02 - Code Breakers: Using CRISPR to Rewrite Genetics
Guide RNAs (gRNA), sometimes called single guide RNA (sgRNA),are the second key component for CRISPR gene editing. Serving as ‘molecular guides’, they direct the Cas enzymes to a specific target site within a genome. Guide RNA molecules consist of two functional parts. The first is a scaffolding sequence where the Cas enzyme binds. The second is an approximately 20-nucleotide sequence which determines where the DNA will be cut. Through base-pairing interactions, this nucleotide
sequence aligns with the target DNA and brings the Cas enzyme to the intended cut site. To ensure proper interactions between the gRNA - CAS complex and the target DNA, there’s an ad- ditional requirement: a nearby DNA sequence known as a protospacer adjacent motif, or PAM, sequence in the organism’s DNA. This is a sequence of two to six nucleotides found immediately downstream of the DNA sequence targeted
Figure 2: Target DNA and PAM site.
Cas9
gRNA
by the gRNA. PAM sequences allow the CAS enzyme to properly bind to, scan, and cut DNA. However, they also limit what DNA regions can be targeted. For example, the popular CRISPR enzyme Cas9 requires a 5′-NGG-3′ PAM sequence (where “N” can be any nucleotide base). This means that for a researcher using Cas9 the genomic locations that can be targeted are restricted to those containing a downstream 5′-NGG-3′ sequence. Luckily, there are a range of CAS enzymes each with its own unique PAM sequences. Multiple CAS nucleases have been identi- fied. Cas9 is a prominent example. Originally discovered in Streptococcus pyogenes, Cas9 quickly gained popularity in CRISPR gene ed- iting due to its high efficiency and accuracy. Other commonly used CRISPR endonucle- ases include Cas3, Cas12, Cas13, casino, SuperFI-Cas9, and Cs7-100.
Binding to target
Double-stranded cut
Figure 3: CRISPR targeting and digestion of DNA.
The target DNA represents the final critical component for the CRISPR experiment. This is
the specific sequence of DNA that researchers aim to modify. During a CRISPR experiment, the CAS- gRNA complex scans an organism’s DNA by unwinding short segments of DNA and comparing these sequences to the gRNA’s 20 bp binding sequence. When the complex reaches a region of DNA that is complimentary, it attaches. Once attached the Cas enzyme cuts the DNA. THE FORK IN THE ROAD: HOMOLOGOUS DIRECT REPAIR AND NON-HOMOLOGOUS END JOINING Once a CAS enzyme has cut through the double stranded DNA, the cell fixes the damaged nucleic acid chain using its intrinsic DNA repair pathways. Researchers design their CRISPR experiments so that the cell repairs the DNA in one of two ways: using non-homologous end joining (NHEJ) or homologous direct repair (HDR). These two repair mechanisms produce different results, allowing researchers to change the target DNA in distinct ways.
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