Direct-seq: employing programmed gRNA scaffold for streamlined scRNA-seq in CRISPR screen

This protocol aimed to combine the single cell RNA-seq (scRNA-seq) with the CRISPR screening assay based on 10x 3’ RNA-seq platform to prole the transcriptome together with genotype at single cell resolution. To achieve this goal, an A/G mixed capture sequence and tRNA sequence are incorporated into the Tetraloop, Loop2 and Tail of the gRNA scaffold. Therefore, cDNA derived from the gRNA transcripts can be captured and barcoded by poly(dT) primer together with the endogenous mRNA and enriched by the nest PCR. With this method, the CRISPR perturbation and its transcriptional readouts can be proled together in a streamlined workow. This method, which we named as Direct-seq, enabled the direct genotyping and expression proling of the CRISPR screening products at single cell resolution on versatile scRNA-seq platforms across different throughput, including 10x 3’ and 5’ RNA-seq, Fluidigim C1, SMART-seq, BGI DNBelab C4 and others.


Introduction
CRISPR-based genome perturbation provides a new avenue to conveniently change DNA sequences, transcription and epigenetic modi cations in genetic screens. However, it's also a big challenge to pro le the transcriptome together with genotype at single cell resolution.
To overcome this challenge, two optimizations are applied to the gRNA scaffold design without negative impact to the genome editing performance. One modi cation is that an A/G mixed capture sequence (a 30-nt sequence, in which one guanine was incorporated with every seven adenosine except for the rst eight leading adenosine, "AAAAAAAAGAAAAAAAGAAAAAAAGAAAAA") was introduced into the Tetraloop, Loop2 or Tail of the gRNA scaffold to mimic the poly(A) tail of transcripts from polymerase II (Pol II). Therefore, gRNA transcripts can be captured and barcoded together with the endogenous mRNA via oligo(dT) from 10x GEM beads. The other modi cation is that a tRNA sequence, which locates at the downstream of the U6 promoter and upstream of the gRNA sequence, was included into the gRNA expression cassette to achieve multiplexed gRNA expression and also provided speci c primer binding sites for the nest PCR.
The following protocol is used to pro le the index gRNAs and endogenous mRNAs simultaneously at single cell resolution based on 10x 3' single cell RNA-seq platform. Single cell capture and mRNA library are prepared following the Chromium User Guide (10x Genomics, CG000184). The speci c enrichment of cDNA from gRNA transcripts and the gRNA library construction are described in details and other alterations are also indicated in this protocol.
For application of the Octo-seq work ow in other single cell RNA-seq platforms, please refer to the Figure1. (1) Long cDNA fragments, which mostly represented mRNA, were size-selected by 0.6x AMPure XP beads. The cDNA binding to the beads was called "long cDNA", and will subject to mRNA library preparation. The "long cDNA" was then eluted in nuclease-free H2O following the 10x protocol.
(2) Short cDNA fragments, which mostly represented the index gRNA, were collected from the supernatant of the 0.6x beads selection, and then followed by a 1.2x size selection. The cDNA binding to the beads was called "short cDNA". The "short cDNA" was then eluted in 25 uL nuclease-free H2O.

3' Gene Expression Library construction
The "long cDNA" was used for mRNA library preparation by following the manual of Chromium Single-Cell 3′ Reagent Kits v3 User Guide.

Index gRNA Library construction
The "short cDNA" was used for the index gRNA library preparation by nested PCR.
(3) The PCR products were combined and puri ed by 0.7-1.0× double-sided size selection (collect the supernatant from the 0.7x beads size selection, and followed by 1.0x beads selection, then collect elution from the beads) and eluted in 80μl of nuclease free water.
(3) The PCR product was cleaned up and size selected with 0.7-1.0x AMPure XP beads.
(4) All eluted DNA was combined, and quanti ed. An aliquot was sent for NGS sequencing using the Illumina platform.

Troubleshooting
(1) 2ng of the mRNA library and the index gRNA library were loaded into Fragment Analyzer to check the library quality. The typical library sizes were as below (Blue: mRNA library; Black: index library). (Figure 2) (2) During index gRNA enrichment procedure, the cDNA size was shown as Figure 3 Time Taken About two days. See "Procedure".

Anticipated Results
The mRNA library is constructed in very standard procedure, and the 10x manual should be strictly followed.
For the index gRNA library, the cDNA from gRNA transcripts were progressively enriched, which can be clearly re ected by the shift of fragment size as shown in the Figure3. The size-shift meets that expectation indicates e cient enrichment of the index gRNA. Figure 1 Work ows of the Direct-seq in different single cell RNA-seq platforms. (a) The suggested procedure to use with the 10x 3' kit; (b) The suggested procedure to use with the 10x 5' kit. The standard protocol should be followed until the pre-ampli cation of cDNA. The supernatant from size-selection contains gRNA sequences and could be speci cally enriched by the nested PCR. The 1st step could use a scaffold-speci c primer, and 2nd step could add the P5 and P7 sequencing adapters; (c) When work with the SMART-seq protocol, the cell barcode and UMI are suggested to be included in the template switch oligos (TSO) to enable sample pooling and eliminate PCR artifacts. The nested PCR is also suggested to make the gRNA library. Figure 2 2ng of mRNA library and index gRNA library were loaded into Fragment Analyzer to check the library quality. The typical library sizes were as below (Blue: mRNA library; Black: index library).