NEW YORK (GenomeWeb) – In a new study published yesterday in Nature, researchers revealed the underlying mechanisms of an RNA-guided genome-editing platform named CRISPR-CasX that is fundamentally distinct from Cas9 and Cas12a.
Meanwhile, in a study published today in Genome Biology, a different team of investigators identified six novel Cas12a orthologs for genome editing in human and mouse cells, some of which utilize simple protospacer adjacent motifs (PAMs) that significantly increase the targeting range for editing in the genomes.
RNA scaffolds play a role in both studies. In the Nature study, published by a team led by Jennifer Doudna at the University of California, Berkeley, eight cryo-electron microscopy structures of CasX in different states of assembly with its guide RNA and double-stranded DNA substrates showed that it has an extensive RNA scaffold and a domain required for DNA unwinding. The Genome Biology study, meanwhile, identified optimized CRISPR RNA (crRNA) scaffolds that can increase the genome editing efficiency of Cas12a.
CasX was first discovered by Doudna and several colleagues in a variety of uncultivated microbes in 2016. It was published in Nature alongside a second similar system dubbed CasY. In this study, she and her team demonstrated that CasX is an RNA-guided DNA endonuclease that generates a staggered double-stranded break in DNA at sequences complementary to a 20-nucleotide segment of its guide RNA, and that it further induces programmable, site-specific genome repression in E. coli and genome editing in human cells.
"Phylogenetic, biochemical and structural data show that CasX contains domains distinct from — but analogous to — those found in Cas9 and Cas12a, as well as novel RNA and protein folds; thus establishing the CasX enzyme family as the third CRISPR-Cas platform that is effective for genetic manipulation," the authors wrote. "The small size of CasX (<1,000 amino acids), its DNA cleavage characteristics, and its derivation from non-pathogenic microorganisms offer important advantages over other CRISPR–Cas genome-editing enzymes."
To determine the precise molecular activity of CasX, the researchers undertook biochemical studies of the wild-type CasX from Deltaproteobacteria (DpbCasX), and found that purified DpbCasX with a single guide RNA is capable of cleaving dsDNA that bears a sequence complementary to the 20-nucleotide guide RNA segment and adjacent to a TTCN PAM. Mapping the cut sites for the target and non-target strands of the DNA showed that DpbCasX generates products with staggered ends about 10 nucleotides in length. This mode of dsDNA cleavage is consistent with the staggered cuts to DNA observed for Cas12a and Cas12b, the researchers said.
Next, to test whether CasX displayed similar non-specific single-strand DNA degradation activity to Cas12a, the researchers incubated single-stranded phage DNA with DpbCasX-guide RNA complexes that target a separate, unrelated dsDNA substrate. They found that trans-ssDNA cutting activity was minimal compared to that observed for Lachnospiraceae bacterium Cas12a (LbCas12a) and another related Cas12b enzyme. This indicated that the presence of a single active site for dsDNA cleavage does not necessarily correspond to target-dependent trans-cleavage activity, hinting at structural and mechanistic differences between these enzyme families.
Further experiments found that CasX is capable of inducing cleavage and gene editing of mammalian genomes in ways that are different than Cas9 and Cas12a. "It is clear that CasX possesses domains analogous to other CRISPR-Cas proteins as well as completely novel domains, as anticipated by its complete sequence dissimilarity from other CRISPR proteins," the authors wrote. "A prominent guide RNA scaffold for CasX structural modelling shows that the guide RNA accounts for about 26 percent of the mass in the CasX-sgRNA binary complex, which is a value that is greater than those observed for other type II or type V CRISPR–Cas effector complexes (about 8 percent in LbCas12a, about 20 percent in Alicyclobacillus acidoterrestris Cas12b, and about 16 percent in SpCas9)."
These findings on CasX's compact size, dominant RNA content, and minimal trans-cleavage activity could enable its development into a third platform for RNA-programmed genome editing, the researchers added. The nuclease could also be used for therapeutic delivery and may be safer than existing genome-editing technologies.
In the second study, a team from China described its search for new Cas12a proteins using the PSI-BLAST program. The researchers identified 21 non-redundant CRISPR-Cas12a loci which had not previously been employed for genome editing, and four Cas12a proteins (BoCas12a, BsCas12a, PbCas12a, and TsCas12a) that had been characterized in another recent study from the same team.
The researchers observed conservation of the crRNA scaffolds in the Cas12a proteins which suggested that they might also recognize the 5' T-rich PAM. To further characterize the PAM requirements, they analyzed the Cas12a cleavage activity on dsDNA substrates bearing 5'-TTN, 5'-TNN, and 5'-NTN PAMs, and found that ArCas12a, BsCas12a, and PrCas12a recognized 5'-TTN PAM for dsDNA cleavage, and that HkCas12a recognized the simple 5'-YYN PAM.
The researchers next explored the capability of these Cas12a orthologs to cleave the target genomic sequences in mammalian cells. They fused each of the 12 synthesized Cas12a genes to two nuclear localization signals at each end, and constructed these complexes into mammalian expression vectors for Cas12a expression in human and mouse cells.
They found that six Cas12a nucleases (ArCas12a, BsCas12a, HkCas12a, LpCas12a, PrCas12a, and PxCas12a) could all facilitate genome editing in both human and mouse genomes with the 5'-TTTN PAM or 5'-TTN PAM. Sanger sequencing further confirmed the capacity of these six Cas12a nucleases to introduce indels at target sites in the mammalian genomes.
The researchers also observed that the crRNA scaffolds carrying nucleotide substitutions had variable effects on the cleavage activity of Cas12a nucleases. To test such effects in mammalian cells, they transfected three naturally existing crRNA scaffolds and two artificial scaffolds into mouse embryonic stem cells with each of the six functional Cas12a nucleases to target the mouse MeCP2 gene. This experiment showed that the highest cleavage efficiency was achieved by the Cas12a nuclease with its cognate crRNA scaffold in most cases.
"As previous studies indicated, crRNA scaffolds could affect or even enhance the targeting activities of CRISPR-Cas systems. Through engineering the nucleotide substitutions at the loop region, we identify a crRNA scaffold that markedly improves the Cas12a-mediated genome editing efficiency," the authors wrote. "The crystal structures of Cas12a-RNA-DNA complex have shown that the nucleotides in the loop region of crRNA scaffold interact with Cas12a residues, indicating nucleotide substitutions in the loop region of crRNA scaffold would affect the activities of Cas12a-crRNA complex."
Further experiments to address the off-target effects of Cas12a showed minimal risks, the team added.