NEW YORK (GenomeWeb) – The Cas9 enzyme is known to have certain problems with binding to off-target DNA sequences, but for the most part, it is still considered to be the gold standard CRISPR enzyme for genome editing.
However, in a new study in Molecular Cell today, researchers from the University of Texas at Austin suggested that the Cas12a enzyme — previously known as Cpf1 — is more precise than Cas9 and could lead to gene editing that's safe enough to be used in humans.
"The overall goal is to find the best enzyme that nature gave us and then make it better still, rather than taking the first one that was discovered through historical accident," Ilya Finkelstein, an assistant professor of molecular biosciences at UT Austin and a co-author of the study, said in a statement.
The researchers used quantitative kinetics to dissect how Acidaminococcus sp Cas12a targets DNA, and they found that the enzyme binds DNA tightly in two kinetically separable steps. First the enzyme recognizes the protospacer-adjacent motif (PAM), and then it propagates the rate-limiting R-loop, leading to inevitable DNA cleavage of both strands. Once those two steps are complete, Cas12a is functionally irreversibly bound to the DNA target sequence.
However, Cas12a also discriminates strongly against mismatches along most of the DNA target sequence. "This result implies substantial reversibility during R-loop formation — a late transition state," the authors wrote.
"It makes the process of base-pair formation more reversible," senior author Rick Russell added in the statement. "In other words, Cas12a does a better job of checking each base pair before moving on to the next one. After seven or eight letters, Cas9 stops checking, whereas Cas12a keeps on checking out to about 18 letters."
The team began by generating a complete kinetic framework for Cas12a on a matched DNA target. They measured rates of DNA binding and dissociation, as well as cleavage of each DNA strand, using a CRISPR RNA-loaded Cas12a and an oligonucleotide DNA target. Through various experiments, the researchers observed that R-loop formation by Cas12a occurred quickly and that the process was largely complete within a few seconds. They further observed that DNA cleavage by Cas12a occurred orders of magnitude faster than dissociation from a matched DNA target, indicating that essentially every complete binding event of Cas12a results in DNA cleavage.
To measure the specificity of Cas12a against mismatches between the CRISPR RNA and target DNA, the researchers then introduced single basepair changes in the DNA at each position throughout the R-loop. They found that when the Cas12a-CRISPR RNA bound to its target DNA, the formation of the R-loop had a later transition state, and more base pairs were formed.
"Indeed, assuming that the R-loop begins to form adjacent to the PAM sequence, the sensitivity of the binding rate to PAM-distal mismatches implies that propagation of the R-loop remains readily reversible until nearly the entire loop is formed," the authors wrote. "Biologically, this late transition state is expected to permit Cas12a to discriminate against mismatches in spite of the irreversible nature of the overall binding process."