Traditional large genome editing techniques employ indirect cloning of large genomes into bacterial artificial chromosome (BAC) plasmid. This technique is rather difficult and protracted. Thus, bypassing the BAC plasmid stage in large genome editing would be a big win for the application.
The CRISPR-cas9 system, first discovered in Streptococcus pyogenes, is an important tool for targeting and modifying specific genomic sequences with high efficacy. The CRISPR-cas9 complex cleaves DNA upstream of the protospacer adjacent motif (PAM) in a sequence specific manner. Currently, the CRISPR system consists of two major components: RNA-guided nuclease (Cas9) and guide RNA (gRNA) complementary to the target sequence of interest. However, till date, only a few kilobases of DNA has been successfully edited.
To overcome this technical hurdle, Tadahiro Suenaga from the Japanese Research Institute for Microbial Diseases led a team of researchers that manipulated the CRISPR-Cas9 system to edit the large HSV-1 (herpes simplex virus 1) genome of about 152-kb. So, how did they do it?
First, Suenaga and colleagues constructed HSV CRISPR-cas9 targeting complex containing human CD8+ antigen using “oligo-DNA primers” complementary to the BbsI restriction sites.
Secondly, they selected guide RNA sequences 20 nucleotides upstream of PAM in either glycoprotein E (gE) or thymidine kinase (TK) genes of HSV.
And finally, they incorporated the complex into human embryonic kidney 293T (HEK 293T) cells. All without incorporating any artificial genes into the HSV genome.
Below is the outcome of their expedition:
- HSV infected HEK 293T cells transfected with glycoprotein E (gE)- targeting cas9/sgRNA (single guide RNA) that incorporated CD8+ antigen showed high proportion of gE-negative cells
- On the contrary, HSV infected HEK 293T cells transfected with mock cas9/sgRNA targeting complex without the CD8+ antigen were drastically low in gE-negative cells
- Almost all positive cas9/sgRNA transfected cells showed mutations in the gE gene, which was absent in the mock transfected cells
- Furthermore, transfecting cells with HSV-1 thymidine kinase (TK)-targeting Cas9/sgRNA yielded many acyclovir (ACV)-resistant HSV-1 mutants.
The team hold that their approach could contribute “to the generation of mutant viruses for therapeutic applications” more effectively and rapidly than other conventional genome editing methods.
Check out this major leap forward in large genome editing at Microbiology and Immunology, July 2014.