Guide RNAs containing universal bases enable Cas9/Cas12a recognition of polymorphic sequences

Abstract

CRISPR/Cas complexes enable precise gene editing in a wide variety of organisms. While the rigid identification of DNA sequences by these systems minimizes the potential for off-target effects, it consequently poses a problem for the recognition of sequences containing naturally occurring polymorphisms. The presence of genetic variance such as single nucleotide polymorphisms (SNPs) in a gene sequence can compromise the on-target activity of CRISPR systems. Thus, when attempting to target multiple variants of a human gene, or evolved variants of a pathogen gene using a single guide RNA, more flexibility is desirable. Here, we demonstrate that Cas9 can tolerate the inclusion of universal bases in individual guide RNAs, enabling simultaneous targeting of polymorphic sequences. Crucially, we find that specificity is selectively degenerate at the site of universal base incorporation, and remains otherwise preserved. We demonstrate the applicability of this technology to targeting multiple naturally occurring human SNPs with individual guide RNAs and to the design of Cas12a/Cpf1-based DETECTR probes capable of identifying multiple evolved variants of the HIV protease gene. Our findings extend the targeting capabilities of CRISPR/Cas systems beyond their canonical spacer sequences and highlight a use of natural and synthetic universal bases.

Disrupting Protein Expression with Double-Clicked sgRNA-Cas9 Complexes: A Modular Approach to CRISPR Gene Editing

“…For example, a recent report by Krysler et al showed how chemically synthesized crRNA with universal bases, or nucleotides with the ability to base pair with all four bases, could be used to allow RNP complexes to target genes with many singlenucleotide polymorphisms (SNPs). 34 For a multi-gene editing assay, another double-clicked sgRNA was prepared containing a Cy5-modified thymine in the tetraloop that corresponded to the Siglec-3 target. This Cy5-modified sgRNA specific for Siglec-3 and an ATTO-550-modified sgRNA specific for Siglec-7 could be independently monitored by their distinct fluorescent properties.…”

Machine learning for design of degenerate Cas13a crRNAs using lassa virus as a model of highly variable RNA target

“…LASV, a viral pathogen and an etiologic agent of a highly lethal hemorrhagic fever (Lassa fever-LF) 18 , has always posed difficulty for the development of nucleic acid based assays due to its high variability with close to 30% divergence on the RNA level 19,20 . An alternative approach using "universal" crRNA for Cas9-based editing of polymorphic genes was shown by Krysler et al 21 That approach utilized nucleotides such as inosine (which pairs with all four RNA nucleotides). In comparison, our approach uses degenerate crRNA sets comprised of standard RNA nucleotides.…”

PAM-less Exonuclease-assisted Cas12a for visual detection of Vibrio Species

“…With PECAN, pathogens with small regions of conserved regions could be detected with cas12a without the presence of a conserved PAM site, which decreases the need for degenerative or polymorphic crRNA [50]. PECAN is applicable to the detection of bacterial or viral species.…”