Optimized minimal genome-wide human sgRNA library
2023年7月18日 · Here we designed a minimal genome-wide human sgRNA library, H-mLib, which is composed of 21,159 sgRNA pairs assembled based on a dedicated selection strategy from all potential SpCas9/sgRNAs in...
Genome-scale CRISPR-Cas9 knockout and transcriptional ... - Nature
2014年12月10日 · This protocol from Feng Zhang's lab enables genome-scale knockout and transcriptional activation screening using the CRISPR-Cas9 system, as sgRNA libraries are constructed and packaged into ...
Pooled AID-seq 助力高通量sgRNA筛选 - 知乎 - 知乎专栏
Pooled AID-seq应用场景. （1）通过pooled AID-seq策略可以 筛选出切割效率最高、脱靶效应最低的sgRNA 进行基因编辑和治疗； （2）其次是用于 CRISPR文库优化：CRISPR文库常用于识别药物敏感性和耐药性的基因，然而，文库可能包含性能不佳的sgRNA，增加了不必要的文库大小并导致强烈的混杂效应。 因此，有必要使用pooled AID-seq策略针对全基因组或一类特殊目标筛选最佳sgRNA减少文库大小和提高文库性能； （3）AID-seq可用作通用工具，以 大规模并行方式 …
This protocol describes the ordering and cloning of pooled, programmed dual sgRNA libraries. As a general strategy, we first order dual-protospacer oligos as an oligonucleotide pool. Oligo pools are PCR-amplified, digested with BstXI/BlpI, gel extracted, and ligated into a similarly digested single sgRNA lentiviral vector.
A benchmark of algorithms for the analysis of pooled CRISPR …
2020年3月9日 · By transducing the sgRNA lentivirus pool into desired cell types (often at a multiplicity of infection much smaller than one to ensure less than one viral particle per cell), high-throughput, parallel loss-of-function genomic perturbations can be carried out to infer their functional relevance.
A genome-wide CRISPR/Cas9 screen to identify phagocytosis
2021年6月21日 · To further evaluate the efficacy of CRISPR mutagenesis, we transduced THP-1 iCas9 (clone A2) with a pool of sgETN-sgRNAs targeting 5 essential genes (TIMELESS, WDHD1, RAD21, SMC3, PLK1) or non ...
Next-Generation Sequencing of Genome-Wide CRISPR Screens
The combination of the simplicity of the CRISPR-Cas9 system, in which the short 20 base pair sgRNA sequence confers the targeting specificity of the Cas9 nuclease, economic oligonucleotide synthesis using pooled microarray synthesis, and the ability to use NGS sequencing for quantifying readout has enabled the use of pooled genome-scale loss-of ...
Pool: CP0022 (sgRNA) - Avana human genome-wide sgRNA pool …
Use the full 20mer (for S. pyogenes Cas9) or 21mer (for S. aureus Cas9) sgRNA target sequence to assess perfect matches to genomic PAM sites. Only consider matches where the "cut site" is properly contained in or touching (at extreme edge of) a target genomic feature loci.
Genome-wide programmable transcriptional memory by CRISPR …
2021年4月9日 · To perform a CRISPRoff pooled screen, we packaged the sgRNA library into lentiviral particles then transduced and selected HEK293T cells such that on average each cell expresses one sgRNA vector. We then transiently transfected this pool of cells with plasmids encoding CRISPRoff and sorted cells that expressed the CRISPRoff protein.
Human CRISPR Knockout Pooled Library (Brunello) - Addgene
Ready-to-use lentiviral pooled library for CRISPR screening in human cells. This backbone contains SpCas9 and unique gRNAs, and can be used to make edits across 19,114 genes in the human genome. In addition to the viral particles, you will also receive purified Human sgRNA library Brunello in lentiCRISPRv2.