Multiplexed orthogonal genome editing and transcriptional activation by Cas12a

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Abstract

CRISPR-Cas9-based combinatorial perturbation approaches for orthogonal knockout and gene activation have been impeded by complex vector designs and co-delivery of multiple constructs. Here, we demonstrate that catalytically active CRISPR-Cas12a fused to a transcriptional-activator domain enables flexible switching between genome editing and transcriptional activation by altering guide length. By leveraging Cas12a-mediated CRISPR-RNA array processing, we illustrate that Cas12a-VPR enables simplified multiplexed knockout and transcriptional activation in vitro and in vivo.

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Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells

Benjamin P. Kleinstiver, Shengdar Tsai, Michelle S. Prew … (2016)

The activities and genome-wide specificities of CRISPR-Cas Cpf1 nucleases 1 are not well defined. We show that two Cpf1 nucleases from Acidaminococcus sp. BV3L6 and Lachnospiraceae bacterium ND2006 (AsCpf1 and LbCpf1, respectively) have on-target efficiencies in human cells comparable with those of the widely used Streptococcus pyogenes Cas9 (SpCas9) 2–5 . We also report that four to six bases at the 3’ end of the short CRISPR RNA (crRNA) used to program Cpf1 nucleases are insensitive to single base mismatches, but that many of the other bases in this region of the crRNA are highly sensitive to single or double substitutions. Using GUIDE-seq and targeted deep sequencing analyses performed with both Cpf1 nucleases, we were unable to detect off-target cleavage for more than half of 20 different crRNAs. Our results suggest that AsCpf1 and LbCpf1 are highly specific in human cells.

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In vivo high-throughput profiling of CRISPR–Cpf1 activity

Hui Kim, Myungjae Song, Jinu Lee … (2016)

A lentiviral library expressing Cpf1 guide RNAs and containing target sequences allows high-throughput profiling of highly active guide RNAs and is the basis for cindel, a webtool to predict the activity at any given target sequence.

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Complex transcriptional modulation with orthogonal and inducible dCas9 regulators

Yuchen Gao, Xin Xiong, Spencer Wong … (2016)

The ability to dynamically manipulate the transcriptome is important for studying how gene networks direct cellular functions and how network perturbations cause disease. Nuclease-dead CRISPR–dCas9 transcriptional regulators, while offering an approach for controlling individual gene expression, remain incapable of dynamically coordinating complex transcriptional events. Here, we describe a flexible dCas9-based platform for chemical-inducible complex gene regulation. From a screen of chemical- and light-inducible dimerization systems, we identified two potent chemical inducers that mediate efficient gene activation and repression in mammalian cells. We combined these inducers with orthogonal dCas9 regulators to independently control expression of different genes within the same cell. Using this platform, we further devised AND, OR, NAND, and NOR dCas9 logic operators and a diametric regulator that activates gene expression with one inducer and represses with another. This work provides a robust CRISPR—dCas9-based platform for enacting complex transcription programs that is suitable for large-scale transcriptome engineering.