CRISPR screening review
CRISPR (clustered regularly interspaced short palindromic repeats) genome editing is a technology that allows scientists to alter the genome of an organism. CRISPR/Cas cuts genes at specific positions, which are usually repaired with errors, knocking out the function of the target gene. The specificity of the cuts is pre-determined by a guide RNA (gRNA). CRISPR screening uses this technology to link key genes or genetic sequences to specific functions or phenotypes. A successful CRISPR screen generates a short list of candidate genes or genetic sequences that appear to participate in producing the physiological effect under investigation. Each identified gene or genetic sequence usually needs to be investigated further using other biological methods to determine if it really produces the effect being studied.
The basic idea of CRISPR screening is to use CRISPR to knock out every gene that could be important, but only knock out one gene per cell. CRISPR is followed by next generation sequencing (NGS) to confirm gene knockout. To perform the knockout, gRNA-encoding oligonucleotide libraries that target important genes are pooled and cloned into lentiviral plasmids. The conventional process uses T4 ligase or homology-based cloning techniques and often involves PCR amplification of gRNA libraries, a common source of library bias. The plasmids are transfected into cells to generate lentiviruses that are used to infect other cells, producing the gRNA used in CRISPR screens.
Vivlion gRNA library technology
The first step of CRISPR screening, making gRNA libraries by incorporating gRNA-encoding sequences into plasmids, can take enormous amounts of time and resources. The 3Cs technology utilized by Dr Manuel Kaulich’s lab provides a rapid method of gRNA library generation which works well with oPools Oligo Pools to encode the gRNA sequences that are inserted into the plasmids . oPools Oligo Pools are pools of custom single-stranded DNA sequences. They provide well-balanced gRNA representation and result in plasmids that produce high quality and uniformly distributed gRNAs. The plasmids are quality controlled using next generation sequencing.
“IDT oPools Oligo Pools are of outstanding uniformity and yield, both of which are key parameters for the generation of Vivlion’s high-quality 3Cs CRISPR libraries.”
—Dr Manuel Kaulich, CSO, Vivlion GmbH
Figure 1. 3Cs CRISPR screening preparation with pooled DNA oligos. Oligo Pools are inserted into the plasmid template as single-stranded DNA (ssDNA) before being heteroduplexed into a double strand and amplified by bacteria.
Demonstrated gRNA diversity
To demonstrate the performance of oPools Oligo Pools, Dr Kaulich and his lab first tested the reproducibility and fidelity of the lentiviral gRNA libraries by targeting 105 human deubiquitinating (DUBs) enzyme genes. They showed high reproducibility between biological replicates of these libraries, testing with R2 values at or above 0.88 at multiple timepoints after transduction, indicating the gRNAs are of high fidelity and reproducibility .
The team also created and tested the largest gRNA libraries to date, containing up to 7.3 x 1010 unique gRNA sequences that target both coding and noncoding regions in the human genome, using these libraries to screen for an antibiotic-resistance phenotype. Together, this work demonstrates fast and robust generation of quality 3Cs CRISPR gRNA libraries with high sequence diversity and minimal bias.