TITLE: GENERATING THE POOLED SINGLE GUIDE RNA LIBRARIES
AUTHORS: Helaine Graziele Santos Vieira, Helen E. King, Renuka Ravi Gupta, Nona Farbehi, hendersa, Vikram Khurana, Gist Croft, Lorenz Studer, Joseph Powell

[DESCRIPTION]
This protocol outlines the designing, cloning, and next-generation sequencing (NGS) of a pooled single guide RNA (sgRNA) library used with CRISPRi technology to knock down (KD) genes within midbrain dopaminergic (mDA) neurons. Dual guides1 have been designed to  target 30 candidate genes that regulate cytoskeletal function, Ca2+ homeostasis, and mRNA metabolism. The causative role of these candidate genes in post-mitotic mDA neurons is yet to be characterised transcriptomically through  single-cell RNA sequencing (scRNA-seq)  Our objective is to elucidate the impact of our chosen gene perturbations in post-mitotic mDA neurons due to CRISPR inactivation with scRNA-seq. 

[BEFORE_START]
Introduction: 
For our dual gRNA libraries, we use the following three elements:
A base Lentiviral Vector: pJR85 
Lentiviral CRISPR guide vector expressing an eGFP-NT2 sgRNA with cs1 incorporated in the loop of the sgRNA constant region. 
The BsmBI sites were removed to allow for programmed dual sgRNA library cloning. 
A insert from: pJR89
This plasmid contains sgRNA constant region and hU6 insert for programmed dual sgRNA cloning.
The sgRNA constant region contains a capture sequence (cs1) in the stem loop for direct capture Perturb-seq

 

 Library oligo pools:
The 10x Genomics compatible sgRNA library oligo pool was designed in silico by Weatherhitt Lab (Garvan) and was ordered from Twist Bioscience.

[STEPS]
SECTION: Synthesizing sgRNA oligonucleotides
1. The library oligo pool was designed in silico by the Weatheritt Lab (Garvan) and was ordered from Twist Bioscience. We utilised two in-silico tools FlashFry4 and CRISPRDO2, for guide design. Per gene, we designed two dual guides containing two sgRNAs targeting the interest gene. These are the protospacer, PS1, and PS2 sequences found in Figure 1 above. We ordered a pool of oligo with PS1-constant region as per Supplementary in Repogle et al. 1 and an example to target TP53 is below:

sgTP53
TCACAACTACACCAGAAGCCACCTTGTTGGAGGGAAGCGTGTCACCGTCGGTTTCAGAGCGAGACGTGTTTGATCTCGGGCCGTCTCAGAAACATGGAAGTCTAGAGCCACCGTCCAGTTTAAGAGCTAAGCTGGCAACACTTTGACGAAGA

where: 
PCR Adapters 002: 
    Fwd -TCACAACTACACCAGAAG 
    Rev - GCAACACTTTGACGAAGA
Red rest. enzyme sites
Photospacer A - AGGGAAGCGTGTCACCGTCG
Photospacer B - AAGTCTAGAGCCACCGTCCA 
CR3/cs1 paper sequence between photospacers-
GTTTCAGAGCGAGACGTGTTTGATCTCGGGCCGTCTCAGAAACATG
SECTION: Synthesizing sgRNA oligonucleotides
2. Prioritised guides that are found to be reported in hCRISPRi3 .
Prioritised guides that are found replicated across CRISPRDO2, FlashFry4 and with high specificity score (Moreno-Mateos2015 On Target) and low off-target score (Hsu2013).
Do not target known common variants found across the Parkinson’s Progression Markers Initiative (PPMI) cohort.
Do not contain enzyme restriction sites BstXI/Blp that are used within the dual guide strategy reported.
Paired dual guides that maximised the distance between respective guides.
SECTION: Generating pooled sgRNA plasmid library using the sgRNA oligonucleotides
3. The oligo Pools were delivered as a lyophilised product pooled in a single tube with a yield of 76.7ng.
Preparation of stock solution of the lyophilised oligo pool:
The oligo pool, ordered from Twist bioscience, contained 30 hit genes with 70 oligos at 152bps.
Prepare a stock solution of the Oligo Pool by resuspending in 10 mM Tris buffer, pH 8.0 to a concentration of at least 20 ng/µl.
Stock solution concentration (ng/ul)=[Total Yield (ng)/ Resuspension volume (ul)]
Use the KAPA HiFi HotStart PCR Kit to perform PCR. Since the oligo pool comes under the 2-100 oligos at 151-200nt, it will require 12-14 PCR cycles according to the manufacturer's protocol. 
Detailed protocol for reconstitution of oligo pool and PCR amplification step can be found in the following link: https://www.twistbioscience.com/sites/default/files/resources/2019-09/Guidelines_OligoPools_%20Amplification_29Aug19_Rev5.1.pdf

SECTION: Generating pooled sgRNA plasmid library using the sgRNA oligonucleotides
4. The pooled sgRNA mDA Neuron Library was cloned according to the following protocol by Weatheritt Lab Cloning of pooled sgRNAs into lentiviral vector.
SECTION: Production of lentiviral supernatant
5. Viral supernatant was produced in accordance with the following protocol:
 
 


SECTION: Titration of lentiviral supernatant
6. Lentiviral titration was done on H9 dCAS9 CRISPRi cells to determine the amount of virus required for an MOI of 0.1-0.3 (10% - 30% BFP positive cells). 
 

SECTION: Transduction of H9 dCAS9 CRISPRi
7. H9 dCAS9 CRISPRi were transduced with the pooled CRISPRi library to  obtain the gDNA (genomic DNA) after lentiviral transduction.
 

SECTION: sgRNA pool NGS sequencing library preparation
8. The genomic DNA was sample prepped for Illumina sequencing