美国纽约大学Neville E. Sanjana研究团队取得一项新进展，他们利用大规模平行Cas13筛选揭示向导RNA的设计原理。相关论文于2020年3月16日在线发表于《自然—生物技术》。
Title: Massively parallel Cas13 screens reveal principles for guide RNA design
Author: Hans-Hermann Wessels, Alejandro Mndez-Mancilla, Xinyi Guo, Mateusz Legut, Zharko Daniloski, Neville E. Sanjana
Abstract: Type VI CRISPR enzymes are RNA-targeting proteins with nuclease activity that enable specific and robust target gene knockdown without altering the genome. To define rules for the design of Cas13d guide RNAs (gRNAs), we conducted massively parallel screens targeting messenger RNAs (mRNAs) of a green fluorescent protein transgene, and CD46, CD55 and CD71 cell-surface proteins in human cells. In total, we measured the activity of 24,460 gRNAs with and without mismatches relative to the target sequences. Knockdown efficacy is driven by gRNA-specific features and target site context. Single mismatches generally reduce knockdown to a modest degree, but spacer nucleotides 15–21 are largely intolerant of target site mismatches. We developed a computational model to identify optimal gRNAs and confirm their generalizability, testing 3,979 guides targeting mRNAs of 48 endogenous genes. We show that Cas13 can be used in forward transcriptomic pooled screens and, using our model, predict optimized Cas13 gRNAs for all protein-coding transcripts in the human genome.