Question

4. The CRISPR-Cas9 system is an important new technique in molecular biology. What is the natural function of this system? Describe how you would use this system to generate a null mutation in another organism (i.e. explain Figure 6-43). How does it work? What is the modification of the method that allows for correction of a mutation (e.g. the mouse crystalline gene)? And lastly, what are the problems with the CRISPR system?

Guide RNA expression Target tracrRNA sequence scaffold Cas9 expression Codon optimized Cas9 NLS = Species- specific Transcription and translation promoter Species- specific promoter Transcription Cas9 Functional chimeric guide RNA (gRNA) Genomic DNA Target Gene of sequence interest Promoter Cas9 binds ORNA target sequence Cas9 Distinct Cas9 nuclease sites cleave target DNA Cas9 Cas9-induced double-strand break at target sequence (d) Nonhomologous end joining Homology-directed repair (HDR) = = Homology Nonhomologous end joining

FIGURE 6-43 Single-nucleotide mutations can be introduced into the genome using an engineered CRISPR-Cas9 system. (a) The genome of a target cell can be modified by expression of the double-stranded DNA endonuclease Cas9 and a guide RNA. Expression of these components can be achieved by transfection with plasmids carrying genes for Cas9 and the guide RNA or by direct injection of Cas9 mRNA and guide RNA. The guide RNA is composed of two parts: a sequence that folds into a hairpin scaffold structure that binds to Cas9, and a sequence of approximately 20 nt corresponding to the targeted site in the genome. Expression of these components can be achieved by transfection with plasmids carrying genes for Cas9 and the guide RNA or by direct injection of Cas9 mRNA and guide RNA. (b) A complex of guide RNA bound to Cas9 is targeted to the genome by base pairing of the guide RNA with the complementary genomic DNA sequence. This structure allows the two distinct nuclease active sites of Cas9 to cleave both strands of the target DNA adjacent to the heteroduplex formed with the guide RNA. (c) By this mechanism, the expression of both Cas9 and a bipartite guide RNA designed to target a specific gene sequence leads to a double-strand cleavage of the target gene. (d) Cleaved DNA can be repaired via a nonhomologous end joining (NHEJ) process, which usually removes a small number of bases at the cleavage site. If the cleavage occurs in a coding sequence, NHEJ will usually inactivate gene function by producing a frameshift mutation. If a ∼100-nt single-stranded DNA segment that spans the sequences flanking the cleavage site is injected along with Cas9 mRNA and the guide RNA, the cleaved DNA can be repaired by homologous recombination (homology-directed repair, HDR). By this mechanism, single base changes can be introduced into the repaired genomic DNA.

Answer 1

In CRISPR system Cas9 is a protein that plays a role of immunological defense system in bacterial cells by cleaving and destroying viral DNA or foreign plasmids in the bacterial cell.

A null mutant can be made using crispr cas9 and small single stranded DNA oligo repair template that is used to insert a long Knock in casette (STOP IN) in exons of target genes. This STOP IN cassette has stop codons in all the reading frames and can lead to frameshifts that give rise to null mutation whatever the reading frame of the insert has.

According to the figure, to introduce a null mutation in genome of target cell double stranded DNA endonuclease cas 9 and guide RNA is expressed. This is done either by transfecting the cells with plasmid containing these genes or directly injecting them into the cell. Complex of guide RNA and Cas9 targets the genome by complementary basepairing. Cas 9 does the targetted double stranded cleavage that is repaired by non homologous end joining mechanism.A single stranded DNA segment introduction will allow single base pair mutation with stop codons causingnull mutation.