CRISPR/Cas-mediated knock-in of DNA sequences enables precise genome engineering for research and therapeutic applications. However, designing effective guide RNAs (gRNAs) and homology-directed repair (HDR) donors remains a bottleneck. Here, we present protoSpaceJAM, an open-source algorithm to automate and optimize gRNA and HDR donor design for CRISPR/Cas insertional knock-in experiments, currently supporting SpCas9, SpCas9-VQR and enAsCas12a Cas enzymes. protoSpaceJAM utilizes biological rules to rank gRNAs based on specificity, distance to insertion site, and position relative to regulatory regions. protoSpaceJAM can introduce ‘recoding’ mutations (silent mutations and mutations in non-coding sequences) in HDR donors to prevent re-cutting and increase knock-in efficiency. Users can customize parameters and design double-stranded or single-stranded donors. We validated protoSpaceJAM’s design rules by demonstrating increased knock-in efficiency with recoding mutations and optimal strand selection for single-stranded donors. An additional module enables the design of genotyping primers for deep sequencing of edited alleles. Overall, protoSpaceJAM streamlines and optimizes CRISPR knock-in experimental design in a flexible and modular manner to benefit diverse research and therapeutic applications. protoSpaceJAM is available open-source as an interactive web tool at protospacejam.czbiohub.org or as a standalone Python package at github.com/czbiohub-sf/protoSpaceJAM.

中文翻译：

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protoSpaceJAM：一个开源、可定制且可通过网络访问的 CRISPR/Cas 插入敲入设计平台


CRISPR/Cas 介导的 DNA 序列敲入能够为研究和治疗应用提供精确的基因组工程。然而，设计有效的向导RNA（gRNA）和同源定向修复（HDR）供体仍然是一个瓶颈。在这里，我们介绍了 protoSpaceJAM，这是一种开源算法，用于自动化和优化 CRISPR/Cas 插入敲入实验的 gRNA 和 HDR 供体设计，目前支持 SpCas9、SpCas9-VQR 和 enAsCas12a Cas 酶。 protoSpaceJAM 利用生物学规则根据特异性、与插入位点的距离以及相对于调控区域的位置对 gRNA 进行排序。 protoSpaceJAM 可以在 HDR 供体中引入“重新编码”突变（沉默突变和非编码序列突变），以防止重新切割并提高敲入效率。用户可以定制参数并设计双链或单链供体。我们通过重新编码突变和单链供体的最佳链选择来证明提高的敲入效率，从而验证了 protoSpaceJAM 的设计规则。附加模块可以设计用于编辑等位基因深度测序的基因分型引物。总体而言，protoSpaceJAM 以灵活和模块化的方式简化和优化 CRISPR 敲入实验设计，以利于多样化的研究和治疗应用。 protoSpaceJAM 在 protospacejam.czbiohub.org 上作为交互式 Web 工具开源提供，或者在 github.com/czbiohub-sf/protoSpaceJAM 上作为独立的 Python 包提供。