Department of Pharmacology, University of Alberta, Edmonton, AB, T6G 2R7, Canada.
The Applied Genomics Core, Office of Research, University of Alberta, Edmonton, AB, T6G 2E1, Canada.
Nat Commun. 2022 Mar 25;13(1):1617. doi: 10.1038/s41467-022-29202-x.
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.
CRISPR/Cas 复合物使在各种生物体中进行精确的基因编辑成为可能。虽然这些系统通过刚性识别 DNA 序列将脱靶效应的可能性降到最低，但这也给识别包含自然发生的多态性序列带来了问题。基因序列中存在遗传变异，如单核苷酸多态性 (SNP)，可能会影响 CRISPR 系统的靶标活性。因此，当试图使用单个向导 RNA 靶向人类基因的多个变体或病原体基因的进化变体时，需要更大的灵活性。在这里，我们证明 Cas9 可以容忍在单个向导 RNA 中包含通用碱基，从而能够同时靶向多态性序列。至关重要的是，我们发现特异性在通用碱基掺入的位点上选择性退化，但在其他方面保持不变。我们证明了这项技术在使用单个向导 RNA 靶向多个自然发生的人类 SNP 以及设计基于 Cas12a/Cpf1 的 DETECTR 探针以识别 HIV 蛋白酶基因的多个进化变体方面的适用性。我们的研究结果将 CRISPR/Cas 系统的靶向能力扩展到了其经典的间隔序列之外，并强调了天然和合成通用碱基的使用。
