a protein to inactivate it. Homologous recombination mediated repair can occur either by a using the homologous chromosome as a template for repair or by providing exogenous DNA with the desired edit, effectively "tricking" the cell into making the repair based on the donor DNA provided. Importantly, at this time, the efficiency of CRISPR gene editing is not 100% so correctly edited cells must be identified. Additionally, scientists are continuing to study the incidence of off-target (non-intended) edits when using CRISPR-based systems.
Order the steps in CRISPR gene editing.
- Cellular DNA repair
- Cas endonuclease binds genomic DNA at location determined by the complementary small guide RNA
- Identification of edited cells
- Cleavage of genomic DNA
- Cas endonuclease and small guide RNA are introduced into cells
- Select a guide RNA sequence
For help, use this link for a review of the steps https://media.hhmi.org/biointeractive/click/CRISPR/ (Click-N-Learn by HHMI Biointeractive).
6, 5, 2, 4, 1, 3
Nobel Prize-Winning Work
In 2020, Emmanuelle Charpentier from the Max Planck Unit for the Science of Pathogens, Berlin, Germany and Jennifer Doudna from the University of California, Berkeley, USA shared the Nobel Prize in Chemistry “for the development of a method for genome editing” having characterized the CRISPR system in prokaryotes and shown that it could be used in other genomes as well (https://www.nobelprize.org/prizes/ch...press-release/).
Inherited or not?
In terms of human gene editing, the distinction between somatic and germline gene editing may be important to both legal and ethical approval of future CRISPR genetic modifications. Any edits made to germ cells will potentially be passed on to offspring during meiosis. However, gene edits created in somatic cells will not be inherited by offspring.
Somatic gene editing is currently being tested in clinical trials, such as for treatment of sickle cell disease.
Human germline gene editing has only been reported by a Chinese scientist who performed gene editing on embryos to create a mutation that is associated with resistance to HIV in 2018.
Questions surround human gene editing
The ability to do something may not necessarily mean that it should be used without restriction. All people, non-scientists and scientists, are considering what CRISPR means for human treatments and gene editing in the future.
A report was released in 2020 with a number of recommendations regarding Heritable Human Gene Editing. You can find them at this link to the National Academies of Sciences, Engineering and Medicine (https://www.nationalacademies.org/our-work/international-commission-on-the-clinical-use-of-human-germline-genome-editing).
Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero DA, Horvath P. CRISPR provides acquired resistance against viruses in prokaryotes. Science. 2007 Mar 23;315(5819):1709-12. doi: 10.1126/science.1138140. PMID: 17379808.
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 Aug 17;337(6096):816-21. doi: 10.1126/science.1225829. Epub 2012 Jun 28. PMID: 22745249; PMCID: PMC6286148.
Thurtle-Schmidt DM, Lo TW. Molecular biology at the cutting edge: A review on CRISPR/CAS9 gene editing for undergraduates. Biochem Mol Biol Educ. 2018 Mar;46(2):195-205. doi: 10.1002/bmb.21108. Epub 2018 Jan 30. PMID: 29381252; PMCID: PMC5901406.