CRISPR/Cas9 Flexibility Slows Gene Editing Process

CRISPR/Cas9 is regarded as the cutting edge of molecular biology technology. CRISPRs (clustered regularly interspaced short palindromic repeats) are segments of prokaryotic DNA containing short repetitions of base sequences. Each repetition is followed by short segments of "spacer DNA" from previous exposures to a bacterial virus or plasmid. CRISPRs are found in approximately 40% of sequenced bacteria genomes and 90% of sequenced archaea. CRISPRs are often associated with cas genes that code for proteins related to CRISPRs.

Since 2013, the CRISPR/Cas system has been used in research for gene editing (adding, disrupting, or changing the sequence of specific genes) and gene regulation. By delivering the Cas9 enzyme and appropriate guide RNAs into a cell, the organism's genome can be cut at any desired location. The conventional CRISPR/Cas9 system is composed of two parts: the Cas9 enzyme, which cleaves the DNA molecule and specific RNA guides that shepherd the Cas9 protein to the target gene on a DNA strand.

Investigators at Uppsala University (Sweden) examined the intracellular search processes of the Cas9 protein, which can be programmed by a guide RNA to bind essentially any DNA sequence. This targeting flexibility requires Cas9 to unwind the DNA double helix to test for correct base pairing to the guide RNA. The investigators studied the search mechanisms of the catalytically inactive Cas9 (dCas9) in living Escherichia coli by combining single-molecule fluorescence microscopy and bulk restriction-protection assays.

Results published in the September 29, 2017, online edition of the journal Science revealed that it took a single fluorescently labeled dCas9 six hours to find the correct target sequence, which implied that each potential target was bound for less than 30 milliseconds. Once bound, dCas9 remained associated until replication.

“Most proteins that search DNA code can recognize one specific sequence merely by sensing the outside of the DNA double helix. Cas9 can search for an arbitrary code, but to determine whether it is in the right place the molecule has to open the double DNA helix and compare the sequence with the programmed code. The incredible thing is that it can still search the entire genome without using any energy,” said senior author Dr. Johan Elf, professor of biological physics at Uppsala University.

“The results show that the price Cas9 pays for its flexibility is time. To find the target faster, more Cas9 molecules searching for the same DNA sequence are needed. The results have given us clues on how we might achieve that kind of solution. The key is in what are known as the PAM sequences, which determine where and how often Cas9 opens up the DNA double helix. Molecular scissors that do not need to open the helix as many times to find their target are not only faster but would also reduce the risk of side-effects."

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