It doesn’t get crispier than CRISPR: An Overview of the Revolutionary Gene Editing Technology

Written By Vaishnavi Kolluru

Clustered regularly interspaced short palindromic repeats (CRISPR) gene editing is a revolutionary technique that allows scientists to “knock out” or “knock in” genes of interest. This technique leverages the natural bacterial defense against bacteriophages; RNA-mediated detection of viral ssDNA initiates the cascade of events ultimately leading to degradation of the foreign ssDNA, thereby preventing integration into the bacterial genome.

We utilize the Cas9 endonuclease to break specific DNA segments in the gene editing process. We insert a plasmid with guide RNA (gRNA) to bind to the area of the genome to knock out / knock in a gene. For knock in experiments, a target DNA sequence to insert in the area of gRNA binding is present next to the plasmid’s gRNA. And of course, the plasmid also has an ampicillin resistance (AmpR) gene (or some other antibiotic resistance gene) to help weed out bacteria that haven’t taken up the plasmid.

These sequences will be inserted by non-homologous end joining (NHEJ) which repairs the double strand break caused by Cas9. For a knock in experiment, the plasmid’s target sequence will be inserted in the genome during the NHEJ process, and the complementary strand will be synthesized by DNA ligase. In addition, homology-directed repair (HDR)* mechanisms also play a role in plasmid integration; the molecular machinery will use homologous regions as guides when inserting elements of the plasmid, leading to increased precision in the integration of genetic material. For a knock out experiment, NHEJ will occur and disrupt the gene that Cas9 broke, so the gene is knocked out with no target DNA from the plasmid to insert as a substitution sequence. 

*Bacteria don’t have HDR since they don’t have homologous chromosomes. But when CRISPR technology is implemented in eukaryotic cells, HDR is a possible mechanism for DNA repair.

Because double strand break repair is very haphazard (due to there being no template strand to guide repair), scientists have developed another technique for CRISPR experiments. This technique uses nickase, which causes single stranded breaks in DNA. This is advantageous over NHEJ and HDR because single strand breaks preserve the complementary strand. DNA ligase can synthesize the necessary elements to reduce net DNA damage. In addition, the nickase technique uses 2 gRNAs (one for each strand) instead of one, which increases the specificity of gene manipulation, thereby improving the precision of the process.

Nickase cutting mechanism.

To further increase precision in knockout experiments, we can use CRISPR interference (CRISPRi), which uses a mutated Cas9 (called dCas9, or “dead” Cas9) to silence genes without damaging the associated DNA. dCas9 has lost its catalytic ability; however, it is still able to bind to the DNA sequence of interest due to its maintained association with gRNA. The dCas9 is fused to the Krüppel associated box (KRAB) domain, which represses transcription by inactivating RNA polymerase, thereby silencing the gene to which dCas9 has bound. With this method, we are able to silence genes without harming the original DNA sequence.

These techniques have many applications in genetic disorders. Gene editing is the future of genetic medicine, but we have a long way to go before this technique is widely used in clinics.

Here are some cool resources to supplement your learning:

PS - little note about Cas9 functionality from Google: “The PAM, also known as the protospacer adjacent motif, is a short specific sequence following the target DNA sequence that is essential for cleavage by Cas nuclease. The PAM is about 2-6 nucleotides downstream of the DNA sequence targeted by the guide RNA and the Cas cuts 3-4 nucleotides upstream of it.” - https://www.synthego.com/guide/how-to-use-crispr/pam-sequence

Vaishnavi Kolluru
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