Genome Editing: The Concept Of CRISPR

An adaptive immune system is found in archaea and bacteria though Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). Thus by using the DNA sequences that are identical to past invaders into the genome a cellular memory of past invaders would be generated. These sequences recognize viral invaders and results in the degradation of the invading sequence and thus functioning as an adaptive immune system for prokaryotes. CRISPR has different phases and the first phase is when a cellular memory is gained of the past invaders. The short sequences of the viral genome are integrated into the CRISPR locus and scientist identified a highly variable genome locus in bacteria. Many non-contiguous repeats are separated by variable sequences called spacers.

These spacers are correlated with phage resistance which leads to the invaders genome being shortened and integrated into the CRISPR loci and this results in a cellular memory of previous infections. CRISPR RNA (CrRNA) is loaded onto the Cas9 protein and the crRNA drives the Cas9 protein to recognize the invader and then to cleave the incoming plasmid. When the crRNA:tracrRNA is bound to the Cas9 and the Cas9 is activated it can cleave invading nucleic acid sequences.

The expression of DNA plasmids is introduced as sgRNA and Cas9 encoding proteins. For proper expression in the targeted eukaryotic cell Cas9 and sgRNA plasmids must be designed. Expression in different organisms is achieved through Cas9 coding sequence codon optimization. The 5’ and 3’ untranslated regions (UTR) is included for the efficient translation of Cas9 mRNA to protein. The promotor is a sequence which recruits RNA polymerase to start transcription at the site of the gene. The sgRNA must be expressed from the correct promotor. A 5’ cap and a 3’ poly-a tail is needed through post-transcriptional modification to ensure appropriate expression of Cas9 in-vitro. The Cas9 protein can also be expressed and purified and complexed with crRNA and tracrRNA to be introduced as an RNA/protein complex. This method eliminates the need to optimize species-specific promotors and UTR’s.

There is two different ways in which you can identify mutations through screens and selections. A genetic screen identifies a successful edit through examination of many individuals. Screening for the molecular lesion generated by the genome edit is a way of screening for mutations caused by CRISPR. An example of a molecular lesion is restriction fragment length polymorphism (RFLP) analysis that takes advantage of the difference in presence of restrictions sites between the mutation generated by using CRISPR/Cas9 editing and wild-type DNA.

Alternatively to RFLP analysis endonuclease-bases assays such as CEL1 and bacterial T7 endonuclease can cleave and recognize single base mismatches, deletions and insertions. Genetic selection is a strategy that utilizes lethal mutations so that individuals that are important will only survive. A selection strategy that uses a template for repair which introduces a gene for drug resistance. The cells that is exposed to the drug after being introduced to Cas9, sgRNA and the HDR template which kills the cells and only the cells which has been successfully been integrated at the desired locus through HDR of the Cas9.

Sterile tobacco (N. tabacum) and the gene of choice is NtPDS and NtPDR6. The effects of mutation in the PDS gene showed a loss of function mutant and thus showed an albino leaf phenotype. The phenotype of the PDS gene can be observed at an early developmental phase. The study showed that four out of nine transgenic plants with the PDS mutation displayed etiolated leaves. The PDS gene showed that mutations identified 9 out of 11 independent transgenic plants. The endogenous gene of tobacco NtPDS has four target sites. The 20 bp target sequence of the gene exon was designed at the upstream of the protospacer adjacent motif at the 3’ end of the target region. The gRNA of the target sites is generated by overlapping PCR and Cas9 were sub cloned into a single expression vector.

The promotor adding SpeI, SacI and Nos terminator, SmaI and SalI was amplified using vector Pcxsn and inserted into both ends of the Cas9 sequence. The Cas9 expression cassette with HindIII and EcoRI was subcloned into the A. tumefaciencies binary vector pORE O4. The PDS gene sequence was amplified from genomic DNA of wild type tobacco. The gRNA expression cassette digested with NotI and EcoRI can be inserted into the same Pore-Cas9 binary vector so that both gRNA and Cas9 can be co-expressed in one vector. The delivery method of the mutation in the plant is done through CRISPR/Cas9 system. The mutations in the PDK6 targeted sites is measured through the frequencies of Cas9/gRNA induced mutations using the intensity of uncleaved-bands to estimate the mutation rates. The estimated rates is determined with the ImageJ software. To calculate to mutation rates in the PDS gene PCR products were cloned to the Peasy-Blunt Zero vector. And then plasmid DNA was extracted from random colonies and the rate was determined based on the rate of clonal amplicons versus total sequenced clonal amplicons (Gao et al, 2014).