The CRISPR–Cas system is a powerful genome editing tool that functions in a diverse array of organisms and cell types. The technology was initially developed to induce targeted mutations in DNA, but CRISPR–Cas has now been adapted to target nucleic acids for a range of purposes. CHOPCHOP is a web tool for identifying CRISPR–Cas single guide RNA (sgRNA) targets. In this major update of CHOPCHOP, we expand our toolbox beyond knockouts. We introduce functionality for targeting RNA with Cas13, which includes support for alternative transcript isoforms and RNA accessibility predictions. We incorporate new DNA targeting modes, including CRISPR activation/repression, targeted enrichment of loci for long-read sequencing, and prediction of Cas9 repair outcomes. Finally, we expand our results page visualization to reveal alternative isoforms and downstream ATG sites, which will aid users in avoiding the expression of truncated proteins. The CHOPCHOP web tool now supports over 200 genomes and we have released a command-line script for running larger jobs and handling unsupported genomes. CHOPCHOP v3 can be found at https://chopchop.cbu.uib.no

Polyadenylation at the 3 ′ ′ ′ ′ ′ -end is a major regulator of messenger RNA and its length is known to affect nuclear export, stability, and translation, among others. Only recently have strategies emerged that allow for genome-wide poly(A) length assessment. These methods identify genes connected to poly(A) tail measurements indirectly by short-read alignment to genetic 3 ′ ′ ′ ′ ′ -ends. Concurrently, Oxford Nanopore Technologies (ONT) established full-length isoform-specific RNA sequencing containing the entire poly(A) tail. However, assessing poly(A) length through base-calling has so far not been possible due to the inability to resolve long homopolymeric stretches in ONT sequencing. Here we present tailfindr, an R package to estimate poly(A) tail length on ONT long-read sequencing data. tailfindr operates on unaligned, base-called data. It measures poly(A) tail length from both native RNA and DNA sequencing, which makes poly(A) tail studies by full-length cDNA approaches possible for the first time. We assess tailfindr's performance across different poly(A) lengths, demonstrating that tailfindr is a versatile tool providing poly(A) tail estimates across a wide range of sequencing conditions.

Highlights d RCP-seq allows the study of both scanning and translating ribosomal complexes d Threading of mRNA through the small ribosomal subunit loads the subunits onto the 5 0 UTR d Processivity of small ribosomal subunits is high while scanning 5 0 UTRs d Direct measurements of initiation redefine optimal start codon context

The design of optimal guide RNA (gRNA) sequences for CRISPR systems is challenged by the need to achieve highly efficient editing at the desired location (on‐target editing) with minimal editing at unintended locations (off‐target editing). Although laboratory validation should ideally be used to detect off‐target activity, computational predictions are almost always preferred in practice due to their speed and low cost. Several studies have therefore explored gRNA‐DNA interactions in order to understand how CRISPR complexes select their genomic targets. CHOPCHOP (https://chopchop.cbu.uib.no/) leverages these developments to build a user‐friendly web interface that helps users design optimal gRNAs. CHOPCHOP supports a wide range of CRISPR applications, including gene knock‐out, sequence knock‐in, and RNA knock‐down. Furthermore, CHOPCHOP offers visualization that enables an informed choice of gRNAs and supports experimental validation. In these protocols, we describe the best practices for gRNA design using CHOPCHOP. © 2021 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Design of gRNAs for gene knock‐out Alternate Protocol 1: Design of gRNAs for dCas9 fusion/effector targeting Support Protocol: Design of gRNAs for targeting transgenic or plasmid sequences Basic Protocol 2: Design of gRNAs for RNA targeting Basic Protocol 3: Design of gRNAs for sequence knock‐in Alternate Protocol 2: Design of gRNAs for knock‐in using non‐homologous end joining Basic Protocol 4: Design of gRNAs for knock‐in using Cas9 nickases

Polyadenylation at the 3’-end is a major regulator of messenger RNA and its length is known to affect nuclear export, stability and translation, among others. Only recently, strategies have emerged that allow for genome-wide poly(A) length assessment. These methods identify genes connected to poly(A) tail measurements indirectly by short-read alignment to genetic 3’-ends. Concurrently Oxford Nanopore Technologies (ONT) established full-length isoform RNA sequencing containing the entire poly(A) tail. However, assessing poly(A) length through basecalling has so far not been possible due the inability to resolve long homopolymeric stretches in ONT sequencing.Here we presenttailfindr, an R package to estimate poly(A) tail length on ONT long-read sequencing data.tailfindroperates on unaligned, basecalled data. It measures poly(A) tail length from both native RNA and DNA sequencing, which makes poly(A) tail studies by full-length cDNA approaches possible for the first time. We assesstailfindr’sperformance across different poly(A) lengths, demonstrating thattailfindris a versatile tool providing poly(A) tail estimates across a wide range of sequencing conditions.