genome wide pertub-seq

 CRISPi + scRNA

https://gwps.wi.mit.edu/

matrix file in H5AD format

https://doi.org/10.25452/figshare.plus.20029387

Genome wide screen targeted n=9867 genes. 

Essential-wide screen targeted n=2285 essential genes. 

Growth phenotypes were measured log2-guide enrichment per cell doubling (gamma)

"The relative homogeneity of CRISPRi reduces selection for unperturbed cells, especially when studying essential genes.  Unlike CRISPR knockout, CRISPRi does not lead to activation of the DNA damage response which can alter transcriptional signatures (Haapaniemi et al., 2018)."

"We use a compact, multiplexed CRISPR interference (CRISPRi) library to assay thousands of loss-of-function genetic perturbations with single-cell RNA sequencing (scRNA-seq) in chronic myeloid leukemia (CML) (K562) and retinal pigment epithelial (RPE1) cell lines."

There are four datasets on SRA:

1. K562 day 8 Perturb-seq (KD8): targeting all expressed genes at day 8 after transduction
2. RPE1 day 7 Perturb-seq (RD7): targeting DepMap essential genes at day 7 after transduction
3. K562 day 6 Perturb-seq (KD7): targeting DepMap essential genes at day 6 after transduction
4. K562 day 8 Perturb-seq (KD8_ultima): scRNA-seq libraries from the KD8 experiment sequenced on the Ultima sequencing platform rather than the Illumina sequencing platform

Potential problem: essential gene cannot be deleted? 

This is good data set for graph controllability, graph neural network analysis. 

genome wide crisp screen in human pancreatic tumor cell line

* Nature medicine, 2017. genome wide SCRIPS screens reveal a Wnt-FZD5 signaling circuit as a druggable vulnerability of RNF43 mutant pancreativ tumaors

https://www.nature.com/articles/nm.4219#supplementary-information
raw reads counts in Excel table
https://www.nature.com/articles/nm.4219#supplementary-information

From the paper, Bayes Factor BF is the confidence of a gene caucses a decrease in fitness. So, high BF indicates decrease in fitness (essential genes), and low BF indicate proliferation.

FZD5 genes shows highest BF factor in 3 pancreatic cancer cell lines, but not in other cells lines. Hence, its essentiality is "context sensitive".

CRISPR in budding yeast

Cut

Fully functional CRISPR/Cas enzymes will introduce a double-strand break (DSB) at a specific location based on a gRNA-defined target sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ), a mechanism which frequently causes insertions or deletions (indels) in the DNA. Indels often lead to frameshifts, creating loss of function alleles.

To introduce specific genomic changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the DSB and a specific edit close to the gRNA PAM site. When a repair template is present, the cell may repair a DSB using homology-directed repair (HDR) instead of NHEJ. In most experimental systems, HDR occurs at a much lower efficiency than NHEJ.

https://www.addgene.org/crispr/yeast/

There different types of CRIPSR/Cas kits: cut, based editing, nick, activate, and interference.

https://benchling.com/pub/ellis-crispr-tools

Editing Genomes to Record Cellular Histories

Whole organism lineage tracing by combinatorial and cumulative genome editing

http://science.sciencemag.org/content/early/2016/05/25/science.aaf7907


http://www.the-scientist.com/?articles.view/articleNo/46173/title/Editing-Genomes-to-Record-Cellular-Histories/&utm_campaign=NEWSLETTER_TS_The-Scientist-Daily_2016&utm_source=hs_email&utm_medium=email&utm_content=30023521&_hsenc=p2ANqtz-_R1n6GFzmrTt34_hkUqUVfwahxXsR3FIUKRDnqbJEghIVaQfXQ6FAd2sYy_HyvmbyFGoI2o9ihE5P2eTgED-mCTiOXyA&_hsmi=30023521

(toread) CRISPR mapping of QTL

http://science.sciencemag.org/content/early/2016/05/04/science.aaf5124

CRISPR/cas 9 systems, Cong and Zheng 2015, Chapter 10

There are 3 known CRISPR systems, types I, II, and III.  Type II CRISPR systems usually only require a single protein, Cas9, to perform the target cleavage. 

Cas9 is a RNA-guided nuclease that is capable of binding to a target DNA and introduce a double strand break in a sequence-specific manner. 

The guide sequence within sgRNA has a length of 20 bp and is the exact complementary sequence of the target site within the genome, sometimes referred to as “protospacer” following the

convention of microbial CRISPR field.

In choosing the target site, it is important to note the requirement of having the “NGG” trinucleotide motif, called protospacer adjacent motif (PAM), right next to the protospacer target on the 3′ end.

The PAM sequence depends on the Cas9 protein employed in the CRISPR-Cas9 system, and hence, the “NGG” PAM sequence applies specifi cally to the Streptococcus pyogenes Cas9 (SpCas9).