Base editor screens link sequence variation and gene function.

Cas12a RNA-guided endonucleases are promising tools for multiplexed genetic perturbations because they can process multiple guide RNAs expressed as a single transcript and subsequently cleave target DNA. However, their widespread adoption has lagged behind Cas9-based strategies due to low activity and the lack of a well-validated pooled screening toolkit. Here we describe the optimization of enhanced AsCas12a (enAsCas12a) for pooled, combinatorial genetic screens in human cells. By assaying the activity of thousands of guides, we refine on-target design rules and develop a comprehensive set of off-target rules to predict and exclude promiscuous guides. We also identify 38 direct repeat variants that can substitute for the wild-type sequence. We validate our optimized AsCas12a toolkit by screening for synthetic lethalities in OVCAR8 and A375 cancer cells, discovering an interaction between MARCH5 and WSB2 . Finally, we show that enAsCas12a delivers similar performance to Cas9 in genome-wide dropout screens but at greatly reduced library size, which will facilitate screens in challenging models.

Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function. Generating such pairs of mammalian cells, however, is laborintensive, time-consuming, and, in some cell types, essentially impossible. Here, we present an approach to create isogenic pairs of cells that avoids single cell cloning, and screen these pairs with genome-wide CRISPR-Cas9 libraries to generate genetic interaction maps. We query the anti-apoptotic genes BCL2L1 and MCL1, and the DNA damage repair gene PARP1, identifying both expected and uncharacterized buffering and synthetic lethal interactions. Additionally, we compare acute CRISPR-based knockout, single cell clones, and smallmolecule inhibition. We observe that, while the approaches provide largely overlapping information, differences emerge, highlighting an important consideration when employing genetic screens to identify and characterize potential drug targets. We anticipate that this methodology will be broadly useful to comprehensively study gene function across many contexts.

Isogenic pairs of cell lines, which differ by a single genetic modification, are powerful tools for understanding gene function. Generating such pairs for mammalian cells, however, is labor-intensive, time-consuming, and impossible in some cell types. Here we present an approach to create isogenic pairs of cells and screen them with genome-wide CRISPR-Cas9 libraries to generate genetic interaction maps. We queried the anti-apoptotic genes BCL2L1 and MCL1, and the DNA damage repair gene PARP1, via 25 genome-wide screens across 4 cell lines. For all three genes, we identify a rich set of both expected and novel buffering and synthetic lethal interactions. Further, we compare the interactions observed in genetic space to those found when targeting these genes with small molecules and identify hits that may inform the clinical uses for these inhibitors. We anticipate that this methodology will be broadly useful to comprehensively study genes of interest across many cell types.Genetic interaction networks can suggest functional roles of uncharacterized genes and capture subtle biological interactions, which may prove critical for interpreting genetic signal from genome-wide association studies of common disease states. Crosses of yeast knockout strains have yielded rich networks of genetic interactions, and have further shown that the shape of the network will change based on growth conditions 1-5 . In mammalian cells, the construction of such networks is orders of magnitude more complicated, due to increased genome size, the diversity of cell types, and numerous technical factors. One approach is to use either RNAi 6 or CRISPR technology 7-11 to screen a library of all possible combinatorial perturbations within a focused gene list. This approach has been used to generate genetic interaction maps for up to hundreds of genes 12 ; however, screening all combinations of protein coding genes in the human genome would require, at bare minimum, approximately 400 million perturbations and 200 billion cells, which is equivalent to 5,000 concurrent genome-wide screens with typical guide libraries and currently exceeds the practical limits of tissue culture. This scale is exacerbated by the diversity of cell types in which to study such interactions.A second, complementary approach to query genetic interactions leverages isogenic pairs of human cells, akin to mutant strains of model organisms, to enable the delineation of a given gene's contribution to phenotypes of interest. Initial gene targeting approaches in human cell lines to create even a single knockout have yielded valuable insights but were quite laborious to generate [13][14][15][16][17][18][19] . Today, CRISPR technology has made cell line engineering possible for a broad range of researchers, but that is distinct from making it easy. Creating the site-specific nuclease for Cas9 is as simple as ordering a short nucleic acid, in contrast to the more expensive and time-consuming task of assembling a customized pair of zinc finger nucleases or TALENs 20 . After design...

CRISPR knockout fitness screens in cancer cell lines reveal many genes whose loss of function causes cell death or loss of fitness or, more rarely, the opposite phenotype of faster proliferation. Here we demonstrate a systematic approach to identify these proliferation suppressors, which are highly enriched for tumor suppressor genes, and define a network of 145 such genes in 22 modules. One module contains several elements of the glycerolipid biosynthesis pathway and operates exclusively in a subset of acute myeloid leukemia cell lines. The proliferation suppressor activity of genes involved in the synthesis of saturated fatty acids, coupled with a more severe loss of fitness phenotype for genes in the desaturation pathway, suggests that these cells operate at the limit of their carrying capacity for saturated fatty acids, which we confirm biochemically. Overexpression of this module is associated with a survival advantage in juvenile leukemias, suggesting a clinically relevant subtype.