In lesson 2.1 Repurposing Bacterial CRISPR for Human Gene Editing you were introduced to the example of sickle cell disease to illustrate the concept and promise of gene editing for human therapeutics, where the causative mutation could be repaired with CRISPR. You also learned about the molecular requirements necessary to harness CRISPR-Cas9 for gene editing: (1) the design of the 20-letter guide sequence, (2) the choice of a matching target sequence in the genome flanked by “NGG” (where “N” can be any letter of DNA), and the use of a repair template that contains the desired edit.

Now, using this information, together with the actual DNA sequence of the mutated beta-hemoglobin gene that causes debilitating sickle cell disease in patients, you will propose a “guide RNA” sequence and “repair template” sequence to serve as starting materials for the therapeutic treatment of this disease. 

In this Benchling file, you can see that the 7th codon of the hemoglobin beta gene, which normally has the sequence “GAG” and codes for the amino acid Glutamic acid (Glu), is mutated to GTG and instead codes for the amino acid Valine. (Remarkably, this single mutation is the entire cause of the sickle cell phenotype in red blood cells.) Your goal is to propose: 

• a 20-letter guide sequence that directs Cas9 to cut somewhere close to the mutation, and 
• a repair template that can be used by human cells, upon experiencing a CRISPR-Cas9-induced DNA break, to convert the mutated sequence back to the healthy sequence. Remember: for Cas9 to work properly, it must target a sequence in the gene that is next to a short motif (called the PAM), that reads “NGG”. 

Refer back to lesson 3.5 Identify and Annotate a Real-life CRISPR-Cas System for introductory information on using Benchling, and propose a “guide RNA” sequence and “repair template” via the discussion or by uploading a file. Feel free to comment on each other’s thoughts.