CRISPR library 101 (Part 2) - how to select and construct a CRISPR library?

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CRISPR library 101 (Part 2) - how to select and construct a CRISPR library?



Whole genome CRISPR library or sub library? Loss of function or gain of function? Negative screening or positive screening? CRISPR library is such a good method for gene screening, but how to select and use it appropriately? In this article, we bring you the classification introduction, selection strategy, construction process and use of CRISPR library, to solve your doubts with one click! (Click to read our CRISPR library 101 (Part I) - What are the popular applications of CRISPR library?)


Classification and selection of CRISPR library


1.Screening coverage (whole genome library or sub library?)

According to the size of the library, it can be divided into whole genome library and sub library. The whole genome library usually designs gRNAs for all coding genes of a species, with a large coverage, but its screening workload is huge. Sub library refers to the combination of a certain type of genes, such as a gene family, a signal pathway, etc. At present, there are many ready-made libraries, such as the whole genome library of human, mouse, and green monkey, and sub library of kinase, nuclear protein, cyclin, metabolism related proteins and epigenetic related proteins. Generally, the library coverage should be selected according to the specific needs, followed by the ready-made library, and finally the smallest library that can meet the screening requirements should be selected to minimize the subsequent screening workload.

Ubigene has 8 off-shelf CRISPR libraries, covering the whole genome (human/mouse), kinase, nuclear protein, and metabolic genes. We also provide one-stop services from plasmid library construction, virus packaging, cell screening and NGS sequencing analysis. Libraries' coverage rate > 99%, uniformity <10. Contact us for more details>>


2.Screening requirements (loss of function, gain of function or others?)

After determining the screening coverage, we need to consider the screening requirements and clarify whether the purpose of the study is to carry out loss of function screening or gain of function screening? The former generally selects CRISPR-KO or CRISPRi system for construction. Generally speaking, the knockout effect of CRISPR-KO is better, but for cell essential genes (lethal after knockout) or non-coding genes (unable to use frameshift mutation to cause KO), CRISPRi system is a better choice. CRISPRi system is a fusion protein composed of catalytically inactive dCas9 and transcriptional repressor, which is cotransfected with gRNA targeting the upstream regulatory region of the gene to achieve gene knockdown. If gain of function screening is needed, the CRISPRa system is selected. The CRISPRa system enhances gene expression by connecting the catalytically inactive dCas9 with the transcription activator and targeting the upstream regulatory region of the gene.

Other requirements: 1) Barcode library: traditional CRISPR pool screening generally requires phenotypes related to cell proliferation or survival, which is conducive to the enrichment of specific cells. However, if it is an inconspicuous phenotype, such as metabolize, tissue homeostasis, etc., it is necessary to analyze the transcriptome of a single cell with the help of single-cell sequencing technology. In this case, when building the library, it may also be necessary to modify the CRISPR backbone and add the corresponding barcode sequence. 2) Base editing library: use the method of base editing (CBE, ABE, etc.) to build a single base mutation (SNVs) library for a gene or some genes.

library

Fig. 1 3different CRISPR librarie

3.Screening purpose (positive screening or negative screening?)

According to different screening purposes, it can be divided into positive screening and negative screening. Positive screening is to exert certain screening pressure on the cells that has successfully integrated sgRNAs, so that only a small number of cells with different phenotypes can survive and enrich key genes. Negative screening is on the contrary. Surviving cells are not target phenotypic cells. It is necessary to compare the abundance of sgRNAs at different time points to find out the difference sgRNAs to determine the key genes. However, negative screening can identify the genes that cause the loss of function of cells. If the screening time is prolonged, genes necessary for cell survival can also be screened. In the application cases mentioned in the previous article, screening driver gene and synthetic lethality gene belongs to negative screening, while screening drug resistance gene and host factor required for viral infection belongs to positive screening.


CRISPR library construction and workflow

1.Library construction: 

Design 3-6 sgRNAs for each gene of a species, synthesize sgRNAs using high-throughput chip synthesis method, and then clone the synthesized sgRNAs into lentiviral vector through Gibson method.
2.Lentivirus packaging and transduction: 

Packaging plasmid library into lentivirus, and infecting target cells with low MOI (generally < 0.3), so as to ensure that one virus particle infects one cell. The number of cells contained in the cell library is generally 200-1000 times the number of all sgRNAs.
3.Cell screening: 

Divide the whole genome knockout cell bank into two parts, one of which is used as the experimental group to apply screening condition, such as virus infection, drug treatment, etc; The other used as the control group. Cells are screened according to their drug resistance, proliferation ability, viability and other phenotypes.
4.NGS sequencing and data analysis: 

genomics are extracted from the cells of the experimental group and the control group, and sgRNA are amplified by PCR, followed by NGS sequencing and bioinformatics analysis.
5.Gene function validation: 

Knockout a single candidate gene by CRISPR/Cas9, or conduct overexpression or complementation experiments to verify gene function.

Features of Ubigene's CRISPR library service:
①Red Cotton CRISPR gene editing designer: 

high throughput automated design of gRNA to ensure gRNA specificity and cutting efficiency.
②Ensure high transformation efficiency: 

use self-developed competent cells, improve transformation efficiency by electro-transformation, and strictly control the number of bacteria ≥ 500X to ensure transformation efficiency. Ensure the library coverage > 99% and uniformity < 10.
③Rich experience in lentivirus packaging: 

ensure the lentivirus titer and purity and the uniformity of the packaging process of mixed plasmid.
④Nearly a hundred Cas9 stable cell lines are available: 

save the time of Cas9 lentivirus infection, and the library screening turnaround is shortened by 3-5 weeks.
⑤Rich experience and sophisticated platform: 

rich experience in cell culture and perfect downstream phenotype analysis platform can provide a variety of cell screening services.
⑥CRISPR-UTM cell line gene editing platform: 

it has accumulated more than 5000 successful cases on more than 200 cell lines, provides gene function validation services, and guarantees the delivery of KO homozygous clones.

Fig 2. CRISPR library construction and screening process


In addition to the eight in-stock CRISPR libraries mentioned above, Ubigene also provides customized services for CRISPR-KO, CRISPRi, and CRISPRa to meet different scientific research needs. Contact us for more details>>


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