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.

VI 型 CRISPR 酶是具有核酸酶活性的 RNA 靶向蛋白，可在不改变基因组的情况下实现特异性和稳健的靶基因敲除。为了定义 Cas13d 引导 RNA (gRNA) 的设计规则，我们针对绿色荧光蛋白转基因的信使 RNA (mRNA) 以及人类细胞中的 CD46、CD55 和 CD71 细胞表面蛋白进行了大规模并行筛选。总的来说，我们测量了 24,460 个 gRNA 的活性，无论是否存在相对于目标序列的错配。击倒功效由 gRNA 特异性特征和靶位点背景驱动。单个错配通常会在一定程度上减少敲低，但间隔核苷酸 15-21 在很大程度上不能容忍靶位点错配。我们开发了一个计算模型来识别最佳 gRNA 并确认其普遍性，测试了针对 48 个内源基因 mRNA 的 3,979 个指导。我们证明 Cas13 可用于正向转录组汇集筛选，并使用我们的模型预测人类基因组中所有蛋白质编码转录本的优化 Cas13 gRNA。