The development of CRISPR-based barcoding methods creates an exciting opportunity to understand cellular phylogenies. We present a compact, tunable, high-capacity Cas12a barcoding system called dual acting inverted site array (DAISY). We combined high-throughput screening and machine learning to predict and optimize the 60-bp DAISY barcode sequences. After optimization, top-performing barcodes had ∼10-fold increased capacity relative to the best random-screened designs and performed reliably across diverse cell types. DAISY barcode arrays generated ∼12 bits of entropy and ∼66,000 unique barcodes. Thus, DAISY barcodes—at a fraction of the size of Cas9 barcodes—achieved high-capacity barcoding. We coupled DAISY barcoding with single-cell RNA-seq to recover lineages and gene expression profiles from ∼47,000 human melanoma cells. A single DAISY barcode recovered up to ∼700 lineages from one parental cell. This analysis revealed heritable single-cell gene expression and potential epigenetic modulation of memory gene transcription. Overall, Cas12a DAISY barcoding is an efficient tool for investigating cell-state dynamics.

基于 CRISPR 的条形码方法的发展为了解细胞系统发育创造了令人兴奋的机会。我们提出了一种紧凑、可调谐、高容量的 Cas12a 条形码系统，称为双作用倒置位点阵列 (DAISY)。我们结合高通量筛选和机器学习来预测和优化 60 bp DAISY 条形码序列。经过优化后，性能最佳的条形码相对于最佳随机筛选设计的容量增加了约 10 倍，并且在不同的细胞类型中都能可靠地执行。DAISY 条形码阵列生成约 12 位熵和约 66,000 个独特的条形码。因此，DAISY 条形码的大小仅为 Cas9 条形码的一小部分，实现了高容量条形码。我们将 DAISY 条形码与单细胞 RNA-seq 结合起来，从约 47,000 个人类黑色素瘤细胞中恢复谱系和基因表达谱。单个 DAISY 条形码可从一个亲代细胞中恢复多达 700 个谱系。该分析揭示了可遗传的单细胞基因表达和记忆基因转录的潜在表观遗传调节。总体而言，Cas12a DAISY 条形码是研究细胞状态动态的有效工具。