One-Day Construction Of Multiplex Arrays to Harness Natural CRISPR Systems

Abstract

CRISPR-Cas systems are prokaryotic immune systems that have proliferated widely not only in bacteria and archaea, but also much more recently, in human biological research and applications. Much work to date has utilized synthetic sgRNAs along with the CRISPR nuclease Cas9, but the discovery of array-processing nucleases now allows the use of more compact, natural CRISPR arrays in heterologous hosts, in addition to organisms with endogenous systems. Unfortunately, the construction of multiplex natural CRISPR arrays remains technically challenging, expensive, and/or time-consuming. This limitation hampers research involving natural CRISPR arrays in both native and heterologous hosts. To address this problem, we present a method to assemble CRISPR arrays that is simple, rapid, affordable, and highly scalable -we assembled 9-spacer arrays with one day's worth of work. We used this method to harness the endogenous CRISPR system of the highly competent bacterium Acinetobacter baylyi, showing that while single spacers are not always completely effective at blocking DNA acquisition through natural competence, multiplex natural CRISPR arrays enable both nearly complete DNA exclusion and genome editing, including with multiple targets for both. In addition to demonstrating a CRISPR array assembly method that will benefit a variety of applications, we also find a potential bet-hedging strategy for balancing CRISPR defense vs. DNA acquisition in naturally competent A. baylyi.CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems are adaptive immunity mechanisms that protect bacteria and archaea against invading nucleic acids, generally by detecting and cutting defined target sequences 1 . CRISPR systems include Cas (CRISPR-associated) proteins, as well as their eponymous arrays of short direct repeats that alternate with similarly short DNA spacers. The spacer array is transcribed into a long pre-crRNA, which is then processed into individual crRNAs (CRISPR RNAs), each composed of a single spacer that is complementary to a particular nucleic acid target, and often a hairpin handle derived from a repeat. These crRNAs bind Cas effector proteins, such as Cas9, or protein complexes, such as CASCADE. Once bound, they guide the effector to complementary DNA or RNA, depending on the system, which the effectors often cleave.In short order, many labs have adapted CRISPR-mediated DNA cleavage for applications ranging from precise genome engineering to genetic circuits 2 to targeted bacterial strain removal 3-6 . Self-spreading CRISPR constructs have also been used to quickly generate homozygous diploid knock-outs (the mutagenic chain reaction) 7 , and preliminary work suggests